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Tesis doctoral

Universidad de Navarra
Facultad de Ciencias

ESTUDIOS SOBRE LA FUNCIN DE LAS CLULAS DENDRTICAS Y


SU APLICACIN EN INMUNOTERAPIA DEL CNCER
STUDIES ON THE FUNCTION OF DENDRITIC CELLS AND THEIR
APPLICATIONS IN CANCER IMMUNOTHERAPY

Natalia Surez Fuentetaja


2011

Servicio de Publicaciones de la Universidad de Navarra


ISBN 978-84-8081-308-2

Universidad de Navarra
Facultad de Ciencias

ESTUDIOS SOBRE LA FUNCIN DE LAS CLULAS DENDRTICAS Y


SU APLICACIN EN INMUNOTERAPIA DEL CNCER

STUDIES ON THE FUNCTION OF DENDRITIC CELLS AND THEIR


APPLICATIONS IN CANCER IMMUNOTHERAPY

Memoria presentada por D


Natalia Surez Fuentetaja para aspirar
al grado de Doctor por la Universidad
de Navarra

El presente trabajo titulado Estudios sobre la funcin de las clulas dendrticas y


su aplicacin en inmunoterapia del cncer ha sido realizado bajo la direccin del Dr.
Ignacio Melero Bermejo en el Departamento de Terapia Gnica y Hepatologa del
Centro de Investigacin Mdica Aplicada (CIMA) de la Universidad de Navarra as
como bajo la supervisin de lvaro Gonzlez Hernndez en el Departamento de
Bioqumica de la Clnica Universidad de Navarra. Mediante este documento doy mi
aprobacin para que sea presentado por D. Natalia Surez Fuentetaja con el fin de optar
al grado de Doctor.

V B del Director del trabajo

V B del Co-Director del trabajo

Dr. Ignacio Melero Bermejo

Dr. lvaro Gonzlez Hernndez

A mis padres
Al amor de mi vida
A mis directores de tesis

AGRADECIMIENTOS
Esta tesis doctoral ha podido realizarse gracias a la participacin y el apoyo de
muchas personas. En este apartado intentar dejar constancia de lo que han aportado
cada una de ellas, esperando no dejarme a nadie en el tintero.
Para empezar, querra agradecer al Departamento de Bioqumica de la Clnica
Universidad de Navarra el haberme dado la oportunidad de realizar una tesis doctoral,
compatibilizando mi formacin como Biloga Interna Residente en Bioqumica Clnica
con la formacin investigadora. Del mismo modo, me gustara agradecer al grupo de
Terapia Gnica y Hepatologa del Centro de Investigacin Mdica Aplicada el
acogerme dentro de sus proyectos de investigacin, como una parte ms del equipo.
Dentro de este grupo me gustara resaltar a mis compaeros de experimentacin: Carlos
Alfaro y Lorena Erro, sin los que no habra sido capaz de realizar todo este trabajo.
Tambin me gustara mencionar al Grupo de Terapia Celular de la Clnica Universidad
de Navarra, que no slo me han enseado a trabajar en entorno GMP, sino que me han
permitido llevar a cabo la preparacin de las vacunas de clulas dendrticas presentadas
en la primera de las publicaciones.
Pasando al lado ms personal quisiera reconocer el valor que ha tenido para m el
apoyo de mis compaeros de residencia y amigos, en especial Sara Martnez, con quien
he pasado muy buenos momentos durante estos aos de aprendizaje y Alicia de Lzar
que me ha acompaado y cuidado en estos ltimos duros meses de escritura. No puedo
olvidar el papel de mi familia y de mi marido, que han estado a mi lado en todo
momento, aconsejndome y escuchndome aunque no entendieran muy bien todo
este gran mundo de las clulas dendrticas.
Por ltimo, pero no menos importante, quiero agradecer profundamente el trabajo
que han desarrollado mi director y codirector de tesis as como las horas que me han
dedicado durante todos estos aos. Esto es fruto de ellos.

NDICE

11

NDICE

11

1- INTRODUCCIN

17

1.1- Consideraciones generales sobre las clulas dendrticas

19

1.1.1- Precursores hematopoyticos y subtipos de clulas dendrticas

19

1.1.2- Activacin y maduracin de las clulas dendrticas

20

1.1.3- Funcin de las clulas dendrticas en el Sistema Inmune:


Inmunidad versus tolerancia

21

1.2- Inmunoterapia del cncer con clulas dendrticas


1.2.1-

Vacunas de clulas dendrticas

1.2.1.1-

22

Obtencin de clulas dendrticas o de sus precursores


de sangre perifrica

1.2.1.2-

22

23

Diferenciacin in vitro a clulas


dendrticas inmaduras

24

1.2.1.3-

Carga antignica de las clulas dendrticas

24

1.2.1.4-

Maduracin de las clulas dendrticas

24

1.2.1.5-

Vas de administracin de la vacuna

25

1.2.1.6-

Migracin in vivo de las clulas dendrticas administradas

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1.2.2-

Ensayos clnicos con clulas dendrticas: estado actual y retos

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1.3- Inmunoterapia combinada

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1.4- Inhibicin de la angiognesis en terapia del cncer

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1.4.1-

VEGF

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1.4.2-

Control de la expresin del VEGF

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1.4.3-

VEGF en el control de las funciones inmunolgicas

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1.4.4-

VEGF y cncer

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1.4.4.1-

Produccin de VEGF por las clulas tumorales y el


estroma tumoral

1.4.5-

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Terapia antiangiognica en cncer

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1.4.5.1- Anticuerpos neutralizantes de VEGF

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1.4.5.2- Inhibidores de VEGFR

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1.5- Desorientacin de las clulas dendrticas en cncer:


papel de la Interleuquina-8

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1.5.1- Quimioquinas

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1.5.2- Regulacin de la funcin de las clulas dendrticas por


quimioquinas

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1.5.3- Interleuquina-8 y cncer

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1.5.4- Desorientacin de la migracin leucocitaria por la


interleuquina-8 producida por los tumores

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1.6- Coestimulacin e inhibicin linfocitaria a travs de las


interacciones CD28/CTLA-4-CD80/86.
Aprovechamiento teraputico del sistema

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1.6.1- Generalidades

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1.6.2- Funcin de CTLA-4 en las clulas T activadas y en los


linfocitos T reguladores

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1.6.3- Ensayos clnicos con anticuerpos monoclonales anti-CTLA-4


en cncer

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1.7- Interacciones entre las clulas dendrticas y los leucocitos


polimorfonucleares

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1.7.1- Inmunidad innata versus adaptativa

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1.7.2- Comunicacin entre neutrfilos y clulas dendrticas

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2- OBJETIVOS

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3- ARTCULOS PUBLICADOS Y MANUSCRITOS ENVIADOS

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3.1-

An immunotherapy strategy based on type-1 dendritic cells


increases circulating interleukin-12 and NK activity while
decreasing circulating endothelial cells.
Alfaro C.; Prez-Gracia J L.; Surez N.; Rodrguez J.; Fernndez M.;
Sangro B.; Martn-Algarra S.; Calvo A.; Redrado.; Agliano A.;
Gonzlez A.; Rodrguez I.; Bolaos E.; Hervs-StubbsS.; Prez-Calvo J.;
Benito A.; Peuelas I.; Richter J.; Martnez-Forero I.; Melero I
Manuscrito enviado

3.2-

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Influence of bevacizumab, sunitinib and sorafenib as single


agents or in combination on the inhibitory effects of vegf on

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human dendritic cell differentiation from monocytes.


Alfaro C.; Surez N.; Gonzlez A.; Solano S; Erro L.; Dubrot J.;
Palazn A.; Hervas-Stubbs S.; Grpide A.; Lpez-Picazo J.;
Grande-Pulido E.; Melero I.; Prez-Gracia J.
British Journal of Cancer, 2009 Abril 7; 100 (7): 1111-9
3.3-

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Carcinoma-derived interleukin-8 retains dendritic cells


intratumorally without causing alteration on their T-cell
stimulation capabilities.
Alfaro C.; Surez N.; Martnez-Forero I.; Palazn A.; Rouzaut A.;
Solano S.; Feijoo E.; Grpide A.; Erro L.; Dubrot J.; Hervs-Stubbs S.;
Gonzlez A.; Prez-Gracia JL.; Melero I.
PloS ONE, 2011 Marzo 14; 6(3): e17922

3.4-

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Synergistic effects of CTLA-4 blockade with tremelimumab


and elimination of regulatory T lymphocytes in vitro and in vivo.
Surez N.; Alfaro C.; Dubrot J.; Palazn A.; Solano S.; Hervs-Stubb S.;
Martnez-Forero I.; Lecanda F.; Prez-Gracia JL.; Gonzlez A.;
Melero I.
International Journal of Cancer, 2010 Septiembre. 129: 347-386

3.5-

143

Dendritic cells take-up and present antigens from viable


and apoptotic polymorphonuclear leukocytes.
Surez N.; Alfaro C.; Prez-Gracia JL.; Martnez-Forero I.; Hervs-Stubbs S.;
Rodrguez I.; Erro L.; Bolaos E.; Palazn A.; Morales-Kastresana A.;
Gonzlez A.; Melero I.
Manuscrito en preparacin

167

4- DISCUSIN GENERAL E IMPLICACIONES FUTURAS

203

5- CONCLUSIONES

209

6- BIBLIOGRAFA GENERAL

213

7- LISTA DE ABREVIATURAS

239

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1- INTRODUCCIN__________________________________

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1.1- Consideraciones generales sobre las clulas dendrticas


1.1.1- Precursores hematopoyticos y subtipos de clulas dendrticas
Las clulas dendrticas (CD) fueron descubiertas hace aproximadamente 40 aos por
Steinman y col. (Steinman y Cohn 1973) como unas clulas adherentes minoritarias, con
capacidad de activar linfocitos T sin experiencia antignica previa. Esta estirpe celular
presentaba prolongaciones citoplasmticas que se asemejaban a las dendritas neuronales
(Steinman y Banchereau 2007).
De los precursores hematopoyticos derivan subpoblaciones de CD con caractersticas
fenotpicas y funcionales diferentes. Segn esto, las CD poseen gran plasticidad ontognica y
funcional. As lo demuestran las diferencias observadas en su origen, en sus marcadores de
diferenciacin leucocitaria, en su distribucin tisular y en la regulacin de la respuesta
inmunitaria (Guermonprez et al. 2002; Shortman y Liu 2002; Ardavin 2003). Adems, el
microambiente en el que se encuentran las CD determina en gran medida sus actividades
funcionales (Wu y Liu 2007). El conocimiento de los subtipos de CD ha progresado de forma
paralela en ratones y humanos. En modelos murinos es ms fcil la experimentacin funcional,
pero an as, es crucial realizar estudios con CD humanas debido a las sutiles diferencias entre
los sistemas inmunolgicos de ambas especies, las cuales pueden ser muy importantes.(Mestas
y Hughes 2004).
Podemos diferenciar dos grandes tipos de CD humanas: CD mieloides (tambin llamadas
convencionales) y CD plasmacitoides. En sangre humana se distinguen los subtipos de CD de
acuerdo con la expresin de tres molculas de superficie celular: BDCA-1 (Blood Dendritic Cell
Antigen 1) tambin conocida como CD1c, BDCA-2 (CD303) y BDCA-3 (CD141) (Dzionek et
al. 2000):
A)

Las CD mieloides se diferencian, a su vez, en dos subtipos segn la expresin

de CD1c y CD141. La subpoblacin de CD mejor equipada para presentar antgenos y activar


linfocitos T citotxicos (LTC) es una poblacin que reside principalmente en rganos linfoides
secundarios. Se ha identificado en base a la expresin de lectinas cCLEC-9 y del marcador
CD141+. La poblacin homloga en ratn se caracteriza por coexpresar CD11c y CD8
(Shortman y Heath 2010). Estas clulas estn ausentes en el ratn que carece del factor de
transcripcin BAFF (B-Cell Activating Factor) y como consecuencia estos ratones tienen
respuestas muy debilitadas de LTC. Dado que en el hombre las clulas CD141+ son residentes

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en el ganglio, se postula que captan antgeno que accede al ganglio de forma soluble o que los
antgenos son transportados por otra estirpe celular que migra desde los tejidos perifricos a los
rganos linfoides. As pues, las CD mieloides CD141+ se caracterizan por una alta capacidad
para capturar antgenos exgenos y presentarlos asociados a molculas presentadoras del
Complejo Mayor de Histocompatibilidad de clase I (CMH I). Este fenmeno es conocido como
presentacin antignica intermediada o cruzada (crosspresentation) (Jongbloed et al. 2010).
Adems, esta poblacin expresa en su superficie celular el receptor de quimioquinas XCR1
(necesario para responder al estmulo de XCL1, secretado por las clulas natural killer (NK) y
linfocitos T CD8+ activados) y la molcula de adhesin Necl2 [protena de unin a la molcula
asociada a linfocitos T restringida a clase I (CRTAM), que se expresa principalmente en las
clulas NK, NK-T y linfocitos T CD8+]. As, las CD mieloides CD141+ estn muy bien
equipadas para la generacin de LTC en la inmunidad celular (Palucka et al. 2010). Las CD
mieloides CD1c tambin pueden presentar de forma cruzada antgenos y secretar IL-12
(Jongbloed et al. 2010; Poulin et al. 2010).
B)

El otro gran subtipo de CD humanas corresponde a las CD plasmacitoides, que

expresan en su superficie el antgeno CD303. Ejercen su actividad principalmente en la


inmunidad antiviral, con una gran capacidad para producir interfern de tipo I (IFN I) en
respuesta a virus (Siegal et al. 1999; Liu 2005). Adems se caracterizan por expresar la cadena
IL-3R (CD123) e ILT-7 (Cao et al. 2006).

1.1.2- Activacin y maduracin de las clulas dendrticas


Las CD son las principales clulas presentadoras de antgeno (CPA) del organismo
(Banchereau y Steinman 1998; Banchereau et al. 2000). Desempean un papel esencial en la
iniciacin, programacin y regulacin de las respuestas inmunolgicas antgeno-especficas,
estimulando eficientemente a linfocitos B y T; son tambin cruciales en el control de linfocitos
NK (Zitvogel et al. 2006). Las CD intensifican la presentacin antignica cuando se induce
sobre ellas un programa de expresin gnica que se conoce como maduracin (o activacin).
Los cambios que definen el fenotipo de las CD maduras son: (i) prdida de la intensa capacidad
fagoctica que tienen las CD en estado inmaduro; (ii) interrupcin del procesamiento antignico;
(iii) cambios en el patrn de receptores de quimioquinas expresados en la membrana, con la
finalidad deorientar su migracin a rganos linfoides secundarios y presentar all el antgeno a
los linfocitos T (Alvarez et al. 2008); (iv) incremento de expresin en la superficie celular, de
molculas del CMH de tipo I y II; (v) aumento en la expresin de molculas de coestmulo
como CD80, CD86 y CD40; (vi) produccin de citoquinas proinflamatorias como IL-12, IL-15

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e IFN- y otros mediadores inflamatorios (Tirapu I. 2005). Este programa de maduracin se


pone en marcha en respuesta a estmulos tales como: (i) citoquinas proinflamatorias (TNF-,
IL-1, IL-12, IFN-, IFN-, etc); (ii) interaccin con linfocitos T colaboradores activados,
mediada por CD40-CD40L (Lanzavecchia 1998) y (iii) tras contacto con molculas
caractersticas de microorganismos como el lipopolisacrido (LPS) o como el cido
desoxirribonucleico (ADN) de origen bacteriano o el cido ribonucleico (ARN) de origen vrico
(Akira et al. 2001; Takeda et al. 2003). Estas seales de activacin y maduracin se transmiten
al interior de las CD generalmente a travs de vas de sealizacin intracelular mediadas por
miembros de la familia NF-B, pero dependiendo del estmulo madurativo, tambin
contribuyen de un modo esencial las rutas de MAP quinasas.
Los mecanismos que regulan la maduracin y los movimientos migratorios de las CD en
los tejidos dependen fundamentalmente del microambiente que rodea a las clulas (Yang y
Carbone 2004; Alvarez et al. 2008) e incluyen quimioquinas y su interaccin con capilares
linfticos (Rouzaut et al. 2010).

1.1.3- Funcin de las clulas dendrticas en el sistema inmune:


Inmunidad versus tolerancia
Las CD son capaces de capturar antgenos, ya sea a travs de pinocitosis, fagocitosis o
endocitosis mediada por receptores de membrana, tales como receptores Fc para
inmunocomplejos, receptores de lectina de tipo C (como por ejemplo DEC-205 y MMR)
(Guermonprez et al. 2002; Trombetta y Mellman 2005), y receptores tipo Toll (TLR),
especficos para patrones moleculares asociados a patgenos (PMAPs) (Takeda et al. 2003) y
otras sustancias tales como HMGB-1 (Apetoh et al. 2007) o el dominio A extra de fibronectina
(Lasarte et al. 2007). Las CD tras capturar a las molculas de su entorno, las internalizan y
procesan, para posteriormente presentarlas en superficie a los linfocitos T. La presentacin
antignica ocurre gracias a la expresin de molculas del CMH de clase I y II cargadas de
pptidos antignicos. Por ello las CD pueden inducir una respuesta inmunolgica antgenoespecfica. Pero la presentacin antignica puede resultar tanto en inmunizacin como en
tolerancia y ello se debe a que la capacidad de las CD para generar una respuesta inmunognica
no es constitutiva (Belz et al. 2002). En condiciones de reposo, en las que no existe inflamacin
o infeccin, las CD estn involucradas en generar y mantener la tolerancia perifrica,
eliminando o inactivando a linfocitos T autorreactivos (Bonifaz et al. 2004), as como activando
a una poblacin linfocitaria minoritaria: los linfocitos T reguladores (CD4+CD25+FoxP3+).

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Existen mltiples evidencias experimentales que indican que estos linfocitos T reguladores
estn encargados de suprimir o inhibir la actividad de los linfocitos T activados (Roncarolo et
al. 2001; Steinman 2003) .
Las CD se encuentran en el organismo en dos estadios diferentes: por un lado estn las
CD inmaduras, que presentan una elevada capacidad fagoctica pero un escaso potencial
activador de linfocitos T, y las CD maduras, que tienen muy desarrollada su capacidad para
migrar desde el foco de lesin o inflamacin a los rganos linfoides secundarios. Una vez all
son capaces de inducir una respuesta celular antgeno-especfica por parte de linfocitos T. Se
piensa que mientras las CD inmaduras son tolerognicas, las maduras son, por el contrario,
inmunognicas (Mahnke et al. 2002; Steinman et al. 2003). Por otra parte, existen estadios
intermedios entre las CD inmaduras y maduras, en los cuales podran no ser totalmente
funcionales y por tanto inducir tolerancia. La tolerancia perifrica que inducen est mediada por
deleccin clonal o por induccin de anergia de clulas T (Mahnke et al. 2002).

1.2- Inmunoterapia del cncer con clulas dendrticas


El objetivo de la inmunoterapia del cncer es reorientar en contra de los tumores los
mismos mecanismos que son capaces de generar una respuesta inmunolgica extremadamente
potente contra los antgenos de bacterias intracelulares y virus (Lopez-Bravo y Ardavin 2008).
Las caractersticas que acompaan a las infecciones por estos patgenos son: destruccin celular
e inflamacin en el tejido afectado, la naturaleza generalmente particulada de los antgenos, su
acceso a tejidos linfoides, as como mecanismos innatos de reconocimiento de patrones
moleculares (Kumar et al. 2011) ausentes en eucariotas y organismos superiores. Del mismo
modo, la muerte de las clulas tumorales puede conllevar la captacin de antgenos liberados al
producirse dao tisular. Esto se piensa que ocurre, por ejemplo, en tumores sometidos a
radioterapia y quimioterapia, aumentando de este modo la transferencia de antgenos a las CD
que se localizan prximas al microambiente tumoral (Melero et al. 2006; Kepp et al. 2011).

1.2.1- Vacunas de clulas dendrticas


Se considera que las CD son excelentes adyuvantes para la vacunacin (Palucka et al.
2010). El importante papel que tienen las CD como principales CPA del organismo, as como su
capacidad dicotmica para activar o inhibir una respuesta del sistema inmune, ha sido explotado

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en el tratamiento del cncer y enfermedades autoinmunes (Marin-Gallen et al. 2010), as como


en la prevencin del rechazo de trasplantes (Figdor et al. 2004). Hasta la fecha, la mayor parte
de las vacunas de CD han sido empleadas para estimular la respuesta inmunolgica, en
particular frente al cncer y el virus de la inmunodeficiencia humana (VIH) (Garcia et al. 2011).

Una vacuna de CD se define como aquella formada por CD cargadas con un antgeno de
inters, por ejemplo un antgeno asociado al tumor (Figdor et al. 2004). Tras su administracin
a los pacientes, la vacuna est pensada para inducir una respuesta celular T especfica frente a
los antgenos del tumor presentados por las CD. El primer estudio clnico de una vacuna de CD
fue publicado en Nature Medicine en el ao 1986 (Hsu et al. 1996). El grupo de R. Levy en la
Universidad de Stanford describi de forma pionera una pauta de vacunacin repetida en cuatro
pacientes con linfoma no-Hodgkin utilizando CD de sangre perifrica y la molcula de
inmunoglobulina producida por el linfoma de cada paciente con el objetivo de inducir
inmunidad antiidiotpica. Desde entonces, la vacunacin con CD se ha aplicado en mltiples
ensayos clnicos en pacientes con neoplasias malignas y en pacientes con infecciones virales
crnicas (por ejemplo VIH) (Miro et al. 2004; Banchereau y Palucka 2005). Se ha demostrado
la capacidad inmunizante de las CD y su seguridad en voluntarios sanos (Dhodapkar y
Bhardwaj 2000).

A continuacin se van a describir los pasos clave en el desarrollo de una vacuna de CD


para inmunoterapia del cncer, as como el estado actual del tema y las dificultades que conlleva
su implantacin asistencial (Palucka et al. ; Figdor et al. 2004; Gilboa 2007; Steinman y
Banchereau 2007):

1.2.1.1- Obtencin de CD o de sus precursores de sangre perifrica


Gracias al trabajo de Inaba, Steinman y col, qued demostrada la capacidad de generar
CD de ratn ex vivo, a partir de precursores hematopoyticos de mdula sea (Inaba et al.
1992). De forma similar, las CD humanas pueden ser obtenidas por diferenciacin in vitro de
precursores hematopoyticos CD34+, y ms comnmente, a partir de monocitos de sangre
perifrica (Banchereau y Palucka 2005; Nestle et al. 2005).

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1.2.1.2- Diferenciacin in vitro a CD inmaduras


Los monocitos se diferencian a CD inmaduras tras 5-7 das en cultivo, en presencia de
factor estimulante de colonias granulocitos-macrfagicos (GM-CSF) e IL-4 (Figdor et al. 2004),
aunque esta ltima citoquina est siendo sustituida en algunos protocolos por IFN- (Gabriele et
al. 2004) e IL-15 (Dubsky et al. 2007) debido a las observaciones de buenos resultados en
modelos preclnicos.

1.2.1.3- Carga antignica de las CD


Este es un paso clave a la hora de disear una vacuna de CD. El objetivo es introducir los
antgenos de inters de manera que se procesen de forma ptima por las maquinarias de
presentacin de clase I y II (Gilboa 2007). El antgeno tumoral puede ser aportado a las CD en
varias formas: (i) aadido exgenamente en forma de pptido, protena, lisado tumoral o restos
de clulas tumorales apoptticas y (ii) mediante transfeccin de material gentico, ya sea ARN
mensajero (ARNm) o ADN complementario (ADNc). Aunque las observaciones originales se
realizaron empleando antgenos sintticos como estmulo, parece que las mezclas antignicas
complejas (qumicamente indefinidas) procedentes del propio tumor, son las que pueden
producir una terapia anticancerosa de mayor eficacia ya que se incluye todo el repertorio
antignico del tumor (Melero et al. 2000). Esto es importante ya que se considera que cada
tumor es distinto en cuanto a las mutaciones que determinan su fenotipo maligno. El problema
es que al optar por el uso de antgenos indefinidos se hace ms difcil la medicin y la
comparacin de las respuestas inmunes, debido a la naturaleza molecular desconocida de los
antgenos inmunodominantes o subdominantes. Referente a la segunda modalidad de carga
antignica, el uso de transfeccin de cidos nucleicos, permite introducir junto con los genes
que codifican para los antgenos, otros genes que confieren una mayor eficiencia funcional de
las CD (Van Nuffel et al. 2010).

1.2.1.4- Maduracin de las CD


La mayor parte de los protocolos de maduracin para las CD diferenciadas in vitro a
partir de monocitos consistan en la incubacin con cuatro reactivos: TNF-, IL-1, IL-6 y
prostaglandina E2 (PGE2) (Gilboa 2007). Curiosamente, no se emplean productos
microbiolgicos como LPS, ARN de doble cadena y otros potentes estmulos experimentados
en ratn, debido a la dificultad para obtenerlos en grado clnico o GMP (Good Manufacturing
Practice). Existen datos que muestran que las CD maduradas solamente con TNF- pueden
convertirse en tolerognicas (Menges et al. 2002). La presencia de PGE2 se basaba en la

24

necesidad de obtener CD maduras que migraran a ganglio (para lo que necesitan expresar
CCR7, que es inducido por PGE2), pero su empleo en la mezcla de estmulos de maduracin ha
sido criticado debido a la capacidad de la PGE2 para favorecer una respuesta de tipo Th2 en el
microambiente tumoral. Esto es as debido a que la PGE2 promueve la secrecin de IL-10 por
las CD (Morelli y Thomson 2003). Debido a la necesidad de una mezcla madurativa eficaz,
Kaliski y col desarrollaron una mezcla, consistente en el empleo de 5 estmulos: TNF-, IL-1,
poly I:C, IFN- e IFN- (Mailliard et al. 2004), que efectivamente maduraba CD con una gran
capacidad inmunognica como inductoras de la respuesta de LTC. Adems posean una elevada
capacidad migratoria in vitro y una intensa produccin de IL-12p70. El poly I:C acta como un
anlogo de ARN viral de doble cadena, estimulando la va TLR3 en los endosomas y RIG-1 en
el citoplasma (Schulz et al. 2005).

Hoy da contina sin haber consenso sobre cual es la mejor combinacin de citoquinas
para madurar las CD. Nuestro grupo emplea para esos fines una mezcla madurativa que consta
de IFN-, TNF- y poly I:C, en grado GMP, obteniendo buenos resultados.

1.2.1.5- Vas de administracin de la vacuna


Con la idea de mejorar la migracin de las CD a los ganglios linfticos, se han estudiado
tanto en humano como en ratn otras vas de administracin alternativas a la intradrmica. As,
por ejemplo, se ha visto que la administracin intravenosa de CD en ratones da lugar a su
acmulo en bazo (Eggert et al. 1999). Para optimizar la va de administracin, se siguen las
siguientes lneas de investigacin: (i) pretratamiento de la zona de la piel en la que se va a
administrar la vacuna de CD, con citoquinas proinflamatorias como en TNF- (Martn-Fontecha
et al. 2003), o agonistas de TLR (Nair et al. 2003) y (ii) la administracin directa de la vacuna
de CD por va intraganglionar (guiada por ecografa). A pesar de las crticas recibidas por la
posibilidad de destruir la estructura del ganglio linftico durante su administracin, se ha
comprobado la eficacia en la migracin de las CD a lo largo de las cadenas linfticas empleando
esta tcnica. Mullins y col demostraron en el ao 2003 que la va de administracin es un
aspecto importante, no slo para obtener una buena respuesta inmune primaria, sino para
determinar la distribucin de linfocitos memoria y para inhibir la invasin de tejidos perifricos
por parte de las clulas tumorales (metstasis). Tambin pueden realizarse administraciones
intratumorales de las CD, pero en este caso la vacuna est habitualmente constituida por CD sin
carga antignica previa, para que stas puedan internalizar los antgenos tumorales en el propio
foco de la lesin maligna (Melero et al. 2000).

25

As pues, parece que podra ser til la combinacin de varias rutas de administracin de
las vacunas de CD en los pacientes con cncer a la hora de inducir una respuesta inmune
sistmica capaz de erradicar los focos tumorales existentes en el organismo (Adema et al. 2005).
Adems, la repeticin de inmunizaciones con las vacunas tambin parece un punto clave
(Ochsenbein et al. 1999; Tirapu et al. 2004). Sin embargo, por el momento no hay consenso en
los protocolos ni en el intervalo entre las dosis, ni en el nmero de repeticiones.

1.2.1.6- Migracin in vivo de las CD administradas


Parece necesario hacer un seguimiento de las CD administradas al paciente para poder
comprobar su capacidad de alcanzar rganos linfoides. Hasta ahora la mejor forma de estudiar
su biodistribucin era empleando un marcaje radiactivo de las CD con 111Indio (Laverman et al.
2006) de forma que mediante gammagrafa se puede seguir a diferentes tiempos el alcance de la
poblacin de CD administradas. Actualmente se estn introduciendo otros mtodos de
seguimiento de las clulas de la vacuna, como es el empleo de partculas de xido
superparamagnticas que permiten observar la migracin celular mediante Resonancia
Magntica Nuclear (RMN) (Frank et al. 2004; Rohani et al. 2010).

1.2.2- Ensayos clnicos con clulas dendrticas: estado actual y retos


La eficacia teraputica de las vacunas de CD est siendo continuamente cuestionada
(Rosenberg et al. 2004; Burgdorf et al. 2008; Rosenberg 2008) Esto encuentra su explicacin en
el limitado nmero de casos con regresin tumoral observada en los diferentes ensayos clnicos
realizados. As, se han planteado crticas a las vacunas de CD argumentando que consiguen
activar un escaso nmero de linfocitos T que realmente responden al antgeno de inters en el
organismo. Segn el Dr Rosenberg, esta diferencia es notoria cuando se comparan los
protocolos de vacunacin con los ensayos que utilizan terapia celular adoptiva con linfocitos T
cultivados y expandidos en el laboratorio (Rosenberg 2008).
Otro punto importante es la deficiencia de criterios objetivos para definir las respuestas al
tratamiento (Rosenberg et al. 2004). As pues, la clasificacin en base a criterios RECIST
(criterios de evaluacin a respuesta en tumores slidos) ha sido recientemente modificada
atendiendo a un ensayo clnico que testaba la eficacia del anticuerpo monoclonal anti-CTLA-4
(Ipilimumab) en pacientes con estadio IV de melanoma, en los que pese a obtenerse una notoria

26

mejora en la supervivencia media de estos pacientes, no haba indicacin temprana de regresin


de las lesiones tumorales (Hodi et al. 2010). Otro ejemplo lo constituye el producto
inmunoterpico Sipuleucel-T, un tratamiento para el cncer de prstata, consistente en la
estimulacin in vitro de leucocitos de sangre perifrica del paciente con una protena de fusin
formada por la fosfatasa cida prosttica y el GM-CSF. Los resultados iniciales no daban
indicios de mejora del paciente en trminos RECIST (criterios radiolgicos), pero han
demostrado un incremento en la supervivencia media de 4 meses en pacientes con cncer de
prstata hormono-resistente (Higano et al. 2009). Debido a estos nuevos tratamientos, donde el
anlisis de la curva de supervivencia mostraba una mejora de los pacientes tras 4-6 meses de
tratamiento, sin respuestas objetivas RECIST, han surgido nuevos criterios de respuesta al
tratamiento que pueden admitir la progresin temprana de la enfermedad ya que se basan en
otros criterios ms adecuados para la evaluacin de ensayos de inmunoterapia (Wolchok et al.
2009; Palucka et al. 2010).

Es importante recordar que existen mecanismos que dificultan la eficacia clnica de la


inmunoterapia. Estos son: (i) cambios que impiden la infiltracin de linfocitos al tejido maligno
(Buckanovich et al. 2008); (ii) sustancias que suprimen a nivel local la funcin de las clulas T
(Lodish et al. 2008) y (iii) prdida de antgenos o de molculas presentadoras de antgeno
(Garrido et al. 1997; Khong y Restifo 2002). Por estas razones, la medicin de respuestas
especficas de clulas T no equivale ni correlaciona directamente con la respuesta clnica. Sin
embargo, las respuestas inmunitarias de clulas T siguen siendo de enorme inters como
parmetro a estudiar en los ensayos clnicos y constituyen el nico correlato vlido de eficacia.
Otra dificultad radica en el avanzado estadio de enfermedad de los pacientes sometidos a
inmunoterapia con CD. Hay una lnea de opinin muy extendida segn la cual la inmunoterapia
(en general) no es muy efectiva en la erradicacin de la enfermedad en estadios avanzados con
elevada carga tumoral, sino que funcionara mejor para tratar la enfermedad mnima residual.
Adems, es bastante difcil obtener buenas conclusiones de ensayos clnicos con un nmero
bajo de pacientes, por lo que sera mejor aumentar la n estadstica mediante la elaboracin de
ensayos multicntricos. A este punto se le aade la dificultad infraestructural de disponer de
salas blancas para la preparacin de productos de terapia celular.

Aunque hoy da los datos de la eficacia en los ensayos clnicos en humanos son modestos,
se est trabajando en una mejora de los mismos mediante estrategias de inmunoterapia
combinada (Melero et al. 2007; Prez-Gracia et al. 2009).

27

1.3- Inmunoterapia combinada


La medicina est avanzando hacia nuevas terapias frente al cncer procurando ser lo ms
agresivo posible con el tumor y evitando efectos secundarios en el paciente. Las principales vas
de inmunoterapia estudiadas consisten en tratamientos combinados buscando un efecto
sinrgico: una terapia convencional como puede ser ciruga, quimioterapia y radioterapia,
combinada con un tratamiento que potencie una respuesta inmunolgica [como puede ser una
vacuna de CD, la transferencia de linfocitos infiltrantes de tumor (TIL), citoquinas
administradas por va intradrmica o anticuerpos monoclonales estimulantes]. La ciruga y la
radioterapia funcionan en el tratamiento de tumores bien localizados, sin embargo, slo son
eficaces como tratamiento paliativo en el caso de cncer ya metastatizado. Los casos con
estadios metastsicos se suelen tratar con quimioterapia, teniendo en cuenta que la toxicidad se
produce no slo en el microambiente tumoral sino tambin a nivel sistmico, pudiendo afectar
al correcto funcionamiento de otros rganos no implicados en la enfermedad. Por estas razones
cada da se da mayor importancia a la inmunoterapia, y aunque no est disponible todava para
ser un tratamiento de primera lnea, se postula que podra beneficiar al paciente oncolgico
actuando de un modo sinrgico con la terapia tradicional.
Podramos clasificar a los diferentes agentes inmunoterpicos en los siguientes grandes
grupos (Prez-Gracia et al. 2009):
A) Vacunas frente al cncer (Gilboa 2004). Incluyendo adyuvantes (Celis 2007), vacunas
de CD (Melief 2008), y CD que han sido modificadas mediante terapia gnica y que
codifican para determinadas biomolculas que tienen como finalidad potenciar la
capacidad de presentacin antignica y aumentar la respuesta del sistema inmune
(Steinman y Banchereau 2007; Nchinda et al. 2008).
B) Transferencia adoptiva de clulas T o NK (Stephan et al. 2007; Terme et al. 2008).
C) Citoquinas administradas sistmica o localmente (Sangro et al. 2005; Parmiani et al.
2007).
D) Inductores de apoptosis de clulas tumorales que utilizan los mecanismos linfocitarios
de citolisis (Tesniere et al. 2008).
E) Activadores/inhibidores de sealizacin, que funcionan a travs de molculas
coestimuladoras formuladas en forma de anticuerpos o protenas quimricas (Flies y
Chen 2007; Melero et al. 2007; Zang y Allison 2007).

28

F) Agentes que causan deplecin o inactivacin de linfocitos T reguladores (Treg) (Zou


2006).
G) Agentes que neutralizan la funcin de clulas mieloides supresoras (Medina-Echeverz
et al. 2011)
H) Agentes quimioterpicos o biomolculas que causan una muerte celular tumoral
inmunognica y una eliminacin de una poblacin linfocitaria reguladora (por ejemplo
Treg) (Casares et al. 2005; Ghiringhelli et al. 2007).
I) Molculas pequeas que interaccionan con enzimas con papel inmunomodulador
como, por ejemplo, la Indolamina 2,3 dioxigenasa (IDO) (Muller et al. 2005; Munn y
Mellor 2007; Gonzalez et al. 2008); arginasa (Zea et al. 2005), u xido Ntrico
Sintasa (NOS) (Gabrilovich y Nagaraj 2009).

1.4- Inhibicin de la angiognesis en terapia del cncer


Uno de los puntos clave en la progresin del cncer es la capacidad de generar nuevos
vasos que irriguen el microambiente tumoral aportndole oxgeno y nutrientes (Ferrara 2002).
La aparicin de nuevos vasos se denomina neoangiognesis y el principal inductor de la misma
es el Factor de Crecimiento Endotelio Vascular (VEGF), cuya expresin es estimulada por
mltiples factores proangiognicos como el Factor de Crecimiento Epidrmico (EGF), el Factor
de Crecimiento Fibroblstico bsico (bFGF), el Factor de Crecimiento Derivado de las
Plaquetas (PDGF)y la IL-1. stos y otros inductores de angiognesis como las angiopoyetinas
estn principalmente regulados por la hipoxia.

1.4.1- VEGF
El VEGF es una glicoprotena homodimrica de 34-46 KDa, producida principalmente
por las clulas del estroma tumoral (Jain 2005) y las propias clulas tumorales. Acta
principalmente sobre clulas endoteliales vasculares y sus precursores, unindose a receptores
especficos con dominios sealizadores con actividad tirosina-quinasa (VEGFR-1 y VEGFR-2).
Esta interaccin activa rutas de transduccin de seales intracelulares, dando lugar a: (i)
proliferacin y migracin de clulas endoteliales vasculares; (ii) supervivencia de clulas

29

endoteliales inmaduras; (iii) incremento de la permeabilidad vascular, aumentando la


produccin de xido ntrico y en consecuencia, induciendo vasodilatacin. Adems de las
clulas endoteliales vasculares, el VEGF tambin ejerce su efecto sobre clulas endoteliales
linfticas y clulas del sistema inmunolgico como monocitos, CD y linfocitos B.
Se han identificado cinco miembros de la familia de VEGF: VEGF-A, -B, -C,-D y el
Factor de Crecimiento de Placenta (PGF) (Chung et al. 2010). VEGF-A parece ser el ms
importante en la angiognesis de vasos sanguneos. El gen que codifica para VEGF-A est
localizado en el brazo corto del cromosoma 6 (6p21.3) y est compuesto por ocho exones. El
splicing alternativo de su ARN da lugar al menos a seis isoformas (Ferrara y Gerber 2001;
Ferrara et al. 2003). Las cuatro isoformas principales son la VEGF121, VEGF165 (la ms
abundante), VEGF189 y VEGF206 (siendo el subndice el nmero de aminocidos de la protena).
Una de las principales diferencias entre las isoformas de VEGF-A es la afinidad que muestran
por la heparina, lo que va a afectar a su adsorcin a la superficie celular y a los proteoglicanos
de heparn sulfato de la matriz extracelular (Chung et al. 2010). Las isoformas del VEGF-A que
tienen una afinidad reducida para la heparina se encuentran en equilibrio: parte en forma soluble
y parte adsorbida a membranas celulares (Achen y Stacker 1998; Ferrara y Gerber 2001). La
forma asociada a membrana puede liberarse por accin de la plasmina que escinde la regin Cterminal del VEGF-A, liberando un VEGF activo. As pues, el VEGF funcional puede surgir
tanto de la produccin celular de isoformas solubles como de la escisin proteoltica de
isoformas asociadas a membranas y a matriz extracelular (Ferrara 2010). VEGF-A se va a unir
preferentemente a los receptores con dominios tirosina-quinasa VEGFR-1 y VEGFR-2, pero las
isoformas con afinidad para la heparina podrn unirse a un tercer receptor Neuropilina-1, cuya
interaccin con ligando resulta en la potenciacin de transduccin de seales a travs de
VEGFR-2. VEGF-B y PGF (tambin relacionados con la angiognesis endotelial vascular) se
unen especficamente a VEGFR-1, mientras que VEGF-C y VEGF-D se unen a VEGFR-3
estimulando la linfoangiognesis (Lohela et al. 2009; Chung et al. 2010) (Figura 1).

1.4.2- Control de la expresin del VEGF


Los factores inducibles por hipoxia (HIF)-1 e HIF-2 son factores de transcripcin
constituidos por una subunidad, HIF1 y HIF2 respectivamente, que dimerizan con la
subunidad (expresada constitutivamente en las clulas). Estos heterodmeros se unen a los
elementos de respuesta a hipoxia (HRE) presentes en los promotores de los genes implicados en
la respuesta a hipoxia (Carroll y Ashcroft 2006). Tienen capacidad para regular la expresin de

30

algunos factores de crecimiento y de activacin oncognica, as como de genes supresores de


tumores como von Hippel-Lindau (VHL). La expresin de HIF-1 est regulada principalmente
por las condiciones de oxigenacin celular. En condiciones de normoxia las subunidades de HIF
se hidroxilan en residuos conservados de asparragina y prolina, y esa seal la reconoce la
ubiquitin E3 ligasa VHL, que las seala para ser degradadas en el complejo del proteasoma. Sin
embargo, en situaciones de hipoxia se inhibe la hidroxilacin de HIF1 y las subunidades HIF
se estabilizan y pueden entrar en el ncleo celular, induciendo la expresin de VEGF y otros
genes de respuesta a hipoxia (Aragones et al. 2009)
En algunos tipos de cncer esta va est constantemente activada produciendo VEGF
debido no slo a la baja presin de oxgeno en el microambiente tumoral, sino a la presencia de
factores de crecimiento (Bardos y Ashcroft 2004) y a la supresin de la expresin o funcin de
VHL (Carroll y Ashcroft 2006).

1.4.3- VEGF en el control de las funciones inmunolgicas


El efecto producido por concentraciones fisiopatolgicas de VEGF sobre el sistema
inmune ha sido estudiado ya desde el ao 1996 por Gabrilovich et al, mediante la obtencin de
CD de cordn umbilical, que tras ser maduradas en presencia de VEGF disminuan su capacidad
de inducir una activacin linfocitaria en experimentos de Reaccin Mixta Linfocitaria (MLR)
(Gabrilovich et al. 1996). Adems, la administracin de VEGF recombinante a ratones (sin
tumor), result en una disminucin en el correcto desarrollo de las CD, asociado a una
acumulacin de clulas supresoras inmaduras Gr1+ de origen mieloide (MDSD) (Gabrilovich et
al. 1998). Este efecto fue parcialmente revertido al introducir en el medio de cultivo inhibidores
del VEGF (Ishida et al. 1998). As mismo, estos autores mostraron la capacidad
inmunomoduladora del VEGF no slo sobre la poblacin de CD, sino tambin sobre los
linfocitos B y clulas inmaduras mieloides en modelos animales (Gabrilovich et al. 1998).
Con experimentacin en ratones, Carbone et al llegaron a comprobar que aquellos
animales expuestos a concentraciones de VEGF similares a las presentes en los pacientes
oncolgicos, desarrollaban una atrofia tmica, que tena como consecuencia un importante
descenso en la poblacin de linfocitos T CD4+ y CD8+, lo que parece ser una de las fuentes de
evasin frente al sistema inmune por parte del microambiente tumoral (Ohm et al. 2003).
Adems, la inmunosupresin de las CD se debe a que el VEGF se une especficamente a los

31

progenitores hematopoyticos CD34+ a travs del receptor VEGFR-1 e inhibe la activacin del
factor de trascripcin NF-B (Dikov et al. 2005), que est asociado a la maduracin de las CD.
Todos estos experimentos demostraron la conveniencia de bloquear al VEGF o a sus
receptores para mejorar la respuesta inmunitaria frente al cncer (Fricke et al. 2007). As,
cuando se emplean inhibidores de la angiognesis junto con inmunoterapia basada en vacunas
de CD, parece que el efecto antitumoral es ms pronunciado y duradero (Osada et al. 2008).
Se han desarrollado frmacos neutralizantes de la va de VEGF, que actan a nivel de la
propia molcula (Bevacizumab) o bien a nivel de la sealizacin intracelular (inhibiendo los
receptores tirosina-quinasas). Estos agentes son testados previamente tanto in vitro como in vivo
(Osada et al. 2008; Alfaro et al. 2009), para posteriormente ser incluidos en ensayos clnicos
con pacientes oncolgicos, demostrando su eficacia solamente cuando se combinan con
quimioterapia (Yang et al. 2003). Algunos autores buscan establecer terapias combinadas de
estos agentes con vacunas de CD (Alfaro et al. 2009).

1.4.4- VEGF y cncer


1.4.4.1- Produccin de VEGF por las clulas tumorales y el estroma tumoral

Sin duda, la patologa en la que mejor se ha estudiado el papel del VEGF es el cncer,
donde desempea un papel decisivo en el desarrollo de la angiognesis tumoral (Ferrara y
Gerber 2001). La mayor parte de las clulas tumorales sobreexpresan VEGF. Estudios de
hibridacin in situ han demostrado la sobreexpresin del ARNm que codifica para VEGF en
muchos tumores humanos (Ferrara et al. 2003). A pesar de que las clulas tumorales son las
principales productoras de VEGF, el estroma asociado al tumor tambin proporciona una
importante cantidad de VEGF en condiciones de hipoxia (Ferrara et al. 2003; Jain 2005). Se ha
demostrado que la inactivacin del gen que codifica para VEGF suprime la angiognesis
tumoral en el modelo Rip-Tag (un modelo gentico bien establecido de insulinoma) (Inoue et al.
2002).
El gen que codifica para VEGF se expresa en una gran variedad de tumores
hematolgicos, como por ejemplo el mieloma mltiple, el linfoma de clulas T, la leucemia
linfoblstica aguda, el linfoma de Burkitt, el linfoma histioctico y la leucemia mieloctica
crnica (Gerber y Ferrara 2003). As mismo, sus receptores VEGFR-1 y VEGFR-2 se expresan

32

en las clulas de algunas lneas de leucemia, encontrndose con mayor frecuencia VEGFR-1
que VEGFR-2.
Otro tipo de tumores en los que se sobreexpresa el VEGF es en el carcinoma renal de
clulas claras (RCC). En esta patologa la sobreproduccin de VEGF viene determinada por una
alteracin en la expresin del gen supresor de tumores VHL. La consecuencia de la inactivacin
del gen VHL es la presencia de tumores muy vascularizados debido a la estimulacin aberrante
de la angiognesis inducida por el descontrol de la respuesta a hipoxia. Del mismo modo, esta
alteracin determina la produccin de VEGF en tumores vasculares de retina y
hemangioblastomas del sistema nervioso central (Lonser et al. 2003).
El VEGF acta conjuntamente con otras sustancias, como las angiopoyetinas y otros
factores de crecimiento, estimulando la formacin de nuevos vasos a partir de vasos
preexistentes, y permitiendo que el tumor reciba oxgeno y nutrientes. Estos mecanismos
tambin son cruciales en el proceso de metastatizacin tanto para la salida de clulas a partir del
tumor primario, como para el anidamiento en el rgano colonizado (Lee et al. 2010).

1.4.5- Terapia antiangiognica en cncer


Actualmente se estn desarrollando un gran nmero de frmacos cuya principal funcin
es la de inhibir los efectos de VEGF. Los frmacos ms prometedores son los que se basan en
anticuerpos monoclonales neutralizantes dirigidos frente al VEGF y los inhibidores de la
actividad tirosina-quinasa de sus receptores en las clulas diana (Figura 1). Este tipo de terapia
est indicada en el carcinoma renal de clulas claras (Rini y Small 2005; Rini et al. 2009) y en
tumores de origen digestivo como el cncer de colon (Geva et al. 2010; Tol y Punt 2010).
A continuacin se comentan brevemente las caractersticas de ambos tipos de terapia
antiangiognica:

1.4.5.1- Anticuerpos neutralizantes de VEGF


El anticuerpo monoclonal recombinante anti-VEGF, ms conocido como Bevacizumab
(Avastin, Genetech). Fue creado a partir de un anticuerpo murino (A.4.6.1) (Kim et al. 1992).
Tiene capacidad para unirse y neutralizar todas las isoformas biolgicamente activas del VEGF,
con una afinidad de unin de aproximadamente 1,8 nM (Rini y Small 2005). Su uso ha sido

33

aprobado por la Food and Drug Administration (FDA) en febrero del 2004, en combinacin con
5-fluorouracilo intravenoso como primera lnea de tratamiento en pacientes con cncer de colon
y recto metastsico (CRC) (Cohen et al. 2007). Adems, Bevacizumab en combinacin con 5fluorouracilo y oxiplatino ha sido aprobado en junio del 2006 como segunda lnea de
tratamiento en pacientes con CRC avanzado o metastsico, que haban sido tratados
previamente con irinotecan y 5-fluorouracilo. Estudios posteriores han definido el beneficio de
su uso en carcinoma renal de clulas claras (de Gramont y Van Cutsem 2005; McDermott y
George 2010), en el cncer avanzado de pulmn de clulas no pequeas en combinacin con
otros agentes teraputicos (Cohen et al. 2007; Di Costanzo et al. 2008), as como en el cncer
de pncreas y cncer de mama (de Gramont y Van Cutsem 2005; Petrelli y Barni 2010).

1.4.5.2- Inhibidores de VEGFR


Estos frmacos actan no slo inhibiendo los VEGFR, sino tambin otros receptores con
dominios tirosina-quinasa de modo promiscuo, incluyendo el receptor del factor de crecimiento
derivado de plaquetas (PDGF) y el factor de crecimiento epidrmico (EGF), implicados en el
crecimiento y divisin celular (Karaman et al. 2008).
Estos receptores de membrana desempean un importante papel en la transmisin de las
seales al citoplasma. Al ponerse en contacto con su ligando, los VEGFR dimerizan y a
continuacin se produce la autofosforilacin de sus dominios citoplasmticos y la activacin de
la actividad enzimtica tirosina-quinasa. Existen mltiples vas de sealizacin intracelular,
incluyendo la va Ras/Raf, PI3K/Akt y protena quinasa C (PKC) (Arora y Scholar 2005). Los
mediadores intracelulares transmiten posteriormente la seal hasta el ncleo, activando la
transcripcin de determinados genes implicados en procesos tales como el crecimiento celular,
la migracin y la diferenciacin (Arora y Scholar 2005).
Entre los diferentes inhibidores destacan los que tienen mltiples dianas enzimticas:
Sunitinib (Sutent, comercializado por Pfizer), inhibidor de VEGFR-2, PDGFR-, FLT3 y c-kit
y Sorafenib (Nexavar, Bayer) inhibidor de Raf quinasa, VEGFR-2, PDGFR-, PDGFR-,
FLT3 y c-kit, ya que han sido los primeros frmacos en mostrar un incremento en la
supervivencia libre de progresin incluso como monoterapia (Jain et al. 2006).

34

Figura 1. Representacin esquemtica de las isoformas de VEGF y las interacciones con sus
receptores. Se sealan (en rojo) las dianas de los agentes antiangiognicos: Bevacizumab,
Sorafenib y Sunitinib. Abreviaturas: PGF, Factor de crecimiento de placenta; VEGF, Factor de
crecimiento endotelio-vascular; Y, tirosina.

1.5- Desorientacin de las clulas dendrticas en cncer: papel


de la Interleuquina-8
1.5.1- Quimioquinas
Las quimioquinas son protenas quimiotcticas de bajo peso molecular (8-11 KDa), que
dirigen la migracin leucocitaria e intervienen en una amplia variedad de procesos fisiolgicos y
patolgicos, fundamentalmente en procesos inmunitarios e inflamatorios. A medida que se iban
descubriendo nuevas quimioquinas, uno de los problemas que surgi fue que varios grupos de
investigacin informaban acerca de la misma molcula pero con distintos nombres. Esto llev a
una confusin entre los cientficos que trabajaban activamente en este campo de investigacin.
As, en el "Keystone Simposium on Chemokines and Chemokine Receptors " realizado en enero

35

de 1999, en Keystone, CO. A. Zlotnik y O. Yoshie propusieron un nombre sistemtico para


todas las quimioquinas y sus receptores basndose en la estructura proteica y en su locus gnico
agrupndolas en cuatro familias: C, CC, CXC y CX3C. Se clasifican de acuerdo a la posicin
relativa de sus residuos cistena en el extremo N-terminal. As, cuando los dos residuos de
cistena estn separados por un aminocido se designan como quimioquinas-CXC y cuando
ambas cistenas estn contigas se les denomina quimioquinas-CC.

Los efectos biolgicos de las quimioquinas se producen por la unin a receptores que
pertenecen a la superfamilia de receptores con siete dominios transmembrana tipo serpina o
rodopsina, la mayora de ellos acoplados a protenas G. Las quimioquinas muestran gran
redundancia en la utilizacin de sus receptores. Segn esto, varias quimioquinas pueden
acoplarse a un mismo receptor y una misma quimioquina puede unirse a varios receptores.

Las quimioquinas participan en la migracin y activacin de leucocitos, en los procesos


de angiognesis, en la produccin de colgeno y en la proliferacin de precursores
hematopoyticos. Tambin participan en guiar de metstasis de clulas malignas (Nguyen et al.
2009)

1.5.2- Regulacin de la funcin de las clulas dendrticas por


quimioquinas
La produccin local (en los tejidos no linfoides) de citoquinas proinflamatorias, como por
ejemplo TNF-, IL-1, IL-6, as como el encuentro con un antgeno, van a promover la
maduracin de las CD y como consecuencia la migracin de las mismas hacia los ganglios
linfticos regionales que es guiada por quimioquinas. Los receptores de quimioquinas se
expresan sobre las CD de forma regulada. As, las CD inmaduras expresan el receptor CCR6 y
responden a la quimioquina CCL20/MIP3/LARC in vitro. Cuando estas clulas maduran,
expresan el receptor CCR7 que es fundamental para la migracin hacia los ganglios linfticos ya
que en los mismos se secretan las quimioquinas CCL20/MIP3/ELC y CCL21/6Ckine/SLC
cuyo receptor es CCR7. Las quimioquinas CCL20 y CCL21 forman un gradiente que hace
migrar a las CD maduras a los ganglios linfticos, donde son capaces de activar clulas T
antgeno-especficas.

36

1.5.3- Interleuquina-8 y cncer


La IL-8 fue descubierta en el ao 1988, y debido a su funcionalidad se le denomin
inicialmente pptido derivado de linfocitos activador de neutrfilos (Lymphocyte Derived
Neutrophil Activating Peptide, LYNAP) (Baggiolini et al. 1989). Posteriormente fue
denominada interleuquina-8 (IL-8) y actualmente CXCL8, ya que fue renombrada por el
Chemokine Nomenclature Subcommittee of the Nomenclature Committee of the International
Union of Immunological Societies (Societies 2001), aunque todava se conserva y usa
frecuentemente el nombre de IL-8, tal y como se har en esta memoria.

La transcripcin del gen que codifica para la IL-8 da lugar primeramente a una protena
de 99 aminocidos, que ser procesada a continuacin para dar la protena activa de 77
aminocidos en el caso de clulas que no pertenecen al sistema inmune, o de 72 aminocidos en
las clulas del sistema inmunitario (Waugh y Wilson 2008). As, la IL-8 est formada por la
unin no covalente de dos monmeros de 72 aminocidos cada uno, formando un homodmero
(Figura 2). Las dos unidades del dmero forman una estructura de 6 lminas- dispuestas de
forma antiparalela. La estructura secundaria de la IL-8 tiene similitud con la que se encuentra en
la estructura cristalina de protenas relacionadas con el factor 4 plaquetario y otras
quimioquinas.

Figura 2. Estructura secundaria de la IL-8. En el esquema se puede observar la estructura


secundaria de un homodmero de IL-8, formado por la unin no covalente de dos cadenas de 72
aminocidos cada una. Imagen obtenida de Protein Data Bank.

Los receptores funcionales identificados para la IL-8 son CXCR1 y CXCR2, unidos en su
regiones citoplasmticas/transmembrana a protena G. Ambos receptores presentan una gran

37

homologa, con un 77% de similitud en su secuencia aminoacdica. La mayor diferencia entre


ambos receptores se encuentra localizada en tres regiones: el dominio N terminal (de unin a
ligando), el cuarto dominio transmembrana y el dominio C terminal. Adems, ambos van a
presentar la caja TATA en la secuencia promotora y un dominio rico en GC en la regin 5
(Stillie et al. 2009). El receptor CXCR1 presenta mayor especificidad por la IL-8 que el receptor
CXCR2, que interacta adems con otras quimioquinas (Waugh y Wilson 2008; Stillie et al.
2009). Tanto la forma monomrica como la homodimrica de la IL-8 son potentes activadores
de CXCR1 y CXCR2, aunque la forma homodimrica ha demostrado tener mayor bioactividad
(Goger et al. 2002).

La IL-8 es secretada por diferentes tipos celulares, siendo los principales productores los
macrfagos. Otros productores importantes de IL-8 son las clulas endoteliales, que retienen la
IL-8 en unas vesculas de almacenamiento, denominadas cuerpos de Weibel-Palade (van
Mourik et al. 2002).

La IL-8 y la protena quimiotctica de monocitos 1 (MCP-1/CCL2) son las quimioquinas


ms importantes para el reclutamiento de leucocitos polimorfonucleares (PMN) y monocitos,
respectivamente. En la inflamacin aguda el infiltrado es inicialmente de PMN. Despus, entre
las 24 y 48 horas predominan los macrfagos. Este proceso de transicin entre ambos tipos de
leucocitos estara relacionado con la cintica y las propiedades funcionales de las quimioquinas
IL-8 y MCP-1 respectivamente. Cuando los PMN y otras clulas son estimuladas por citoquinas
inflamatorias, la IL-8 es producida tempranamente, y durante 24 horas, recluta y activa
localmente ms PMN. Una prolongada produccin de IL-8 puede generar una alta
concentracin de esta quimioquina en los vasos, lo cual inhibe la adhesin de PMN al endotelio
y la extravasacin de los PMN por sobresaturacin del estmulo quimiotctico (Simonet et al.
1994). Del mismo modo, parece que la IL-8 retiene las CD que se encuentran en el
microambiente tumoral inhibiendo su migracin a ganglio linftico (Feijoo et al. 2005). Este
fenmeno ocurre muy posiblemente por un fenmeno de desensibilizacin de sus receptores por
internalizacin que establece un mecanismo regulatorio (Alfaro 2011; Alfaro et al. 2011). Es
interesante destacar que la estimulacin de neutrfilos con citoquinas inflamatorias durante
varias horas produce selectivamente MCP-1 y no IL-8, observndose adems que el complejo
IL-6-sIL-6R puede activar a las clulas endoteliales para secretar ms IL-8 y MCP-1.

38

Estas interrelaciones entre citoquinas y quimioquinas se producen en todos los procesos


biolgicos donde intervienen estas protenas, y es lo que hace tan dificultoso su estudio in vivo.

A pesar de que los granulocitos neutrfilos son las principales clulas diana de la IL-8,
hay varios tipos celulares (como clulas endoteliales, macrfagos, mastocitos, queratinocitos,
CD, etc.) que tambin van a ser capaces de responder a IL-8 gracias a la expresin de sus
receptores en la membrana (Stillie et al. 2009).

El papel de las quimioquinas IL-8 y MCP-1 en el reclutamiento de leucocitos es doble.


En primer lugar, actan sobre el leucocito mientras que rueda (rolling) sobre las clulas
endoteliales en el tejido inflamado. Este rodamiento se convierte en una unin estable al
inducirse un cambio conformacional en molculas de adhesin conocidas como integrinas
leucocitarias, cuyos ligandos se expresan en el endotelio inflamado (Butler et al. 2008). Ello
permite a los leucocitos atravesar el vaso sanguneo, pasando entre o a travs de las clulas
endoteliales, fenmeno conocido con el nombre de extravasacin o diapdesis (Springer 1994).
En segundo lugar, las quimioquinas dirigen la migracin de los leucocitos a lo largo de un
gradiente de concentracin quimiotctico.

La IL-8 al ser producida en el sitio de infeccin establece la mxima concentracin en el


punto de mayor necesidad de la actividad defensiva de los leucocitos. La unin de las
quimioquinas a proteoglicanos de la matriz extracelular y de las superficies endoteliales
proporciona un sustrato sobre el que se fija el gradiente de concentracin de modo que no se
disipe rpidamente (Culley et al. 2003). El gradiente de concentracin de IL-8 entre un polo y
otro de la clula es el que distribuye los receptores, reorienta el citoesqueleto y dirige la
direccin de la migracin celular (Rot y von Andrian 2004).

Las quimioquinas IL-8, MCP-1 y el pptido del complemento activado C5a estimulan a
sus clulas diana (principalmente neutrfilos) de modo que se incrementa su capacidad
germicida. Concretamente, los neutrfilos que han sido expuestos a la IL-8 y a TNF- se
activan para producir un estallido respiratorio (una serie de reacciones que generan radicales
libres de oxgeno y xido ntrico, y la liberacin del contenido lisosomal) contribuyendo as
tanto a la defensa del husped como a la destruccin del tejido.

39

Muchas clulas tumorales humanas tienen la capacidad de producir IL-8 con o sin estrs
proinflamatorio. As la IL-8 juega un papel muy importante en los procesos de angiognesis y
metstasis (De Rossi et al. 2000; Xie 2001). Los experimentos que llevan a estas conclusiones
se basan en estudios comparativos de tumores que no expresan esta quimioquina y variantes que
han sido transfectadas para expresar IL-8. La relevancia de la IL-8 en los mecanismos
patognicos del cncer se ha experimentado analizando la progresin de los transfectantes en
ratones inmunodeficientes y por tanto se ha obviado la implicacin del sistema inmunitario
adaptativo. Esto es posible en xenoinjertos de tumores humanos en ratones inmunodeficientes
porque la IL-8 es capaz de estimular los receptores CXCR1 y CXCR2 de ratn.

La expresin de la IL-8 se ha detectado en mltiples cnceres humanos, incluyendo


leucemia mieloide aguda, leucemia linftica crnica de clulas B, tumores cerebrales, cncer de
mama, cncer de colon, cncer de crvix, cncer gstrico, enfermedad de Hodgkin, cncer de
pulmn, melanoma, mesotelioma, cncer de ovario, adenoma pituitario, cncer de prstata,
carcinoma de clulas renales y en tumores de tiroides (Xie 2001). Posiblemente es una
propiedad comn de la mayor parte de las enfermedades neoplsicas.

Se han publicado resultados obtenidos en diferentes estudios clnicos, que identifican la


expresin de IL-8 como un factor que ensombrece el pronstico de los pacientes, lo que
probablemente refleja la actividad protumoral de la sustancia (Xie 2001; Yuan et al. 2005). Por
ejemplo, varios grupos indican que la expresin de IL-8 se correlaciona con la progresin ms
rpida de la enfermedad en pacientes con melanoma (Hensley et al. 1998; Nurnberg et al. 1999;
Singh et al. 1999; Singh y Varney 2000; Payne y Cornelius 2002; Gabellini et al. 2009; Singh et
al. 2010; Singh et al. 2010). La ascitis y/o el plasma de los pacientes con cncer de ovario
contienen niveles de IL-8 significativamente ms altos si se comparan con pacientes con
trastornos ginecolgicos benignos (Radke et al. 1996; Merogi et al. 1997; Ivarsson et al. 1998;
Nowak et al. 2001; Lokshin et al. 2006; Ohata et al. 2008). Adems, la IL-8 tambin se ha
relacionado con la malignidad del cncer de prstata (Inoue et al. 2000; Araki et al. 2007;
Seaton et al. 2008). Se han publicado artculos con conclusiones similares tras el estudio de
muestras de pacientes con cncer de pulmn (Chen et al. 2003; Luppi et al. 2007; Millar et al.
2008), as como una mayor expresin del ARNm de la IL-8 en macrfagos infiltrantes en el
tumor (Yuan et al. 2002; Chen et al. 2003). Considerando est informacin de modo global,

40

parece que la expresin de IL-8 puede servir como un factor pronstico de la progresin de
varios tipos de cncer humano (Shahzad et al. 2010).

El papel de la IL-8 en la angiognesis, el crecimiento tumoral y la quimiotaxis


leucocitaria de monocitos y macrfagos ha sido demostrado en varios tumores (Koch et al.
1992; Xie 2001). Los primeros estudios funcionales utilizaron tejidos homogeneizados de
tumores que atraan quimiotcticamente a las clulas endoteliales in vitro y eran capaces de
inducir la neovascularizacin corneal cuando se implantaba en la superficie ocular de roedores
(Koch et al. 1992). Posteriormente se comprob que la adicin de anticuerpos policlonales que
neutralizaban la funcin de la IL-8 inhiba significativamente estas respuestas vasculares in vivo
(Arenberg et al. 1996), de modo que la neutralizacin de IL-8 podra tener un gran futuro como
estrategia teraputica.

En este sentido se ha demostrado la expresin de receptores de la IL-8 en las clulas


endoteliales. En particular, las clulas endoteliales microvasculares, que expresan el receptor
CXCR1. Por consiguiente la interferencia farmacolgica con el receptor constituye una
alternativa teraputica factible (Zigler et al. 2008; Singh et al. 2010).

Cabe resaltar, adems, que tanto el estroma tumoral como las clulas malignas producen
factores solubles y expresan protenas de membrana que deterioran las funciones inmunitarias
que podran destruir al tumor (Gabrilovich 2004; Zitvogel et al. 2006; Zitvogel et al. 2008). La
disrupcin de las funciones quimiotcticas que gobiernan la migracin de los linfocitos
efectores o de las CPA pueden conformar un conjunto de mecanismos de escape del tumor al
sistema inmunitario cuya importancia solamente empezamos a entender en la actualidad. Estos
mecanismos aunque complejos y multifactoriales nos ofrecen nuevas dianas teraputicas en
oncologa.

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1.5.4- Desorientacin de la migracin leucocitaria por la


Interleuquina-8 producida por tumores

Adems de la inhibicin de la vascularizacin, la produccin de quimioquinas por parte


del tumor es posible que trastorne los mecanismos de migracin celular inherentes al
funcionamiento del sistema inmunitario. En pacientes portadores de neoplasias avanzadas
observamos una acumulacin de IL-8 en el plasma y por consiguiente en los tejidos (Feijoo et
al. 2005). Como resultado, pueden hipotticamente derivarse las siguientes consecuencias:
1) Desensibilizacin de los receptores para esta quimioquina, de modo que se producira
una falta de respuesta a la misma. Esto de producirse, podra trastornar la migracin
de neutrfilos para formar pus en focos de infeccin y por tanto contribuir a la
situacin de inmunodeficiencia secundaria que presentan los pacientes con cncer
avanzado.
2) Determinar una alteracin de otras funciones distintas a la migracin leucocitaria,
como podran ser las funciones de macrfagos o CD. Por tanto, la IL-8 podra actuar
como un mecanismo de escape del tumor al sistema inmunitario al estropear
directamente el funcionamiento de las clulas del sistema inmunitario.
3) Desorientar las rutas de migracin celular necesarias para el encuentro entre
linfocitos T y CD. En este sentido existen distintas posibilidades: (i) por un lado es
posible que las CD presentes en el organismo se encuentren desensibilizadas por
exposicin crnica a IL-8; (ii) por otro lado las CD administradas exgenamente y en
principio sensibles a IL-8, quedarn a merced de gradientes quimiotcticos que las
retendran en tejidos donde no son de utilidad (Feijoo et al. 2005) o impediran su
migracin a rganos linfoides secundarios. Estos fenmenos podramos englobarlos
en el concepto de inmunodesorientacin que cinstituira un mecanismo ms de
escape del tumor al sistema inmunitario y que podra depender de IL-8 y/o de otras
quimioquinas.

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1.6- Coestmulo e inhibicin linfocitaria, a travs de las


interacciones CD28/CTLA-4-CD80/86. Aprovechamiento
teraputico del sistema.
1.6.1- Generalidades
CD28 es la principal molcula coestimuladora en la activacin de las clulas T
(Greenwald et al. 2005; Sharpe 2009). La activacin de los linfocitos T depende en gran medida
de la estimulacin de CD28 por las molculas CD80 y CD86 (tambin conocidas como B7.1 y
B7.2 respectivamente) presentes principalmente en la superficie de las CPA. CD28 es una
glicoprotena de membrana de 44 KDa, con una secuencia de 202 aminocidos, codificada por
un gen situado en el brazo largo del cromosoma 2. Estructuralmente est formado por una
porcin extracelular que posee un dominio nico, homlogo a los dominios de la regin variable
de las inmunoglobulinas (IgV), una regin hidrofbica transmembrana y una cola
citoplasmtica corta. El residuo de cistena en la porcin extracelular le va a permitir formar
homodmeros, aunque tambin coexiste en la membrana formando monmeros. Pese a que la
expresin de CD28 es constitutiva, su presencia en la membrana se incrementa transitoriamente
despus de la activacin de las clulas T, seguida por una disminucin cuando se produce su
unin con el ligando.

CD28 cumple un amplio espectro de funciones. Uno de los efectos ms importantes es el


incremento en la produccin de IL-2 junto con otras citoquinas como IL-4, IL-5, IL-13, IFN-,
TNF- y GM-CSF. Estas citoquinas van a actuar como factores de crecimiento, ejerciendo una
accin tanto autocrina como paracrina. As mismo, disminuye el umbral de respuesta de los
linfocitos T a su antgeno especfico, requiriendo por tanto un menor umbral del nmero de
TCRs que necesitan ser estimulados para obtener una activacin linfocitaria completa
(Lanzavecchia y Sallusto 2001). Adems, CD28 previene la apoptosis y ayuda a mantener la
supervivencia celular, regulando positivamente la transcripcin del gen Bcl-xL (Boise et al.
2010).

El antgeno asociado al linfocito T citotxico (CTLA-4 o CD152) es estructuralmente


homlogo a CD28, y se encuentran prximos en el genoma. CTLA-4 debe su nombre a haber
sido originalmente identificado como el cuarto ADNc de una genoteca derivada del ARNm de

43

clulas T citotxicas murinas (Sagerstrom et al. 1993; Henao J 1999). Su analoga con CD28,
tanto en la localizacin cromosmica como en la organizacin exn-intrn, sugiere que ambos
genes provienen de un ancestro comn. Tanto CD28 como CTLA-4 contienen una regin
altamente conservada (un hexapptido, MYPPPY) en el sitio de unin a CD80 y CD86. CTLA4, se expresa en superficie como dmero o monmero y consta de un nico dominio extracelular
tipo IgV, una regin transmembrana y una cola citoplasmtica.

De este modo, CTLA-4 se expresa en los linfocitos T efectores (donde alcanza una
expresin mxima a las 48-72 horas despus de la activacin del linfocito T), as como en las
clulas Treg, una poblacin linfocitaria inmunorreguladora que coexpresa CD4+CD25+FoxP3+.
La principal diferencia en la expresin de CTLA-4 entre ambas poblaciones est en que en las
clulas reguladoras la expresin es constitutiva, sin embargo la expresin sobre los linfocitos
efectores solamente es inducida tras la activacin celular por antgeno. Otra diferencia est en el
trfico intracelular de CTLA-4, ya que en las Treg se expresa constitutivamente en la superficie
celular, mientras que en la poblacin efectora tiene una seal de retencin en los grnulos
secretores (Linsley et al. 1996; Iida et al. 2000).

En un principio a CTLA-4 se le atribuy un papel similar al de la molcula CD28 en


cuanto a la activacin de las clulas T, pero los hallazgos experimentales le adjudican
fehacientemente un papel regulador negativo de la activacin linfocitaria (Krummel y Allison
1995; Hodi 2007; Wolchok y Saenger 2008). Prueba de ello es el cuadro clnico de los ratones
knock out para CTLA-4 con un fenotipo autoinmune muy severo, que resulta en la muerte del
animal entre las 4-8 semanas de vida extrauterina (Tivol et al. 1995; Waterhouse et al. 1995;
Friedline et al. 2009). El mecanismo responsable de la funcin de CTLA-4 parece estar
relacionado con la asociacin de esta molcula a la fosfatasa SHP2, compitiendo por molculas
de sealizacin intracelular como fosfatidil inositol-3-quinasa (PI3K).

Sin embargo, parece que el mecanismo competitivo de la unin de CD28 a sus ligandos
es el ms importante ya que en ratones genticamente deficientes en CTLA-4, un transgen de
CTLA-4 sin la cola citoplasmtica protege del fenotipo autoinmune (Sharpe 2009).

Se ha observado la existencia de interrelaciones coordinadas entre la expresin y la


funcin de CD28 y CTLA-4: as pues, la presencia en la membrana y el estmulo de CD28 son

44

esenciales para la expresin mxima y la regulacin eficiente del ARNm de CTLA-4 (Tai et al.
2007). Asimismo, los antagonistas de CD28 bloquean la induccin de CTLA-4 en respuesta a
antgeno. Es ms, los ratones dobles knock out para CD28 y CTLA-4 no presentan
autoinmunidad (Buhlmann et al. 2003), lo que es compatible con la hiptesis de que el principal
papel de CTLA-4 es limitar la funcin de CD28.

CTLA-4 se encuentra en linfocitos T activados, en niveles 10 a 100 veces menores que


los correspondientes a CD28, pero gracias a que la afinidad de CTLA-4 en su interaccin con
CD80 y CD86 es de 20 a 50 veces ms alta, esta molcula va a competir eficazmente con CD28
por la unin a CD80 y CD86 en las CPA (Rudd et al. 2009). En relacin a sus ligandos (CD80 y
CD86), ambos poseen una regin extracelular con dos dominios tipo inmunoglobulina (Ig) (uno
variable y otro constante), una porcin transmembrana y una cola citoplasmtica corta (Henao J
1999). Los genes que codifican para CD80 y CD86 se encuentran en el cromosoma 3, prximos
entre s. Adems, ambas molculas tienen solamente un 25% de homologa en su secuencia,
especialmente en la cola citoplasmtica, donde CD86 tiene 3 potenciales sitios de fosforilacin
por la enzima PKC, indicando que esta molcula puede tener propiedades de sealizacin. De
hecho, CD80 y CD86 transmiten seales inhibidoras cuando se expresan sobre linfocitos T
(Freeman et al. 1995; Greenwald et al. 2005; Sharpe 2009).

Recientemente se ha observado que la molcula de CTLA-4 expresado en clulas T es


capaz de secuestrar transcelularmente el CD80 y CD86 expresado por las CD. CTLA-4
internaliza al CD80 capturado de la CPA y se produce la degradacin lisosmica de CD80 y
CD86 en el interior del linfocito T. Este mecanismo determinara una menor capacidad
coestimuladora de las CD que han interaccionado con linfocitos Treg o linfocitos T activados
(Qureshi et al. 2011)

La cintica de expresin y el significado funcional de CD80 es diferente al de CD86, as


pues CD80 comienza a detectarse a las 24 horas de la activacin y alcanza niveles mximos a
las 48 y 72 horas despus de la estimulacin, mientras que CD86 experimenta una regulacin
positiva ms rpida, detectndose a las 6 horas y llegando a una expresin pico a las 24 horas.
Asimismo, los hallazgos experimentales indican que CD86 es la molcula coestimuladora
primaria responsable del inicio de las respuestas de los linfocitos T y la provisin de la ayuda a
las clulas B por parte de linfocitos T cooperadores. En cambio, CD80 parece predominar en las
respuestas inflamatorias crnicas, como se ha observado en algunos modelos de enfermedades

45

autoinmunes (Greenwald et al. 2005). Lanier y col. corroboraron que tanto CD80 como CD86
proveen seales coestimuladoras eficientes a los linfocitos T para la proliferacin, la produccin
de IL-2 y la generacin de clulas citotxicas (Lanier et al. 1995; Greenwald et al. 2005; Zang y
Allison 2007).
La regulacin de CD80 y CD86 es ejercida de manera diferente por diversos factores
(Greenwald et al. 2005). El entrecruzamiento del receptor antignico de las clulas B y el
estmulo de la va de sealizacin de CD40/CD40L inducen la expresin de CD80 y CD86,
mientras que la ligacin del receptor FcR II en monocitos las regula negativamente. La IL-4 es
un potente inductor de CD86 y en menor grado de CD80. La IL-10 bloquea la expresin de
CD80 y CD86 en macrfagos peritoneales y regula negativamente a CD86, pero no a CD80, en
clulas dendrticas humanas. El IFN- tambin exhibe efectos diversos sobre la expresin de
CD80 y CD86: incrementa la expresin de CD86 en linfocitos B, macrfagos peritoneales y
monocitos de sangre perifrica, al tiempo que hace una modulacin positiva de CD80 en estas
ltimas clulas, mostrando sorprendentemente una accin contraria sobre la expresin de este
ligando en macrfagos peritoneales.
Adems de con CD28 y CTLA-4, CD80 tambin interacciona con la molcula PD-L1
(B7-H1) presente en la superficie de las CPA (Freeman et al. 1995; Butte et al. 2007; Butte et
al. 2008). Parece ser que la unin de CD80 a este receptor transmite seales inhibidoras al
interior del linfocito T que expresa CD80 tras la activacin por antgeno (reverse signaling).

1.6.2- Funcin de CTLA-4 en las clulas T activadas y en los linfocitos


T reguladores
Es muy posible que la distribucin espacial y temporal de la expresin de CTLA-4 sea
crtica para la regulacin de las respuestas inmunitarias. La expresin de CTLA-4 es
prcticamente indetectable en linfocitos T en reposo, pero su induccin es uno de los fenmenos
ms tempranos en la activacin de clulas T (Korman et al. 2006). CTLA-4 sigue unas rutas de
trfico intracelular muy complejas, debido a que se une a protenas adaptadoras de clatrina del
tipo de AP-1 y AP-2. Segn esto, la molcula de CTLA-4 es movilizada a vesculas
intracelulares (grnulos secretores) que se localizan en la proximidad del Centro Organizador de
Microtbulos (MTOC) que se sita polarizado hacia la sinapsis inmunolgica, cuando se
produce una estimulacin a travs del receptor especfico de antgeno (Korman et al. 2006). De
hecho, cuanto ms potente es el estmulo a travs del receptor de antgeno TCR, ms
eficientemente se produce la traslocacin de CTLA-4 hacia la sinapsis inmunolgica (Korman

46

et al. 2006). La transfeccin de clulas T con CTLA-4 carente del tallo citoplsmico o con
mutaciones en el mismo, proporciona evidencia en el sentido de que la cola citoplasmtica de
CTLA-4 no es siempre necesaria para inhibir la funcin linfocitaria y que la competicin con
los ligandos que comparte con CD28 (CD80 y CD86) puede ser el mecanismo ms importante
de inhibicin mediado por CTLA-4 (Figura 3). Hay dos modelos diferentes, pero no
mutuamente excluyentes, para intentar explicar los efectos de CTLA-4 cuando se estimula por
sus ligandos: (i) el modelo del umbral y (ii) el modelo de atenuacin (Chambers et al. 2001). En
el modelo del umbral, CTLA-4 incrementa la seal necesaria a travs de TCR y CD28, evitando
que las clulas se activen completamente tras recibir bajas intensidades de estimulacin. Segn
el modelo de atenuacin habra una inhibicin de linfocitos T ya ptimamente activados. Es
muy posible que ambos fenmenos coexistan. Los linfocitos T con un TCR transgnico en
ratones CTLA-4-/- permiten estudiar el papel de CTLA-4 en la regulacin de las respuestas
antgeno-especficas. Tales estudios muestran que el efecto inhibitorio de CTLA-4 es mayor tras
un segundo contacto con el antgeno, que en la respuesta inmune primaria. Adems, se observa
un importante papel de CTLA-4 tanto en la poblacin de linfocitos T CD4 como CD8 (Korman
et al. 2006). Todo ello indica que la estimulacin crnica de linfocitos T resulta en una
elevacin de la expresin de CTLA-4. Es por ello que el bloqueo de CTLA-4 ofrece un
mecanismo potencial para aumentar la respuesta inmunolgica de los linfocitos T ya
estimulados frente a antgenos del tumor, bien reduciendo el umbral de activacin y/o
potenciando la linfoproliferacin y adquisicin de funciones efectoras.

Figura 3. Mecanismo de competicin de CTLA-4 por los ligandos de CD28 en la superficie de la


clula dendrtica. Representacin esquemtica de las interacciones de CD28 y CTLA-4 con los ligandos
que comparten. Las flechas de mayor grosor indican interacciones de mayor afinidad. El color verde de
las flechas indica seales de coestmulo, el color rojo de las flechas indica seales de coinhibicin.

47

Una minoritaria, aunque importante, subpoblacin linfocitaria que expresa CTLA-4 es la


de los linfocitos Treg. Los linfocitos Treg expresan constitutiva e intensamente CTLA-4 en su
superficie celular (Salomon et al. 2000). Se caracterizan por coexpresar CD4, CD25 y GITR y
dependen de la expresin de un factor de transcripcin llamado FoxP3 (forkhead box P3) para
su desarrollo y funcin. Las clulas Treg son capaces de suprimir la linfoproliferacin de otras
clulas T de manera no especfica de antgeno, aunque la activacin de las clulas Treg en s
misma si que es dependiente de antgeno (Korman et al. 2006). Se ha comprobado que in vivo
su actividad inmunosupresora requiere de factor de crecimiento transformante (TGF)- o de IL10 (Fahlen et al. 2005). Adems, parece que CTLA-4 est implicado en las actividades
inmunosupresoras de las clulas Treg, aunque no en todos los casos (Korman et al. 2006). Se ha
demostrado la presencia de linfocitos Treg infiltrantes de tumor (TIL) en cncer de ovario,
mama, pncreas, carcinoma de pulmn y melanoma metastsico (Korman et al. 2006).
Cabe resaltar que la unin de la molcula de CTLA-4 de los linfocitos T efectores a
CD80 y CD86 en las CPA induce la produccin de la enzima indoleamina 2-3 dioxigenasa
(IDO) en las CD por un mecanismo dependiente de IFN- autocrino. IDO adems de promover
la expansin de linfocitos Treg (Chung et al. 2009), ejerce una actividad inmunosupresora
mediada por la privacin de triptfano del medio y la produccin de metabolitos que son
proapoptticos sobre linfocitos T (Fallarino et al. 2003).

1.6.3- Ensayos clnicos con anticuerpos monoclonales anti-CTLA-4 en


cncer
El grupo de Allison es pionero en la realizacin de experimentos en ratones mediante el
bloqueo de CTLA-4 empleando anticuerpos monoclonales para inducir el rechazo de tumores
trasplantados experimentalmente (Leach et al. 1996; Peggs et al. 2008). Este fenmeno
teraputico se intensifica notablemente mediante terapia combinada con vacunas tumorales (van
Elsas et al. 1999; Hodi et al. 2008). El mecanismo de accin del efecto antitumoral es una
respuesta mediada por linfocitos T CD8 y CD4. Se ha postulado que el efecto principal consiste
en una liberacin del efecto de frenado que CTLA-4 ejerce sobre estos linfocitos. Se ha podido
objetivar tambin un mecanismo dependiente del control de los linfocitos Treg por CTLA-4
(Peggs et al. 2008).
Existen dos anticuerpos monoclonales humanos anti-CTLA4 que estn siendo empleados
actualmente en diversos ensayos clnicos en fase II/III: Ipilimumab y Tremelimumab. En

48

melanoma, el empleo de estos anticuerpos induce de un 5-20% de respuestas clnicas duraderas


(Ribas 2008; Saenger y Wolchok 2008; Kirkwood et al. 2010). Recientemente ha sido
publicado un ensayo clnico en el que queda patente el beneficio en cuanto a supervivencia en
pacientes con melanoma en estadio IV de la enfermedad (Hodi et al.). El frmaco se est
desarrollando para el tratamiento de otras neoplasias como es el cncer de prstata, vejiga y
pulmn (Theoret et al. 2007; Madan y Gulley 2010).

1-7- Interacciones entre las clulas dendrticas y los leucocitos


polimorfonucleares

1.7.1- Inmunidad innata versus adaptativa

Los neutrfilos desempean un papel muy importante en la inmunidad innata en la


captura y en la muerte de patgenos extracelulares como primera lnea de defensa (Ludwig et al.
2006; Megiovanni et al. 2006). En el momento en que tiene lugar una infeccin, los neutrfilos
migran desde el torrente sanguneo hasta el foco de inflamacin gracias al gradiente de
estmulos quimiotcticos existente. Tales citoquinas son secretadas por clulas endoteliales y
fibroblastos del microambiente lesionado (Ludwig et al. 2006). Los neutrfilos poseen en su
superficie receptores especficos para el reconocimiento y captura de patgenos, como
receptores del tipo del receptor de manosa, scavenger receptors, TLRs, receptores Fc y los
receptores del complemento (Ludwig et al. 2006). Su funcin principal es la de matar a los
patgenos localmente a travs de la fagocitosis y de la liberacin del contenido de grnulos
intracelulares (Megiovanni et al. 2006). Sin embargo, puede que las protenas y especies
reactivas de oxgeno producidas durante el ataque a patgenos, sean capaces de lesionar el
tejido circundante (Ludwig et al. 2006). Una vez han actuado, los neutrfilos son eliminados del
microambiente inflamado por apoptosis y a continuacin fagocitados por los macrfagos
residentes (Ludwig et al. 2006). Un mecanismo importante es la capacidad de los neutrfilos
que mueren para formar redes protruyendo su propio ADN. Las redes de ADN y protenas
asociadas actan atrapando de modo adherente a microorganismos y sus toxinas (Borregaard ;
Brinkmann et al. 2010).

49

Existen evidencias de que los neutrfilos no slo participan en la inmunidad innata, sino
que pueden interpretar un papel directo en la inmunidad adaptativa reclutando clulas del
sistema inmune tales como linfocitos T y CD. Los neutrfilos son capaces de migrar desde el
foco inflamatorio a una zona prxima al ganglio linftico (Miyazaki et al. 2004) donde entrarn
en apoptosis y pueden ser captados por las CD, que de ese modo sern capaces de presentar los
antgenos capturados por los neutrfilos a los linfocitos T. El grupo de Megiovani demostr en
el ao 2006 que los neutrfilos eran capaces de transferir antgenos directamente a las CD
(Megiovanni et al. 2006). Para ello realiz un cocultivo de neutrfilos (pre-expuestos a C.
albicans) y CD. Tales CD fueron posteriormente capaces de activar a linfocitos T especficos
para C. albicans, del mismo modo que si las levaduras hubieran estado en contacto directo con
las CPA.

1.7.2- Comunicacin entre neutrfilos y clulas dendrticas

En condiciones normales, los neutrfilos y las CD estn localizados en diferentes


compartimentos (torrente sanguneo y tejidos perifricos respectivamente). Tras una infeccin
microbiana, los neutrfilos producen citoquinas proinflamatorias que van a atraer a las CD
inmaduras, probablemente para ayudar en el aclaramiento de antgenos, as como para impulsar
la induccin de la inmunidad adaptativa. Se ha demostrado recientemente que los neutrfilos y
las CD interactan fsicamente (van Gisbergen et al. 2005; Megiovanni et al. 2006). La unin
de los neutrfilos a las CD inmaduras provoca su maduracin, demostrada por el incremento en
la expresin en superficie de molculas como por ejemplo el CMH clase II (HLA-DR), CD86 y
CD40. Otros autores, en cambio, han observado que la llegada de neutrfilos procedentes de un
ambiente asptico a ganglios linfticos de ratn determina paradjicamente una inhibicin en la
maduracin de CD (Eken et al. 2008). Segn otros autores, los neutrfilos son capaces de
inducir la produccin de IL-12 por las CD maduras, que resultar en una mayor capacidad para
activar linfocitos Th1 y LTC (Megiovanni et al. 2006). La capacidad de madurar a las CD viene
determinada por la produccin de factores como TNF- por los neutrfilos, as como por el
contacto entre ambos tipos celulares regulado por toda una serie de receptores expresados tanto
por los neutrfilos como por las CD. Entre estas molculas que median la interaccin
intercelular destacan: CD18 (van Gisbergen et al. 2005), CD11b (ambos forman el
heterodmero denominado Mac-1) y CEACAM1 en los neutrfilos, que se unir a DC-SIGN en
la superficie de las CD.

50

DC-SIGN es una lectina tipo C que se expresa sobre CD diferenciadas a partir de


monocitos e in situ en CD de la piel, mucosas, amgdalas, ganglios linfticos y bazo
(Geijtenbeek et al. 2000). DC-SIGN tiene una alta afinidad por residuos de manosa, que
posiblemente media la adhesin al endotelio a travs de ICAM-2, y a linfocitos T a travs de sus
gricoprotenas ICAM-3 (Figura 4). DC-SIGN se considera un receptor especfico para motivos
Lewis X, que se expresan en altos niveles en la superficie de los neutrfilos (Appelmelk et al.
2003). Tales estructuras de Lewis X estn presentes en una fraccin de las protenas de
superficie de los neutrfilos.
.
Mac-1 (CD11b/CD18) es una molcula que contiene motivos Lewis X en los neutrfilos
sobre ambas cadenas: la cadena alfa (CD11b) de 160 KDa y la cadena beta (CD18) de 100 KDa
(Skubitz y Snook 1987; Stocks et al. 1990). Aunque Mac-1 posee ambas cadenas, la unin de
DC-SIGN a Mac-1 parece que se produce principalmente a travs de la cadena alfa (van
Gisbergen et al. 2005). Para predecir como DC-SIGN es capaz de unirse tanto a CEACAM1
como a Mac-1, se han realizado estudios de inmunoprecipitacin de ambas protenas con
anticuerpos especficos. Mac-1 se expresa en general en todos los leucocitos mieloides, pero
DC-SIGN solamente se va a unir a Mac-1 presente en la superficie de los neutrfilos pero no en
otros leucocitos ya que solamente en neutrfilos adquiere motivos Lewis X. Esto supone que
una glicosilacin especfica de la estirpe leucocitaria determina la interaccin entre neutrfilos y
CD. Por otra parte, la interaccin dependiente de DC-SIGN entre PMN y DC permite a los
PMN inducir la maduracin de CD y predispone a la induccin de respuestas Th1. Esto
demuestra que DC-SIGN y Mac-1 definen una nueva va de la adhesin celular entre las DC y
los PMN. Lewis X y el ligando de hidratos de carbono relacionadas con DC-SIGN denominado
Lewis Y, tambin estn presentes en el CEA, un antgeno de las clulas tumorales que se
expresa en altos niveles en adenocarcinomas (van Gisbergen et al. 2005). Hay estudios
histolgicos que ponen de manifiesto contactos ntimos entre DC inmaduras (DC-SIGN+) y
clulas de adenocarcinoma. As pues, la interaccin de DC-SIGN con CEA puede estar
relacionada con la evasin inmune de las clulas de adenocarcinoma.

Las relaciones de las CD con otras estirpes leucocitarias es muy posible que sea de gran
importancia para entender sus mecanismos de actuacin en condiciones de salud y enfermedad.
Adems de sus interacciones con linfocitos T y NK (Zitvogel et al. 1996; Pallandre et al. 2008),
es muy posible que interrelaciones de CD con macrfagos y leucocitos polimorfonucleares sean

51

crticos en la fisiologa de las CD, aunque este campo se ha comenzado a explorar


recientemente. Cmo explotar estas interacciones celulares en terapia sigue siendo un campo
inexplorado.

Figura 4. Esquema de las interacciones moleculares de DC-SIGN. Adems de las protenas humanas
autlogas sealadas, DC-SIGN es receptor/correceptor para biomolculas de mltiples microorganismos
patgenos. Figura publicada en Dendritic cells and C-type lectin receptors: coupling innate to adaptive
immune responses.van Vliet S.; Garca-Vallejo JJ and van Kooyk Y. Immunology and Cell Biology 86,
580-587.2008. Abreviaturas: CEA, Antgeno Carcinoembrionario; CEACAM, Carcinoembryonic
Antigen-Related Cell Adhesion Molecule; DC-SIGN, Dendritic Cell-Specific Intercellular adhesion
molecule-3-Grabbing Non-integrin; ICAM, molclas de adhesin intercelular; MGL, Macrophage
Galactose-Type Lectin; MUC1, mucina 1.

52

2- OBJETIVOS

53

2.1- Objetivos
Los objetivos del presente trabajo de investigacin son los siguientes:
1- Comprobar la eficacia y seguridad clnica del tratamiento combinado con una vacuna de
clulas dendrticas (basada en la estimulacin in vitro de las mismas con lisado tumoral
autlogo previamente procesado) y el pretratamiento con un agente quimioterpico
(ciclofosfamida).
2- Realizar estudios encaminados a potenciar el efecto de las clulas dendrticas in vitro e
in vivo mediante el empleo de anticuerpos monoclonales inmunoestimulantes.
3- Estudiar factores tumorales que reprimen la funcin inmunoestimuladora de las clulas
dendrticas.
4- Estudiar experimentalmente las interacciones funcionales entre clulas dendrticas y
leucocitos polimorfonucleares, definiendo su posible papel en inmunologa/
inmunoterapia del cncer.

55

3- ARTCULOS PUBLICADOS Y MANUSCRITOS


ENVIADOS

57

An immunotherapy strategy based on type-1 dendritic cells increases


circulating interleukin-12 and NK activity while decreasing circulating
endothelial cells.

C. Alfaro

1,6

, JL. Prez-Gracia 2, N. Surez

1,3

, J. Rodriguez 2, M. Fernndez de Sanmamed

1, 2

B. Sangro , S. Martn Algarra , A. Calvo , M. Redrado , A. Agliano , A. Gonzlez , I.


Rodrguez 1, E. Bolaos

1,6

, S. Hervas-Stubbs1, J. Prez-Calvo 6, A. Benito 7, I. Peuelas 8, J.

Richter 8, I. Martinez-Forero 1 and I. Melero 1,2,6

Gene Therapy and Hepatology Unit. Centro de Investigacin Mdica Aplicada.2 Oncology
Department. Clnica Universidad de Navarra.3 Biochemistry Department. Clnica Universidad
de Navarra.4 Hepatology Department. Clnica Universidad de Navarra.5 Oncology Division.
Centro de Investigacin Mdica Aplicada. 6 Cell Therapy Unit. Clnica Universidad de
Navarra.7 Radiology Department. Clnica Universidad de Navarra.8 Nuclear Medicine
Department. Clnica Universidad de Navarra.University of Navarra, Pamplona, Spain.
1

Manuscrito enviado

59

An immunotherapy strategy based on type-1 dendritic cells increases circulating


interleukin-12 and NK activity while decreasing circulating endothelial cells.
C. Alfaro

1,6

, JL. Prez-Gracia 2, N. Surez

1,3

, J. Rodriguez 2, M. Fernndez de Sanmamed

1, 2

B. Sangro 4, S. Martn Algarra 2, A. Calvo 5, M. Redrado 5, A. Agliano 5, A. Gonzlez 3, I.


Rodrguez 1, E. Bolaos

1,6

, S. Hervas-Stubbs1, J. Prez-Calvo 6, A. Benito 7, I. Peuelas 8, J.

Richter 8, I. Martinez-Forero 1 and I. Melero 1,2,6


1

Gene Therapy and Hepatology Unit. Centro de Investigacin Mdica Aplicada.2 Oncology Department.
Clnica Universidad de Navarra.3 Biochemistry Department. Clnica Universidad de Navarra.4
Hepatology Department. Clnica Universidad de Navarra.5 Oncology Division. Centro de Investigacin
Mdica Aplicada. 6 Cell Therapy Unit. Clnica Universidad de Navarra.7 Radiology Department. Clnica
Universidad de Navarra.8 Nuclear Medicine Department. Clnica Universidad de Navarra.University of
Navarra, Pamplona, Spain.

ABSTRACT
This study assesses the biological effects and safety of dendritic cell (DC) immunizations
combined with GM-CSF, Peginterferon and cyclophosphamide pre-treatment in patients with
solid tumors. Twenty-four patients with metastatic cancer received two cycles of four daily
immunizations with monocyte-derived DC. DC were incubated with autologous tumor lysate,
IFN-, TNF- and poly I:C. One dose was delivered intranodally, under ultrasound control, and
the rest intradermally. Cyclophosphamide (day -7), GM-CSF (days 1-4) and Peginterferon (days
1 and 8) completed each cycle. Extensive immunological and imaging studies were performed
to reveal the biological effects of the treatment. Pre-treatment with cyclophosphamide decreased
regulatory T cells to levels observed in healthy subjects. Treatment induced sustained elevations
of Interleukin-12 in serum and increased NK activity in peripheral blood. Circulating
endothelial cells (CEC) decreased in 17/18 patients and circulating tumor cells (CTC) markedly
dropped in 6/19 cases. IFN--ELISPOT among freshly isolated peripheral blood T lymphocytes
responding to DC+tumor lysate were observed in 4/11 cases. Tracing DC migration with 111Inscintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of
patients, while intradermal injections almost constantly showed arrival of a small fraction of DC
to draining inguinal lymph nodes. Five patients experienced disease stabilization. One patient
with completely resected disease before treatment has not relapsed after 12+ months. This
combinatorial immunotherapy strategy is feasible and suggests potential activity in patients with
minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.

61

INTRODUCTION
Dendritic cells (DC) present antigen to nave and memory T lymphocytes (Steinman y
Banchereau 2007; Melief 2008). DC artificially presenting tumor antigens are efficacious antitumor vaccines for mouse transplanted tumors (Melief 2008; Palucka et al. 2011). Many DCbased clinical trials have been performed in cancer patients with evidence of increased immune
responses and clinical activity (Su et al. 2003; Avigan et al. 2004; Fay et al. 2006; Melief 2008;
Okada et al. 2011), but efficacy is unfortunately lower than that one observed in mouse models
(Mayordomo et al. 1995; Nair et al. 2000). A key difference might be that cancer patients
present multiple immunosuppressive regulatory mechanisms (Rabinovich et al. 2007), such as
the augmentation of CD4+CD25+FoxP3+ regulatory T cells (Treg) (Dranoff 2005; de Vries et al.
2011). DC for clinical trials are most often differentiated in cultures from monocytes with GMCSF and IL-4 (Schuler 2010). Other protocols have substituted IL-4 by IFN- (Gabriele et al.
2004) or IL-15 (Dubsky et al. 2007) with encouraging preclinical results (Palucka et al. 2011).
DC become highly immunogenic, as opposed to their steady state tolerogenic model (Steinman
2010) when they sense in their microenviromment inflammatory cytokines and/or the presence
of moieties denoting microbial infection such as viral nucleic acids (Melief 2008).
DC can be artificially manipulated to present tumor antigens either in the form of defined
protein sequences (Hsu et al. 1996; Thurner et al. 1999) or as antigenic material obtained from
autologous tumor cells (Nestle et al. 1998). Defined tumor antigenic sequences facilitate
experimental assessment of tumor immunity (Aarntzen et al. 2008), but likely miss tumor
specific mutations that ought to be drivers of the malignant phenotype.
DC are also important regulators of the activity of Natural Killer (NK) cells (Walzer et al.
2005). These cells lyse tumor target cells in an MHC-unrestricted fashion, produce
proinflammatory mediators and regulate angiogenesis (Yao et al. 1999). NK cells also play an
important role in the orchestration of the adaptive immune response (Vivier et al. 2011). IL-12
(Borg et al. 2004) and some surface-attached receptor-ligand pairs have been found to be
involved in the DC-NK interplay (Walzer et al. 2005; Ebihara et al. 2010).
Autologous tumor lysates have been used as a source of antigen (Palmer et al. 2009; Schwaab et
al. 2009). The abundance of immunosuppressive factors (Hatfield et al. 2008; Tirapu et al.
2008; Alfaro et al. 2009) is a potential drawback of freeze/thaw tumor lysates as antigen
sources. Some of such factors should be thermo labile and therefore pre-heating the lysates to
boiling temperatures (Speidel et al. 1997) might deactivate such immunosuppressants, while
preserving the primary aminoacid sequences of the polypeptide antigens (Speidel et al. 1997).

62

Kalinski et al. have reported that the DC optimal at inducing cellular immunity are those
activated by IFN- and those which have sensed viral RNA or viral RNA analogues (Kalinski y
Okada 2010). Such DC were termed type-1 DC (Kalinski y Okada 2010) since they are
powerful producers of IL-12, migrate to lymph nodes guided by their expression of CCR7 and
induce immune responses dominated by Th1 and cytotoxic T lymphocytes (Okada et al. 2011).
Type-1 DC are also powerful at activating NK cells (Ebihara et al. 2010).
We have tested the safety and biological activity of immunotherapy based on type-1 DC for
advanced cancer patients. Our objective was to imitate the strong immunity that typically occurs
following an acute viral infection, by attaining sustained antigen presentation in lymphoid tissue
by DC which are endowed with type-1 features.

PATIENTS AND METHODS


Eligibility
Patients were required to have a diagnosis of metastatic cancer non-amenable to standard
treatment, adequate hematological and hepatic function, ECOG status < 2 and adequate access
to tumor tissue for lysate production. Patients were excluded in the event of relevant
concomitant diseases, including other tumors, infections or need to receive immunosuppressant
treatment. All patients signed informed consent and the trial was approved by the Ethical
Committees of the participating institutions and Spanish regulatory boards (AGEMED and the
Ethical Review Board of our Institution). The trial was registered in www.clinicaltrials.gov
(NCT00610389).
Study design and statistical analysis
The main objective was response rate assessed by RECIST criteria. Secondary clinical
objectives were progression-free survival, overall survival and toxicity, according to CTC
criteria version 3.0. Sample size was calculated using Simons Minimax two stage method for
P0=0.05 and P1=0.25, using error probability limits of =0.05 and =0.10. It was estimated that
at least 25 evaluable patients were required. Under these conditions, observation of at least 3
responses was required to confirm a 25% response rate (P1). Descriptive statistics were used to
present response rate, clinical characteristics and toxicity. For comparisons, unpaired Students
t-tests or MannWhitney U tests were performed, using Prism software (Graph Pad Software).
Patient evaluation
At baseline, complete blood tests and imaging studies were performed. Clinical evaluation and
blood tests were repeated on days 15 and 29 of each cycle during the first two cycles and every

63

6 weeks thereafter. Imaging tests were repeated every 12 weeks during treatment. Follow-up
after treatment discontinuation was performed every 3 months.
Dendritic cell production
Monocytes selected by CD14+ immunomagnetic selection (Miltenyi Biotec) (Mazzolini et al.
2005) were differentiated to DC by incubation with GM-CSF and IL-4 for 7 days using GMP
standard procedures (Mazzolini et al. 2005). DC were exposed to autologous tumor lysate
generated by 5 rounds of freezing/thawing and 10Gy irradiation with a 5 min-long heating step
at 100 C during the first thawing step. DC-loading with lysate was carried out at 200-100 g/ml
of protein during 2h and later DC were matured with clinical-grade tumour necrosis factor-
(TNF-; 50 ng/ml; Boehringer Ingelheim, Ingelheim, Germany), IFN- (1,000 IU/ml;
Schering-Plough, Kenilworth, NJ, USA) and poly I:C (20 mg/ml; Ampligen, Bioclones, Tokai,
South Africa) for 24 to 48 hours.
FACS analyses and ELISAs
Before immune staining, cells were incubated with PBS/human IgG (50 g/ml; Beriglobina P;
Behring, Barcelona, Spain) for 10 min on ice to block Fc receptors. Subsequently, DCs (105)
were washed in cold PBS and incubated 15 min at 4C with specific FITC and PE-labeled mAb
for CD80, CD83, CD86, CCR7, B7H1 and CD40 (BD Biosciences, Erembodegem, Belgium).
Treg were analyzed by intracellular FoxP3 staining following CD4 and CD25 surface
immunostaining (BD Biosciences). NK cells were identified as CD56+ CD3-negative
lymphocytes. Samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences).
Concentrations of IL-12 and IFN- were assessed by commercial sandwich ELISA kits (BD
Biosciences).
NK Cell Cytotoxicity Assays
Cytotoxic activity of NK cells against K562 cells was measured by standard 5h sodium
51

chromate (51Cr)-release assay. Target cells (106) were labelled with 50 Ci of

51

Cr

(PerkinElmer, Boston, MA, USA) for 1 h at 37 C, and labelled cells were then washed and
resuspended in RPMI 1640 (Invitrogen, Paisley, UK) containing 10% fetal bovine serum (FBS)
from Invitrogen. Isolated PBMCs from different days before and after the treatments were used
as effector cells. These cells were resuspended in the same medium and placed at various
effector:target ratios (E:T). Labelled target cells were added to each well at a concentration of 3
x 103 cells/well for a total volume of 0.2 ml per well. After 5 h incubation, release of 51Cr into
the supernatant was quantified with a microplate scintillation counter (Packard TopCount,
PerkinElmer). The percentage of cytotoxicity was calculated as the percent

64

51

Cr release using

the equation: (experimental release - spontaneous release)/ (maximum release - spontaneous


release) x 100.

IFN- ELISPOT assay


Multiscreen HA plates (Millipore, Bedford, MA, USA) were coated with 15 g/ml of
monoclonal anti-human IFN- (1-D1K; Mabtech, Stockholm, Sweeden) in PBS overnight at 4
C. Unbound Ab was removed by three washings with PBS and plates were blocked with RPMI
1640 (Invitrogen) supplemented with 10% FBS (Invitrogen) for 1 h at 37 C. The medium was
aspirated and effector cells (2x105) were seeded in triplicates in RPMI/10% heat-inactivated
human serum type AB (Bio Whitaker Lonza, Basel, Switzerland). PBMC, from one day before
each treatment and three days after them, were used as effector cells. Stimulator cells were
autologous DC loaded with tumor lysates (50-250 g/ml) or tetanus toxoid (5 Lf units/ml) and
maturated with TNF- (50 ng/ml), IFN- (1000 U/ml) and poly I:C (20 g/ml) for 48 h in
AIM-V medium (Bio Whitaker Lonza). DC (2 x 104) were cultured with the effector cells.
Control wells contained unloaded DC and effector cells alone stimulated with Concanavaline A
(20 g/ml). Cells were incubated at 37 C in 5% CO2 in a water-saturated atmosphere. After a
culture period of 20 h, cells were removed by six washings with PBS/0.05% Tween 20 (PBS/T).
Captured cytokine was detected by incubation for 3 h at 37 C with biotinylated mAb antihIFN- (7-B6-1; Mabtech, Stockholm, Sweden) at 1 g/ml in PBS with 0.5% fetal calf serum
(PBS-0.5% FCS). After washing the cells six times with PBS/T, Streptavidin-Alkaline
Phosphatse (1/1000; Mabtech) was added for 2 h at room temperature. Unbound complex was
removed by washing plates as before, and IFN- spots were detected at sites of secretion with
BCIP/NBT substrate (Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped with tap
water and spots were visually analyzed. To calculate the number of T cells responding to a
particular antigen, the mean numbers of spots induced by DC alone were subtracted from mean
spot numbers induced by antigen-loaded DC.
Assessment of Circulating Endothelial Cells and Circulating Tumor Cells
Blood samples (10 ml) before initiation of the therapy (basal sample), after a first cycle of DC
injections (Post I), and once treatment was completed (Post II) were collected. 1.5 ml were used
for FACS analysis and the rest of the blood for RNA isolation. The response was measured by
flow cytometry by enumerating circulating endothelial cells (CECs) and progenitors (CEPs).
Complete blood was incubated with 10% goat normal serum diluted in PBS with 2% BSA and
3% EDTA for 30 min at 4 C to block unspecific signal. Samples were incubated for 30 min at
4C with anti-CD31-FITC (BD Pharmingen), anti-VEGFR-2-PE (R&D, Minneapolis, MN,
USA), anti-CD133-APC (Miltenyi Biotec) and anti-CD45-PerCP (BD Pharmingen); or 7AAD

65

(7-Aminoactinomycin D (Sigma). Anti-CD45 was used to exclude hematopoietic cells, whereas


7AAD was used to exclude apoptotic and dead cells. After antibody incubation and red cell
lysis, at least 1x106 cells per sample were acquired with a FACSCalibur flow cytometer (BD
Biosciences). Following acquisition, appropriate gating was used to exclude dead cells, platelets
and debris, and analyses were considered as informative when adequate numbers of events (i.e.,
>50, typically 100200) were collected in the relevant gates. Percentages of stained cells were
determined and compared to appropriate negative controls defined as nonspecific background
staining. For RNA isolation, cDNA synthesis and Real-time PCR, the blood samples were
centrifuged at 2,500 rpm for 8 min. RNA from nucleated cells was isolated with the QIAamp
RNA blood kit (Qiagen, Dusseldorf, Germany) following the manufacturers instructions.
Reverse transcription of 1 g of RNA was performed with Superscript II (Invitrogen) according
to the manufacturers instructions. RT-PCR was performed in a 7300 Real Time PCR system
(Applied Biosystems, Carlsbad, CA, USA) using the SYBR Green PCR Master Mix (Applied
Biosystems, Foster City, CA, USA). The relative expression of each gene was normalized with
GAPDH. The list of primers used is shown in Supplementary table 1.
111

In labelling and scintigraphy

DC migration was tracked in vivo by scintigraphy. DCs were labelled with 500-700 Ci of 111Inoxinate for 15 min at room temperature. Cells were washed twice, resuspended in saline and
mixed with the non labelled DCs. Scintigraphic and Single Photon Emission Computed
Tomography (SPECT) studies were performed in a hybrid system (Symbia Truepoint,
SIEMENS TM, Munich, Germany) using a fast acquisition protocol. Images were taken 4, 24,
48 and 72 h post-intranodal injection.

RESULTS
Treatment plan
The treatment schedule is presented in Figure 1A. Patients underwent apheresis to obtain
peripheral blood leukocytes and received a single dose of cyclophosphamide 600 mg/m2 (day 7). DC were administered in two cycles of four daily immunizations, separated by 3-6 weeks.
The first dose was delivered inside an inguinal lymph node, under ultrasound control and the
rest intradermally in the opposite upper thigh. GM-CSF 100 g/24 h (days 1-4) and
Peginterferon 80 mg (days 1 and 8) were injected subcutaneously in the upper thigh region.
Patients could receive additional DC doses contingent to the presence of clinical benefit and DC
availability according to investigators criteria.

66

Rationale for the design of the vaccination strategy


The vaccination protocol was created based on previous preclinical work developed by our
group and on previous clinical studies (Mazzolini et al. 2005). Boiling lysates from mouse and
human tumors changed the protein patterns as visualized by SDS-PAGE (Supplementary Figure
1A). Experiments in mice bearing transplanted CT26 colon carcinomas indicated that series of
four daily immunizations with tumor lysate-loaded DC achieved complete tumor rejections in a
number of cases (7/14). Freezing and heating to boiling the tumor lysates used to load DC for 5
min provided a survival advantage to the treated mice (Supplementary Figure 1B). Lysates at
1:10 dilution, even if pre-heated, decreased the proliferation of human T cells co-cultured with
mature fully allogenic DC (Supplementary Figure 1C) (Hatfield et al. 2008; Tirapu et al. 2008)
indicating the presence of actively suppressing factors that were not completely destroyed by
heating. Based on these preclinical models, DC were cultured with pre-heated autologous tumor
lysates and matured with TNF-, IFN- and poly I:C, in order to induce differentiation of type-1
DC (Kalinski y Okada 2010). Cyclophosphamide was administered to decrease regulatory T
cells and their immunosupresive effects. Daily DC vaccinations were planned to ensure
sufficient bioavailability of antigens at lymph nodes. The first dose of DC in each cycle was
given intranodally while the rest were given intradermally in the opposite thigh. This permitted
independent observations on both routes and maximized the number of responding lymph
nodes. Patients received GM-CSF and Peginterferon to potentiate DC functions and survival,
and to enhance overall immunity (Schwaab et al. 2009). GM-CSF and Peginterferon were
administered in the territory draining to the same lymph nodes to enhance and sustain DC
performance.

Patient characteristics and treatment administration


From May 2008 to September 2010, 31 patients were included. Twenty-two patients received
two cycles of vaccination and two received just one cycle of vaccination, because they died
before the second one. Seven patients did not complete the planned treatment because of change
of decision or death. Patient characteristics are presented in Table 1. Patients are color and
number-coded to facilitate their follow-up through the figures. Histological diagnosis were:
colorectal cancer (9 patients, 38%), melanoma (5 patients, 21%), hepatocellular carcinoma (4
patients, 17%), renal cell carcinoma (3 patients, 13%), cholangiocarcinoma (2 patients, 8%) and
carcinoid tumor (1 patient, 4%). Fifteen patients were male (62%) and 9 female (38%).
Treatment was administered as planned in 24 patients. Three patients received additional
vaccinations.

67

Clinical efficacy and toxicity


Five patients presented disease stabilization (21%). One patient was rendered free of metastatic
disease when the surgical biopsy to obtain antigen was performed in a case of a solitary brain
metastasis of melanoma. Interestingly, the melanoma patient without macroscopic evidence of
disease upon treatment has not relapsed yet, after at least 12 months of follow-up.
Systemic toxicity included grade 1 and 2 fever (17 patients, 71%), asthenia (10 patients, 29%),
and pain at the injection site (6 patients, 25%) (Table 1). All toxicities lasted few days and no
grade 3 or 4 events were recorded. One patient presented a perineal abscess that evolved to a
Fourniers gangrene three weeks after receiving the first cycle and died from it. Although the
patient did not present neutropenia and the doses of cyclophosphamide administered were
relatively low, a potential relationship of the event with treatment induced immunosupression
could not be fully excluded. No clinical autoimmunity or changes in a battery of auto-antibodies
were observed.
Effect of cyclophosphamide administration on the number of regulatory T-cells
Patients showed a sustained decrease in the percentage of CD4+CD25+FoxP3+ Treg cells in
peripheral blood (Figure 1B). The decline was more evident in patients with the highest pretreatment levels. Frequencies of Treg around < 5% of total PBMCs were maintained.
Type-1 DC produce IL-12, show bright expression of costimulatory molecules and
increase circulating IL-12 and NK activity
Subcultures of the DC that we used for vaccinations indicated that large quantities of IL-12
were produced as a result of maturation (Figure 2A). The maturation cocktail also clearly
enhanced the surface expression of CD80, CD83, CD86, CD40 and the chemokine receptor
CCR7 that is crucial to guide migration toward draining lymph nodes. All these features of
mature DC were related to the induction of type-1 immunity (Figure 2B and Supplementary
Figure 2).
All patients experienced an important rise in circulating IL-12 that was sustained during each of
the two immunotherapy cycles (Figure 3A and Supplementary Figure 3A). NK activity was
enhanced in 11/17 patients in the first cycle and in 8/17 in the second cycle (Figure 3B and
Supplementary Figure 3B). In 6/17 of the evaluated patients the percentage of NK cells (CD3CD56+) increased markedly (Figure 3B). No antibodies directed to surface proteins of a panel of
eight tumor cell lines encompassing the histological origins of the patients neoplasias were
observed (data not shown), indicating lack of induction of humoral responses to tumor surface
antigens. Evaluation of the tumor specific cellular immunity when tumor lysates are used as

68

immunogens is challenging. Sufficient material for these experiments was available in 11 cases
and increases in IFN--ELISPOT reactivity from freshly isolated PBMC to DC loaded with
autologous tumor lysate (without any antigen-driven pre-culture) were observed in four out of
those eleven cases (Figure 3C).
Treatment effects on circulating endothelial cells (CET), and circulating tumor cells
(CEC)
The well-known anti-angiogenic effects of IL-12 (Mazzolini et al. 2001; Romagnani et al.
2001) supported the evaluation of the number of CEC as a surrogate marker for angiogenesis
and vasculogenesis(Mancuso y Bertolini 2010). CEC were evaluated in 18 patients, and a
dramatic decrease in circulating CD45-CD31+VEGFR-2+ endothelial cells was noted in most
cases (Figure 4A).
The increase in NK activity that we observed could also result in lysis of CTC. In samples from
19 patients quantitative RT-PCR tests with primers for mRNAs selectively expressed by tumor
cells were performed to comparatively evaluate the presence of CTC before and after treatment.
In six of the 19 cases (32%) marked decreases of CTC were observed, including two
hepatocellular carcinoma patients (Figure 4B). Results are summarized in Supplementary Table
2.
DC migration following intranodal and intradermal injections
With the first intranodal injection and the first subcutaneous DC dose, 18 patients received a
tracing dose of 106 DC labelled with 111Indium oxynate (Feijoo et al. 2005). This allowed using
scintigraphy to monitor the anatomic biodistribution of the tracing dose (Figure 5A). SPECT
permitted clear identification of lymph node anatomy (Figure 5B). The biodistribution from
intranodal and subcutaneous injections is summarized in Supplementary Table 3. In 11 of the 18
patients studied (61%), the intranodal injections reached deeper lymph node chains and constant
arrival of a small fraction of the radioactive tracing isotope to draining lymph nodes from
subcutaneous injections was observed.

DISCUSSION
Immunotherapy has shown activity in several types of human cancers (Finn 2008). Cancer
vaccines, adoptive T cell therapy and immunostimulatory monoclonal antibodies are among the
most relevant strategies. Careful observation of biological effects induced by immunologicalbased strategies is required to improve current paradigms and to develop the most promising
combination regimens. This DC-based clinical trial tested a number of innovative features such

69

as the maturation cocktail, the daily administration, cyclophosphamide pre-treatment and the
accompanying cytokines.
Our results indicate that pre-treatment with cyclophosphamide decreased Treg cells, mainly in
patients with higher baseline numbers. There is controversy regarding the optimal dose and
schedule (Ghiringhelli et al. 2007) to achieve these selective Treg-decreasing effects of
cyclophosphamide that are related to low content of ATP in this lymphocyte subset (Zhao et al.
2010). The decreasing effects on Treg cells are likely to involve cyclophosphamide but may also
involve combined effects of the strategy and of IL-12 as described (Cao et al. 2009).
Repeated daily immunizations were chosen to deliver antigen to lymph nodes during several
days, thereby mimicking the conditions expected for a replicating virus causing an acute
infectious disease. When we designed our trial, we thought of the evidence suggesting that
exogenous DC need to transfer the antigen to lymph node-resident DC (Shortman y Heath
2010), which are hypothesized to be main actual performers of antigen presentation (Poulin et
al. 2010). In addition, the incoming DCs, such as those that we injected into the patients, would
stimulate lymph node-resident DC with secreted proinflammatory cytokines and microbialdenoting molecules that they may carry such as poly I:C.
GM-CSF was administered at a site near each DC injection to sustain DC viability and enhance
crosspresentation (Ferrantini et al. 2008). Patients received IFN- to promote antigen
presentation at the tumor microenvironment and to foster cytotoxic lymphocyte responses
(Bracci et al. 2007; Schwaab et al. 2009; Okada et al. 2011).
No objective radiological responses were observed. However, five patients showed disease
stabilization. These patients presented rapid disease progression before entering the trial.
Another patient who underwent a complete resection of metastatic disease has not relapsed after
at least 12 months. Yet, no clear conclusions on efficacy can be drawn based on these data, and
randomized data are needed. The decrease in the number of CEC and CTC clearly supports that
this strategy might be especially relevant in the setting of minimal residual disease.
Consequently, a randomized clinical trial in colorectal cancer patients following complete
surgical resection of liver metastasis is ongoing at our institution.
Treatment was well tolerated. The most frequently observed toxicities were grade 1 and 2 fever,
asthenia and pain at the injection site. These reactions are probably related to the endogenous
and exogenously administered inflammatory mediators. One patient died of a Fourniers
gangrene that developed from a perineal abscess but relation to treatment is doubtful.
Amazingly high concentrations of IL-12 were observed in the plasma of all patients. The type-1
DC used in the study produced IL-12 and thus are very likely to be the main source of IL-12 in
the patients organism although maybe not the only one. IL-12 has been used as an efficacious
anticancer agent but systemic administration at high doses was because of serious toxicity
(Alatrash et al. 2004). IL-12 is a powerful NK activating factor (Trinchieri 2003) that in our

70

treatment would act in concert with Peginterferon to strongly raise NK activity. As a result of
NK activity and the downstream IFN-CXCL10 axis, angiogenesis and vasculogenesis are
inhibited (Romagnani et al. 2001). Indeed, we observed a clear decline of circulating endothelial
cells that likely denotes these trains of phenomena (Mancuso y Bertolini 2010). The decline of
circulating endothelial cells (Mancuso y Bertolini 2010) could be also interpreted as a result of
NK activity (Yao et al. 1999), activities of type I interferons(Bracci et al. 2007) and
cyclophosphamide (Wong et al. 2010). Type-1 DC are known to produce type I IFN and control
NK functions through direct cell to cell contact (Ebihara et al. 2010; Kalinski y Okada 2010).
Indeed, IL-12 and other surface molecules are involved in this cross-talk of NK and DC (Borg
et al. 2004; Ebihara et al. 2010). In previous works we have tested dendritic cells adenovirally
engineered to produce IL-12 (Mazzolini et al. 2005). It is of note that the production of IL-12 in
DC matured with our cocktail is superior to that achieved by adenovirus-mediated transfection.
Tumor lysates were chosen as a source of antigen because such mixtures contain individual
tumor antigens and because of technical feasibility under GMP. Lysates were pre-heated at 100
C to counteract immunosuppressive compounds, but thermo-resistant components with
suppressive effects certainly persist. A feasible alternative is to transfect mRNA encoding tumor
antigens (Su et al. 2003), a procedure that offers the possibility of adding mRNAs or siRNAs
that up-regulate DC immunostimulatory functions (Bonehill et al. 2008). No direct clinical
comparison between RNA transfection and tumor lysates is available.
Regarding DC distribution upon injection, our data match those obtained by the group of Carl
Figdor and Jolanda de Vries (Verdijk et al. 2009). We noticed no obvious hardening
inflammatory reactions at the vaccination sites in any of the cases, even in the second cycle.
This may reflect the immunosuppressive mechanisms in the advanced cancer patients
(Rabinovich et al. 2007). Although in some cases peripheral blood lymphocytes obtained posttreatment showed some degree of reactivity to tumor lysate-loaded DC, the intensity of these
adaptive cellular responses is probably too weak as to permit meaningful effects on the
established tumor masses.
Indeed, our data suggest that our combined treatment would turn on immunosuppressive
mechanisms such as B7-H1 (PD-L1) (Brahmer et al. 2010) expression by antigen presenting
cells and possibly tumor cells. Integrating agents that tamper with these immunosuppressive
pathways in this therapeutic approach is considered important (Brahmer et al. 2010). Even if
our procedure normalized percentages of regulatory T cells, a transient but more drastic
reduction is likely to be required for efficacy.
In summary, we have developed a DC vaccination strategy that incorporated several novel
elements. The treatment was feasible and well tolerated and induced tumor specific cellular
immunity, as well as several relevant biological effects, including reduction of regulatory Tcells, high concentrations of IL-12, decreases in CET and CEC. This regimen deserves further

71

study in situations of minimal residual disease and in combination with other


immunotherapeutic approaches. Such studies are ongoing and being developed in our
institution.

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76

FIGURAS_______________________________________
(An immunotherapy strategy based on type-1 dendritic cells increases circulating interleukin-12
and NK activity while decreasing circulating endothelial cells. C. Alfaro, JL. Prez-Gracia, N.
Surez et al. Manuscrito enviado)

77

Figure 1
A

78

Figure 1. Therapeutic strategy and its effects on regulatory T cells


(A) Schematic representation of a cycle of the combined immunotherapy strategy represents the
time line of a cycle of treatment and the procedures of DC manipulation.
(B) Individual follow up of the percentages of regulatory T cells in peripheral blood of the
colour-coded patients before and after each cycle of combined type-1 DC-based
immunotherapy. Insets represent meanSD of the series of patients.

79

80

Table 1. Patient characteristics.


Abbreviations: A, anaemia; As, asthenia; AP, articular pain; B, bone; BR, best clinical
response; Carc, carcinois tumor; ChC, cholangiocarcinome; CRC, colorectal carcinoma; ECT,
electrochemotherapy; F, fever; FG, Fourniers gangrene; GA, gluteal abscess; HCC,
Hepatocellular carcinoma; IFN, interferon; IL-2, interleukin-2; K, kidney; L, liver; LAK,
limphokyne-activated killer cells; Lu, lung; LN, lymph node; M, melanoma; Me, mediastinum;
NA, Not available; NED, No evidence of disease; NED (*), without macroscopic disease at the
beginning of the vaccines; P, pancreas; PD, progressive disease; Pe, peritoneum; PSI, pain at
site of injection; QT, Chemotherapy; RCC, renal cell carcinoma; RE, radioembolization; RLN,
regional lymph node; RPLN, retroperitoneal lymph node; RT, radiotherapy; S, spleen; SC,
subcutaneous tissue; SD, stable disease; sr, suprarenal; st, soft tissue; Surg; surgery; T,
thrombocytopenia; TACE, trans-arterial chemoembolization; TKI, tyrosine kinase inhibitors;
TTP, time to progression; V, vomiting.

81

Figure 2.
325

325

300

300

275

275

250

250

225

225

IL-12 (ng/ml/10 cells)

200
175
150
125
100

175
150
125
100

75

75

50

50

25

25

Immature DC
Mature DC

200

IL-12 (ng/ml/106 cells) / 48h

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Immature DC

Mature DC

Patient

B
CD80
1400
1200
1000
800
600
400
200

CD83

Immature DC
Mature DC

40

Immature DC
Mature DC

30

MFI

MFI

150

20

100
10

50

S1 S2 2

9 10 11 12 13 14 15 17 18 19 22 23 24

CCR7
10

S1 S2 2

Healthy
donor

Patient

Healthy
donor

9 10 11 12 13 14 15 17 18 19 22 23 24

Patient

B7H1

Immature DC
Mature DC

4000

Immature DC
Mature DC

MFI

MFI

3000

2000

1000

S1 S2 2

Healthy
donor

9 10 11 12 13 14 15 17 18 19 22 23 24

Patient

S1 S2

Healthy
donor

82

10 11 12 13 14 17 21 22 24

Patient

Figure 2. Phenotypic features of type-1 DC.


(A) Production of IL-12 to the supernatant by subcultures of the DC used for treatment in
individual samples of colour-coded patients (right) and meanSD (left).
(B) Surface expression assessed by flow cytometry of the indicated surface markers on DC from
individual patients represented as the mean intensity of fluorescence (after subtracting
background imunofluorescence by isotype-matched control monoclonal antibodies.

83

Figure 3

84

Figure 3. Immunotherapy treatment increases circulating IL-12, NK activity and T cell


reactivity.
(A) Individual concentrations of serum IL-12 p70 in the indicated days of each immunotherapy
cycle (left). The meanSD values are provided in the right bar diagram.
(B) (left) individual follow-up pre and post the first immunotherapy cycle of NK cytotoxicity
assessed at 5:1 effector target ratios on freshly isolated peripheral blood PBMC in 51Chromium
release assays against K562 target cells. The black and white bar graph represents the meanSD
(right) corresponding to percentages of CD3-CD56+ NK cells in PBMC before and after the first
immunotherapy cycle. The black and white bar graph represents the meanSD.
(C) ELISPOT activity of freshly isolated PBMC from the indicated colour-coded patients at the
given time points pre and post each immunotherapy cycle. PBMCs were exposed to thawed
mature DC that had been loaded with autologous tumor lysate. The right graph represents the
meanSD

85

Figure 4

86

Figure 4. Effects on circulating endothelial cells and circulating tumor cells.


(A) Individual follow-up of circulating endothelial cells in the peripheral blood of color-coded
patients. The right box plot groups the data.
(B) Normalized quantitative RT-PCR results of the indicated genes in blood samples drawn at
the indicated time-points from patients in whom a clear decrease in circulating tumor cells was
observed. The list of primers used is shown in supplementary Table 1 and supplementary Table
2 shows the results in all the patients evaluated.

87

Figure 5
A

4h

48h

24h

72h

Figure 5. Biodistribution of type-1 dendritic cells from intranodal and intradermal


injections.
(A) Scintigraphic images of In-Labelled tracing doses of DC at different time points following
intranodal injection (time 0) and intradermal injection in the opposite thigh 24 h later.
(B) Shows CT-SCA anatomical correlate with the radioactive signal super imposed.

88

Supplementary Figure 1

89

Supplementary Figure 1. Heating tumor lysates and preclinical efficacy.


(A) Silver-stained SDS PAGE of tumor lysates obtainded from CT26 mouse tumors and tumors
from two representative patients obtained following five freezing/thaweing cycles with or
without heating to 100C in the first cycle. Overlay profiles depict changes that aremarked with
arrows(B) Follow up of tumor growth in BALB/c grafted and treated with DC pulsed with tumor
lysate as indicated in the scheme. The fraction of animals completely rejecting their tumors is
provided in each line.
(C) Two representative experiments of alloreactive proliferation (measured by 3H-thymidine
incorporation) of human T lymphocytes induced by DC such as those produced for the clinical
trial in the presence or absence of the indicated tumor lysates with or without preheating at 100
C. DC to T cell ratios are provided in the horizontal axes.

90

Supplementary Figure 2
CD86

Immature DC
Mature DC

MFI

1000

500

S1 S2 2

10 11 12 13 14 15 17 18 19 22 23 24

Patient
CD40
200

Immature DC
Mature DC

MFI

150

100

50

S1 S2 2

9 10 11 12 13 14 15 17 18 19 22 23 24
Patient

Suplementary Figure 2. Other co-stimulatory molecules on the surface mature DC from


the patients.
Continuation of figure 2, showing the surface expression of CD40 and CD86.

91

Supplementary Figure 3

Supplementary figure 3. Circulating IL-12, NK activity and NK cell percentages in the


second cycles of treatment.
Continuation of data shown in figure 3 for the first cycle.

92

Supplementary Table 1

PATHOLOGY

GENE
AFP

Hepatocellular
carcinoma

MAGE-1
MAGE-3
MART-1

Melanoma
TYR
CEACAM5
Colon carcinoma

CK19
CK20
CK7

Renal cell carcinoma

CA9
MMP9

PRIMER SEQUENCES
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv
Fw
Rv

GTTCCAGAACCTGTCACAAG
CTTTGTTTGGAAGCATTCAACTGC
ACAGAGGAGCACCAAGGAGAAG
AGTTGATGGTAGTGGGAAAGGC
CTCCAGCAACCAAGAAGAGG
GCAAGGAACTGGAAGCTTTG
GCTCATCGGCTGTTGGTATT
ATAAGCAGGTGGAGCATTGG
ACTTACTCAGCCCAGCATCATTC
ACTGATGGCTGTTGTACTCCTCC
CTGTCCACCAAGATCAAGCA
GCGACCACATAGGGAGAAAA
GGTCAGTGTGGAGGTGGATT
TCAGTAACCTCGGACCTGCT
CACCTCCCAGAGCCTTGAGAT
GGGCCTTGGTCTCCTCTAGAG
ATTCCACTGGTGGCAGTAGC
GGGTGGGAATCTTCTTGTGA
CTGCGCCTGCGCAACAATGG
CTCGGCAGGGAAACGGTGGC
GACAAGAAGTGGGGCTTCTG
GCCATTCACGTCGTCCTTAT

Suplemmentary Table 1. List of primers.


Fw: Forward sequence. Rv: Reverse sequence. Human genes: AFP, Alpha fetoprotein; MAGE1, Melanoma antigen gen family member 1; MAGE-3, Melanoma antigen gen family member
3; MART-1, Melanoma antigen recognized by T cells 1; TYR, Tyrosinase; CEACAM5,
Carcinoembryonic antigen-related cell adhesion molecule 5; CK19, Cytokeratin 19; CK20,
Cytokeratin 20; CK7, Cytokeratin 7; CA9, Carbonic anhydrase 9; MMP9, Matrix
metalloprotease 9.

93

Supplementary Table 2
Pathology

Patient
19
21

MELANOMA
22
23
2
HEPATOCELLULAR
3
CARCINOMA
4

10
COLON
CARCINOMA

11

12

13

14

15
16
RENAL CARCINOMA 17
18

Gene

Basal

MART-1
TYR
MART-1
TYR
MART-1
TYR
MART-1
TYR
AFP
MAGE-1
MAGE-3
AFP
MAGE-1
MAGE-3
AFP
MAGE-1
MAGE-3
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CEACAM5
CK19
CK20
CA9
CK7
CA9
CK7
CA9
CK7

94

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

Post 1st DC
cycle
1,004
0,518
1,288
1,207
1,137
0,799
6,881
1,753
0,003
0,762
0,460
ND
ND
ND
1,194
5,970
5,045
0,626
0,032
0,122
1,686
0,795
0,548
0,524
0,732
0,128
3,158
2,865
99,090
2,301
2,345
9,344
2,236
1,691
2,264
0,857
0,778
1,281
1,262
1,142
0,807
0,106
0,061
0,253
7,601
3,035
0,194
0,166
1,116
0,820

Post 2nd DC
cycle
2,626
3,880
0,778
0,939
2,539
1,915
2,754
2,240
ND
ND
ND
0,100
0,775
0,392
2,508
8,035
8,439
2,010
0,036
0,294
0,872
0,900
0,742
0,360
0,336
0,300
1,918
1,361
17,377
8,585
9,757
72,277
2,134
2,044
0,913
5,593
4,172
0,168
0,768
0,734
1,494
0,609
0,333
0,099
3,488
4,541
ND
ND
0,648
193,985

Supplementary Table 2.
Monitorization of circulating tumor cells (CTCs) in patients using specific primers for each
tumor type and RT-PCR analysis. Data indicate fold-change compared to basal values. ND: Not
determined due to lack of appropriate sample.

95

Supplementary Table 3

Patient
N
3
4
5
6
7
8
9
11
12
13
14
17
18
20
21
22
23
24
TOTAL

INTRANODAL (distribution to deep


pelvic/iliac lymph nodes)
NO
NO
YES ++
YES ++
NO
YES +++
NO
NO
YES +++
YES +++
NO
YES +++
NO
YES ++
YES +++
YES ++
YES ++
YES +++
YES / NO = 11 / 7

+++
++
+

Migration in 4h
Migration in 24h
Migration in 48h

96

INTRADERMAL (distribution to
local/regional lymph nodes)
YES ++
YES +++
YES ++
YES +
YES +++
YES +++
YES +++
YES ++
YES ++
YES +++
YES +++
YES ++
YES +++
NO
NO
YES +++
YES +++
YES +++
YES / NO = 16 / 2

59 %
37 %
4%

Influence of bevacizumab, sunitinib and sorafenib as single agents or


in combination on the inhibitory effects of vegf on human dendritic cell
differentiation from monocytes.

C. Alfaro

1,6

; N. Surez

2,6

; A. Gonzlez 2.; S. Solano 1; L. Erro 1; J. Dubrot 1; A. Palazn 1; S.

Hervas-Stubbs 1; A. Grpide 3; J.M. Lpez-Picazo 4; E. Grande-Pulido 4; I. Melero

1,5,7

; J.L.

Prez-Gracia 3,7.

Gene Therapy and Hepatology Division, CIMA, Universidad de Navarra, Pamplona 31008,
Spain; 2 Biochemistry Department, Clnica Universidad de Navarra, Universidad de Navarra,
Pamplona 31008, Spain; 3 Medical Oncology Department, Clnica Universidad de Navarra,
Universidad de Navarra, Pamplona 31008, Spain; 4 Clnical Research Department, Pfizer Inc,
Madrid 28006, Spain; Universidad de Navarra. 5 Internal Medicine Department, Clnica
Universidad de Navarra, Universidad de Navarra, Pamplona 31008, Spain.

British Journal of Cancer, 2009 Abril 7; 100 (7): 1111-9

97

British Journal of Cancer (2009), 1 9


& 2009 Cancer Research UK All rights reserved 0007 0920/09 $32.00

www.bjcancer.com

Full Paper

Influence of bevacizumab, sunitinib and sorafenib as single agents


or in combination on the inhibitory effects of VEGF on human
dendritic cell differentiation from monocytes


















































C Alfaro1,6, N Suarez2,6, A Gonzalez2, S Solano1, L Erro1, J Dubrot1, A Palazon1, S Hervas-Stubbs1, A Gurpide3,


JM Lopez-Picazo3, E Grande-Pulido4, I Melero*,1,5,7 and JL Perez-Gracia3,7
1

Gene Therapy and Hepatology Division, CIMA, Universidad de Navarra, Pamplona 31008, Spain; 2Biochemistry Department, Clnica Universitaria de
Navarra, Universidad de Navarra, Pamplona 31008, Spain; 3Medical Oncology Department, Clnica Universitaria de Navarra, Universidad de Navarra,
Pamplona 31008, Spain; 4Clinical Research Department, Pf izer Inc., Madrid 28006, Spain; 5Internal Medicine Department, Clnica Universitaria de
Navarra, Universidad de Navarra, Pamplona 31008, Spain

Vascular endothelial growth factor (VEGF) inhibits differentiation and maturation of dendritic cells (DC), suggesting a potential
immunosuppressive role for this proangiogenic factor. Bevacizumab, sorafenib and sunitinib target VEGF-mediated angiogenesis and
are active against several types of cancer, but their effects on the immune system are poorly understood. In this study, VEGF and
supernatants of renal carcinoma cell lines cultured under hypoxia were found to alter the differentiation of human monocytes to DC.
Resulting DC showed impaired activity, as assessed by the alloreactive mixed T-lymphocyte reaction. Bevacizumab and sorafenib, but
not sunitinib, reversed the inhibitory effects of VEGF, but not of those mediated by tumour supernatants. Dendritic cells matured
under the influence of VEGF expressed less human leukocyte antigen-DR (HLA-DR) and CD86, and this effect was restored by
bevacizumab and sorafenib. Finally, tumour-cell supernatants decreased interleukin-12 (IL-12) production by mature DC, and such
inhibition was not restored by any of the tested drugs, delivered either as single agents or in combination. The deleterious effects of
tumour-cell supernatants were mainly mediated by thermostable molecules distinct from VEGF. These results indicate that inhibition
of the differentiation of monocytes to DC is a multifactorial effect, and that they support the development of combinations of
angiogenesis inhibitors with immunological modulators.
British Journal of Cancer advance online publication, 10 March 2009; doi:10.1038/sj.bjc.6604965 www.bjcancer.com
& 2009 Cancer Research UK
Keywords: dendritic cells; renal cell carcinoma; VEGF; bevacizumab; sunitinib; sorafenib

Angiogenesis is a critical step in the development of solid tumours


and their metastasis, and its blockade has become a major target of
cancer therapy (Kerbel and Folkman, 2002). Renal cell carcinoma
(RCC) was the first human tumour in which inhibition of
angiogenesis, mediated by bevacizumab, a monoclonal antibody
(mAb)-neutralising vascular endothelial growth factor (VEGF),
showed clinical benefit (Yang et al, 2003a). Subsequently, RCC has
remained a platform for the development of antiangiogenic
compounds. Sunitinib, a tyrosine kinase inhibitor of VEFG
receptor (VEGFR), c-kit and platelet-derived growth factor
receptor, was superior to interferon-a (IFN-a) in a randomised
phase III trial as first line treatment of RCC (Motzer et al, 2007),
and sorafenib, another tyrosine kinase inhibitor that targets
*Correspondence: Professor I Melero, Gene Therapy and Hepatology
Division, CIMA, Clnica Universitaria de Navarra and Medical School,
University of Navarra, Avda Pio XII, 55, Pamplona 31008, Spain;
E-mail: imelero@unav.es
6
These authors contributed equally to this work
7
These authors will share senior authorship
Received 28 October 2008; revised 23 January 2009; accepted 10
February 2009

Ras-activated factor (RAF) and VEFGR, also showed clinical


benefit in a phase III trial in which it was compared with placebo
as second-line treatment of this disease (Escudier et al, 2007a).
Bevacizumab, in combination with IFN, was also superior to single
agent IFN in another phase III trial (Escudier et al, 2007b).
Even though the main mechanism of the action of drugs that
inhibit VEGF-related pathways is antiangiogenesis, an effect of
VEGF inhibitors on the immune system has been reported. In an
earlier study, Gabrilovich et al (1996) obtained dendritic cells (DC)
from umbilical cords and described an inhibition of their ability to
induce T-lymphocyte proliferation, assessed by the mixed
lymphocyte reaction (MLR), when they were matured in supernatant cell cultures containing VEGF. This effect was partially
reverted by anti-VEGF antibodies, showing that VEGF was
probably the cause of inhibition of DC-induced proliferation.
The same authors showed that VEGF inhibited the development of
DC and increased B lymphocytes and immature myeloid cells in
animal models (Gabrilovich et al, 1998). The proposed mechanism
of action was the interference with the activation of nuclear factorkB transcription factors (Ohm et al, 1999). Using the same model,
this group also showed that the addition of anti-VEGF antibodies
increased the number and activity of DC in mice implanted with
subcutaneous tumours (D459 and MethA sarcoma), although

VEGF blockade and dendritic cell differentiation


C Alfaro et al

2
without any therapeutic effect on tumour growth (Gabrilovich
et al, 1999). Nevertheless, a combined treatment with anti-VEGF
antibodies and DC pulsed, with p53 peptides that contained
specific mutations of the implanted tumours, produced a sustained
antitumour effect, indicating potential synergism between antiangiogenic drugs and DC-based immunotherapy treatments for
cancer (Gabrilovich et al, 1999). Other groups have suggested that
angiogenic factors at the tumour microenvironment differentiate
DC that display features of vascular cells and are immunosuppressive (Conejo-Garcia et al, 2004). In addition, VEGF has
been described to mediate immunosuppression (Ohm et al, 2003),
which depends on the inhibition of differentiation and migration
of thymic lymphocyte progenitors from the bone marrow
and subsequent reduction of the numbers of CD4 and CD8
thymocytes, which can be reverted when exposure to VEGF ceases.
Cancer therapeutic vaccines formulated with DC artificially
presenting tumour antigens are being extensively tested in clinical
trials in several tumour types (Vulink et al, 2008), including RCC
(Dannull et al, 2005). The possibility to overcoming potential
interactions of VEGF on the immune system through clinically
available angiogenesis inhibitors led us to evaluate the influence of
such drugs on DC differentiation from monocytes. A recent report
has documented the inhibitory effects of sorafenib, but not of
sunitinib, on DC maturation (Hipp et al, 2008). Maturation is the
gene expression programme that renders DC capable of mediating
T-cell expansion and activation (Steinman, 2008). Maturation is
triggered by microbial biomolecules (such as, lipopolysaccharide
(LPS) or bacterial DNA), proinflammatory cytokines and ongoing
T-cell responses. In spite of the reports on the effects of VEGF on
DC, little is known about the effects of these compounds on the
differentiation of DC from myeloid precursors. In this study, the
effects of soluble VEGF and of VEGF-containing supernatants from
RCC cells were assessed on monocytes to DC differentiation
cultures. The influence of bevacizumab, sorafenib and sunitinib on
such cultures was defined.

MATERIALS AND METHODS


DC generation and maturation
Dendritic cells were generated from filter buffy coats (FBC)derived monocytes donated by healthy human donors (Meyer et al,
2005). The protocol for obtention of FBC was approved by the
Ethics Committee of our institution and was carried out on donors
who gave informed consent. Isolated mononuclear cells were
subjected to positive selection using anti-CD14-conjugated paramagnetic beads and purified using the AutoMACS system
according to the manufacturers instructions (Miltenyi Biotec,
Bergisch Gladbach, Germany). Purified monocytes were cultured
for 7 days in AIM-V serum-free media (Gibco-BRL, Gaithersburg,
MD, USA), supplemented with granulocyte-macrophage colonystimulating factor (GM-CSF) (1000 U ml1; Leukine, Berlex,
Richmond, CA, USA) and interleukin-4 (IL-4, 500 U ml1; R&D
Systems, Minneapolis, MN, USA). Cytokines were added every 2
days. Dendritic cells were matured with clinical-grade tumour
necrosis factor-a (TNF-a; 50 ng ml1; Boehringer Ingelheim,
Ingelheim, Germany), IFN-a (1,000 IU ml1; Schering-Plough,
Kenilworth, NJ, USA) and poly I:C (20 mg ml1; Ampligen,
Bioclones, Tokai, South Africa) for 48 h.
Maturation was confirmed by assessing increases in the
immunofluorescence of CD80, CD83, CD86 and human leukocyte
antigen-DR (HLA-DR), as well as variations in CD1a. Flow
cytometric analysis for immature and mature DC was performed
at days 7 and 9, using a FACSCalibur Flow Cytometer (Becton
Dickinson, San Diego, CA, USA). Purified anti-VEGFR-1 and
VEGFR-2 mAbs were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA) and purified anti-VEGFR-3 mAbs from
British Journal of Cancer (2009), 1 9

R&D Systems. Indirect immunofluorescence was performed with


anti-mouse IgG1-FITC (Dako, Glostrup, Denmark). Cell viability
was confirmed by the trypan blue exclusion test. Recombinant
human VEGF 165 (25 ng ml1, R&D Systems), bevacizumab
(rhumAb-VEGF, Avastn, 1 mg ml1, Roche, Basel, Switzerland),
sorafenib (Nexavar, 10 ng ml1, Bayer, Berlin, Germany) and
sunitinib (Sutent, 10 ng ml1, Pfizer, New York, NY, USA) were
used during DC differentiation. For experiments with supernatant
of RCC line cultures, we added 1 : 5 (v/v) ratio of the RCC-10
supernatant to the culture medium of DC.

Cell culture and reagents


Parental von Hippel Lindau (VHL)-negative RCC-10 cells,
originally cultured from a clear cell renal adenocarcinoma case
(Krieg et al, 2000; Cuevas et al, 2003), and a clone derived by stable
transfection of the VHL gene (VHL 53), were kindly provided by
Dr Luis del Peso (CSIC-UAM, Madrid, Spain). The cells were
maintained in an RPMI 1640 medium with GLUTAMAX-I
(Invitrogen, Carlsbad, CA, USA). The culture medium was
supplemented with 100 units ml1 penicillin, 100 mg ml1 streptomycin and 10% foetal bovine serum. To perform hypoxic
experiments, hypoxia was induced by exposing cell cultures to
1% oxygen for 24 48 h, using a hypoxic workstation (Cuevas et al,
2003).

MLR
Dendritic cells were cultured in 96-well plates in an AIM-V
medium. A total of 2  105 lymphocytes from a distinct donor were
added on day 9 at different Tcell/DC ratios (80 : 1, 40 : 1 and 20 : 1).
After 3 days, the [methyl-3H]thymidine uptake was determined by
the addition of 1 mCi of [methyl-3H]thymidine (25 Ci mmol1;
Amersham, Uppsala, Sweden) for 16 20 h. At the end of this
labelling time, the [methyl-3H]thymidine uptake was determined
by transferring cells to 96-well filter microplates (Unifilter-96
GF/C, PerkinElmer, Boston, MA, USA) and adding 25 ml of liquid
scintillation (Microscint O, PerkinElmer) to measure radioactivity.
Technical controls were from phytohemagglutinin (PHA)-stimulated human lymphocyte populations from a single individual.

Cytokine measurement
Renal cell carcinoma culture supernatants (106 cells/ml) were
assayed for VEGF production at 24 and 48 h with an enzyme-linked
immunoabsorbent assay (ELISA) kit (R&D Systems). Interleukin6, IL-8, IL-10, TNF-a and IL-12 were simultaneously analysed by
microparticle-based flow cytometry (Cytometric Bead Array) in
supernatant samples of DC cultures at baseline and on day 2,
according to the manufacturers instructions (BD Bioscience, San
Jose, CA, USA).

Indoleamine 2,3-Dioxygenase (IDO) activity measurement


Indoleamine 2,3-Dioxygenase (IDO) activity in DC culture supernatants was measured by high-performance liquid chromatography (HPLC). The samples were deproteinised by mixing 100 ml
supernatant and 100 ml of 30% (w/v) trichloroacetic acid. After
centrifugation (4 min at 10 000 g), 25 ml of the supernatant was
added to a vial containing Na2HPO4 buffer of 125 ml (HPLC:
Hewlett-Packard Series 1100; Agilent Technologies Inc., Santa
Clara, CA, USA) using a 5 mM column C18 Symmetry Shield
(Waters Corporation, Milford, MA, USA). The liquid chromatography parameters were injection volume: 100 ml; flow:
0.8 ml min1; stop time: 15 min; maximum pressure: 350 bar. The
mobile phase was 15 mM sodium acetate (pH 4) with 27 ml l1
acetonitrile. The IDO activity is proportional to the [kynurenine]/
[tryptophan] ratio. Kynurenine concentration was measured at
& 2009 Cancer Research UK

VEGF blockade and dendritic cell differentiation


C Alfaro et al

C
on
tro
ls
up
R
er
C
na
C
-1
ta
nt
0
co
nt
R
ro
C
l2
C
-1
4
0
h
co
R
nt
C
ro
C
l4
-1
0
8
hy
h
po
R
C
x
ia
C
-1
24
0
h
hy
po
VH
xi
a
L+
48
53
h
co
nt
VH
o
l2
L+
4
53
h
co
VH
n
to
L+
l4
53
8
h
hy
po
VH
x
L+
ia
24
53
h
hy
po
xi
a
48
h

VEGF (pg ml1)

1200
1000
800
600
400
200
0

20 000

20:1
40:1
80:1

16 000
12 000
8000
4000

C
c
ni

ith

PB

um
iz

be
va
c

l 1
)
ge
ta
llo
ou

(1
ab

(1
ab
m
zu

be
va
ci

g
00

g
00

m
re
53
L+

53

+
0

L+

-1
C
Su

p.
V

C
Su
p.
R

m
l 1
)

96
ov
ed

ov
ed
m
re

53
L+

Su
p.
VH

H
p.
V

h
24

h
96

h
C
C
Su

Su
p.
R

p.
R

-1

-1

re

re

VH
p)
Su
t(
Su

an

ov
ed

L+

ov
ed

53

0
-1
C
C
R
p)

Su
rn
at
Su

pe

24

1:

1:

tro
on
C
t(
an
rn
at
pe
Su

0
l

3H-Thymidine uptake
(c.p.m. )

Figure 1 Tissue culture supernatants from RCC cells contain VEGF and suppress differentiation of MLR-stimulating DC from monocytes. (A) Vascular
endothelial growth factor concentrations measured by ELISA in the 24 and 48 h conditioned media from B80% confluent cultures of the indicated cell lines.
Renal cell carcinoma-10 is a renal cell carcinoma with VHL deficiency. VHL 53 is a stable transfectant recovering the VHL expression. Cultures were
carried out in normoxia (21% O2) or hypoxia chambers (1% O2) as indicated. HT-29 human colon carcinoma cell supernatants were used as control
supernatant. (B) Lymphocyte proliferation at the indicated DC to allogenic PBL ratios, as induced by DC differentiated for 7 days from monocytes in the
presence of GM-CSF and IL-4 with or without 1 : 5 (v/v) of the indicated conditioned media. After differentiation, DC were matured for 48 h with TNF-a,
IFN-a and poly I:C. Conditioned media were removed when indicated by three washes. Anti-VEGF mAb (bevacizumab) was added at the indicated
concentrations for the duration of the differentiation culture. Mature DC differentiated without additives were used as positive control. Data represent
means.d. from three experiments. ***Po0.001, statistical analyses were performed by the Kruskal Wallis statistical test.

360 nm and tryptophan at 285 nm, with a spectrophotometer


coupled to HPLC. A standardised curve to measure kynurenine
and tryptophan concentrations was plotted and assessed.

Arginase activity assay


Cell lysates were tested for arginase activity by measuring the
production of urea derived from L-arg. A volume of 100 ml of cell
lysate was treated with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA). The resulting lysate was added to 100 ml
of an activation solution (Tris-HCl 25 mM, pH 7.4, MnCl2 5 mM).
The mixture was heated for 10 min at 561C, mixed with 100 ml of
0.5 M arginine and incubated for 30 min at 371C. The reaction was
interrupted with 200 ml of an acid solution (H2SO4/H3PO4/H2O;
1 : 3: 7 (v/v/v)) and the samples were boiled for 45 min with 20 ml of
9% (v/v) a-isopropiophenone in absolute ethanol. The enzymatic
activity was assessed by colorimetric reaction in a spectrophotometer at 540 nm using a standard curve.

Fractions of RCC by dialysis and filtration


Renal cell carcinoma supernatant was fractionated to determine
the molecular weight of the inhibitory molecules using centrifugal
& 2009 Cancer Research UK

filters (Microcon, Millipore, Billerica, MA, USA) of 10, 30, 50 and


100 kDa. The collected fractions were tested on monocytes
differentiating to DC by incubation with IL-4 and GM-CSF for
7 days. The only fraction that did not produce the inhibition of
full differentiation of DC from monocytes was below 10 kDa. To
know the more exact the molecular weight, the RCC supernatant
was included in a 3.5 kDa dialysis cassette (Slide-A-Lycer Dialysis
Cassette; 3500 MWCO; Pierce, Rockford, IL, USA) for 2 h at room
temperature and overnight at 41C under continuous stirring.
Thereafter, the supernatant with moieties of molecular weight over
3.5 kDa was fractionated using the same process. To assess
thermostability of the putative inhibitory factor, RCC supernatants
were boiled at 1001C for 5 min.

RESULTS
RCC culture supernatants contain abundant VEGF that is
increased by hypoxia
Vascular endothelial growth factor levels were measured in the
culture supernatant from RCC-10 and VHL 53 tumour cell lines
under normal and hypoxic (1% O2) conditions (Figure 1A). Renal
British Journal of Cancer (2009), 1 9

VEGF blockade and dendritic cell differentiation


C Alfaro et al

4
Tcell: DC ratio

100 000
90 000
80 000
70 000
60 000
50 000
40 000
30 000
20 000
10 000
0

C
-1
0
be
Su
va
p
ci
1:
zu
5
m
ab
Su
(1
p
+
g
so
m
ra
l 1
fe
)
ni
b
(1
Su
0
p
ng
+
su
m
l 1
ni
tin
)
Su
ib
(1
p
+
0
be
ng
va
m
ci
l 1
zu
)
m
Su
ab
p
/
s
+
or
be
af
en
va
ci
ib
zu
m
ab
Su
/s
un
p
+
iti
ni
so
b
ra
fe
ni
b/
su
O
ni
nl
tin
y
al
ib
lo
ge
ni
c
PB
L

80:1 40:1
20:1

Su
p

C
on
tro
l

3H-Thymidine uptake
(c.p.m.)

100 000
90 000
80 000
70 000
60 000
50 000
40 000
30 000
20 000
10 000
0

Tcell: DC ratio

VE
G
F

VE

VE
G
F

(2
5

ng

m
l 1
)

F
(1
00
be
ng
va
VE
ci
m
z
l 1
G
um
F
)
ab
+
be
(1
va
g
ci
zu
m
l 1
m
ab
)
VE
(
10
G
F
0
+
g
so
m
ra
l 1
f
en
VE
)
i
b
G
(1
F
0
+
ng
so
ra
m
fe
l 1
ni
)
b
VE
(1
G
0
0
F
ng
+
su
m
ni
l 1
tin
VE
)
i
b
G
(1
F
0
+
ng
su
m
ni
l 1
tin
ib
)
(1
00
ng
m
l 1
)

80:1 40:1
20:1

C
on
tro
l

3H-Thymidine uptake
(c.p.m.)

Figure 2 Vascular endothelial growth factor and RCC supernatants during monocyte differentiation to DC inhibit MLR-stimulating activity of resulting DC.
Vascular endothelial growth factor-mediated inhibition is reversible by bevacizumab and sorafenib. (A) CD14 monocyte cultures as in Figure 1 were set
up in the presence of VEGF and the indicated VEGF inhibitors. In all the experiments, TNF-a, IFN-a and poly I:C were added to induce DC maturation for
48 h, including the control culture. (B) Anti-VEGF agents in different combinations are tested at the indicated concentrations to reverse the inhibition in the
MLR from mature DC that had been differentiated in the presence of RCC supernatants. Mature DC without VEGF or RCC supernatants were used as
positive controls. Microcultures of allogenic PBL without DC are plotted as negative controls. Results in panels a and b represent the means.d. from four
different experiments.

cell carcinoma-10 lacks VHL expression, whereas VHL 53 is a


transfected revertant for this gene that controls the hypoxia
response. Exposure to hypoxia for 48 h stimulated VEGF secretion
in both cell lines. Vascular endothelial growth factor production
under hypoxia was higher by RCC-10 than by VHL 53, despite
the absence of the VHL expression. The supernatant of RCC-10
under 48 h hypoxia was chosen for subsequent experimentation.

Human DC differentiated in the presence of VEGF or RCC


supernatants show impaired ability to induce T-cell
proliferation, as assessed by MLR
Dendritic cells were differentiated from CD14 monocytes for 7
days by GM-CSF and IL-4 in the presence of either supernatants
from RCC cultured under hypoxic conditions (Figure 1B) or
from VEGF (Figure 2A), leading to a strong inhibition of their
ability to induce allogenic T-cell proliferation, as determined by
the primary MLR. This inhibition did not take place if tumour-cell
supernatants were removed at 24 h, but remained if supernatants
were washed after 96 h of culture (Figure 1B). The supernatants
British Journal of Cancer (2009), 1 9

from other cell lines in Figure 1 showed similar effects (data not
shown).

Bevacizumab and sorafenib reverse the effects induced by


VEGF on DC activity. Neither of the drugs, as single agents
or in combination, reversed the inhibitory effects of RCC
culture supernatants
The addition of bevacizumab or sorafenib restored the MLR of DC
differentiated in the presence of VEGF to baseline levels, whereas
sunitinib did not (Figure 2A). As the activity of indoleamine
2,3-dioxygenase (Munn et al, 2002) or arginase (Ochoa et al, 2007)
can decrease the ability of DC to stimulate T cells, we tested the
activity of both enzymes in DC differentiated under the effects of
VEGF, without finding evidence of increased activity even in the
presence of pharmacological antagonists (Supplementary Figure 1).
The decrease in MLR stimulating activity caused by RCC culture
supernatants could not be restored by bevacizumab, sorafenib or
sunitinib, delivered either as single agent or in combination
(Figures 1B and 2B). Therefore, it can be concluded that VEGF is
& 2009 Cancer Research UK

VEGF blockade and dendritic cell differentiation


C Alfaro et al

5
CD1a

75

75

50
25

B 100

50

50

50
25
0

0
A

75

25

0
A

CD11c

100

75

25

CD1a

MFI

100

MFI

100

MFI

MFI

CD11c

**
CD83

CD80

40
MFI

30
20
10
0

CD83

75

55
50
45
40
35
30
25
20
15
10
5
0

50
MFI

50

MFI

MFI

CD80
55
50
45
40
35
30
25
20
15
10
5
0

25

0
A

***
HLA-DR

CD86

MFI

75
50
25
0
A

**

**

***

**

HLA-DR

100

70
60
50
40
30
20
10
0

75
MFI

100

MFI

MFI

CD86
55
50
45
40
35
30
25
20
15
10
5
0

50
25
0

**

**

A: Immature dendritic cell (iDC)


A: Immature dendritic cell (iDC)

B: Mature dendritic cell (mDC)

B: Mature dendritic cell (mDC)

C: mDc+RCC-10 Supernatant (Sup) 1:5

C: mDC+VEGF (25 ng ml1)

D: mDC+Sup RCC-10/Bevacizumab (1 g ml1)

D: mDC+VEGF/Bevacizumab (1 g ml1)
E: mDC+VEGF/Sorafenib (10 ng
F: mDC+VEGF/Sunitinib (10 ng

ml1)

ml1)

E: mDC+Sup RCC-10/Sorafenib (10 ng ml1)


F: mDC+Sup RCC-10/Sunitinib (10 ng ml1)
G: mDC+Sup RCC-10 removed 24 h
H: mDC+Sup RCC-10 removed 96 h

Figure 3 Vascular endothelial growth factor and RCC supernatants added to DC differentiation cultures inhibit maturation-induced surface markers on
DC: effects of bevacizumab, sorafenib and sunitinib. Dendritic cells obtained from monocytes in 7-day cultures with GM-CSF and IL-4 were analysed by
surface immunostaining and flow cytometry for the indicated leukocyte differentiation antigens before and after maturation in the presence of TNF-a, IFN-a
and poly I:C. As indicated, recombinant VEGF (A) or RCC supernatants (B) were added during differentiation. When indicated, bevacizumab, sorafenib or
sunitinib was also added during the differentiation cultures. Values are expressed as means.e.m. of four different experiments. **Po0.01,***Po0.001,
statistical analyses were performed by the Kruskal Wallis statistical test. In all cultures set up with CD14 cells, the expression of CD14 was lost, so at the
end of the cultures o1% cells were CD14 .

not the only or main mediator of the deleterious effect played by


RCC supernatants on DC differentiation from monocytes.

VEGF and RCC supernatants induce changes in the surface


phenotype of DC
We determined the phenotype of DC differentiated with and
without recombinant VEGF or RCC supernatants. Dendritic cells
were maturated with a cocktail containing TNF-a, IFN-a and poly
I:C (all manufactured under good manufacturing practice conditions). We assessed the surface expression of CD1a, CD11c, CD80,
CD83, CD86, HLA-DR and CD14 using flow cytometry analyses
(Figures 3A and B). The most relevant effects induced by VEGF on
mature DC were marked decreases in the intensity of CD11c, CD86
and HLA-DR. These effects were completely reversed by the
addition of bevacizumab and sorafenib. Sunitinib also restored the
normal expression of CD11c, but not of CD86 and HLA-DR.
Supernatants from RCC decreased the expression intensity of
& 2009 Cancer Research UK

CD11c, CD83, CD86 and HLA-DR to some extent . In these cases,


bevacizumab and sorafenib restored CD86 and HLA-DR but not
CD83. Renal cell carcinoma supernatants, but not VEGF, led to DC
cultures in which cells were more adherent and presented spread
morphology, which was more reminiscent of macrophages under
phase contrast-microscopy (Supplementary Figure 2).
It is of note that DC differentiated with VEGF did not repress the
MLR stimulating activity when added to third-party DC/T cell
cultures, thus revealing the fact that VEGF yields less-active DC
rather than suppressive DC (Supplementary Figure 3).

Addition of VEGF or RCC supernatants during DC


maturation did not alter the MLR-promoting activity of DC
In contrast to monocyte to DC differentiation cultures, neither
VEGF nor RCC supernatants altered the MLR-stimulating activity
of DC if they were added to the culture during the 48 h of
maturation as induced by the combined effects of TNF-a, IFN-a
British Journal of Cancer (2009), 1 9

VEGF blockade and dendritic cell differentiation


C Alfaro et al

6
Tcell:DC ratio

3H-Thymidine uptake, (c.p.m.)

80:1
40:1
20:1

80 000
70 000
60 000
50 000
40 000
30 000
20 000
10 000
ng
Be
m
va
l 1
ci
zu
)
m
ab
VE
(1
G
g
F
+
m
So
l 1
ra
)
fe
ni
b
VE
(1
G
0
F
ng
+
m
Su
l 1
ni
)
tin
ib
(1
0
ng
m
l 1
)
R
C
C
-1
0
Su
p
O
1:
nl
5
y
al
lo
ge
ni
c
PB
L

(1
00

m
at
ur
at
io
n
VE
G
F

VE
G
F

VE

du

rin

du
rin
g

at
ur
at
io

(2

ng

l 1
)

C
on
tro
l

Figure 4 Vascular endothelial growth factor and RCC supernatants, when added during maturation, cannot inhibit the MLR-stimulating activity of already
differentiated DC. Vascular endothelial growth factor and RCC supernatants were added to differentiated DC during the 48 h maturation culture with TNF-a,
IFN-a and poly I:C. When indicated, VEGF-inhibiting drugs were also added. The mitogenic activity on allogenic T cells of DC from the different conditions was
monitored. Mature DC without further additives were used as positive control.

and poly I:C (Figure 4). This indicates that once differentiated, DC
are less sensitive to the effects of VEGF or RCC supernatants with
regard to activation/maturation, at least when triggered by these
powerful maturation-inducing agents.

RCC culture supernatants, but not VEGF, decrease the


production of IL-12 by mature DC
We determined concentrations of IL-12, IL-10, IL-6 and IL-8 in
cultures of immature and mature DC, using microparticle-based
flow cytometry assays. It is interesting that the presence or absence
of VEGF during differentiation did not change the production of
IL-6, IL-8, IL-10 and IL-12. It is surprising that the addition of
sunitinib during DC differentiation dramatically reduced IL-12
and, to a lesser extent, IL-10 concentrations (Figure 5A and
Supplementary Figure 4, showing dose dependency). Such effects
did not occur when DC differentiation cultures were exposed to
either bevacizumab or sorafenib, suggesting a mechanism of action
that is dependent not on VEGF-R but rather on an alternative
pathway (Figure 5A).
We also measured the same cytokines on DC from cultures
either conditioned or not with (1 : 5 (v/v)) hypoxic renal carcinoma
cell-culture supernatant. The supernatant from the cell lines
inhibited IL-12 production almost completely, and such inhibition
was maintained in spite of the addition of antiangiogenic drugs.
The supernatant did not alter the levels of the other assessed
cytokines (Figure 5B), further indicating the functional viability of
the cultures and suggesting immunomodulatory effects.

The effects of RCC culture supernatants on DC activity are


mainly mediated by small thermostable molecule/s and
cannot be reversed by VEGF pharmacological antagonism
As we could not revert the inhibition of MLR activity caused by
RCC culture supernatants with either of the antiangiogenic
British Journal of Cancer (2009), 1 9

compounds, we fractionated the RCC-10 supernatant to determine


the molecules that could cause this effect.
Filtration fractions showed that small molecule/s of o10 kDa
suppressed the MLR activity of DC (Figure 6). Further fractionation
by dialysis pre-excluding molecules o3.5 kDa showed that the
activity was partially recovered when molecules 43.5 kDa were
excluded. Accordingly, most of the inhibitory activity can be traced
to the 3.510 kDa range. Moreover, such inhibitory activity persisted
after heating the supernatant for 5 min at 1001C (Figure 6).
These results conclude that small soluble molecules distinct
from VEGF (described as a moiety in the 35 45 kDa range) are the
main mediators of decreased T-cell-stimulating activity in DC
differentiated from monocytes in the presence of RCC culture
supernatants.

DISCUSSION
The treatment of metastatic kidney cancer has rapidly evolved
during the last few years, and a large number of targeted molecules
have either already shown efficacy in phase III trials or are
in advanced stage of clinical development, replacing former
immunotherapeutic approaches (Schrader and Hofmann, 2008).
Nevertheless, immunotherapy may retain a role in the treatment of
this disease. It must be remembered that the only treatment that
can induce cure in some patients with metastatic renal cell
cancer is high-dose intravenous IL-2 (Yang et al, 2003b;
McDermott et al, 2005). Moreover, immunotherapy with vaccines
is the only treatment that has shown clinical efficacy as an adjuvant
treatment of resected RCC (Jocham et al, 2004). Finally, a large
number of treatments based on the modulation of immunotherapy
are being developed (Melero et al, 2007) and some of them
are being tested in patients with metastatic RCC, suggesting
that, in the near future, combinations of targeted agents with
new immunotherapeutic approaches might become a reality
& 2009 Cancer Research UK

VEGF blockade and dendritic cell differentiation


C Alfaro et al

7
Immature DC (iDC)
Mature DC (mDC)
mDC+VEGF (25 ng ml1)
mDC+VEGF/Bevacizumab (1 g ml1)
mDC+VEGF/Sorafenib (10 ng ml1)
mDC+VEGF/Sunitinib (10 ng ml1)

(pg ml1)

A 20 000
15 000
10 000
5000
1500
1200
900
300
200
100
0
IL-12

IL-6

IL-10

IL-8

Control
RCC-10 supernatant (Sup) 1:5
Sup+bevacizumab (1 g ml1)
Sup+sorafenib (10 ng ml1)
Sup+sunitinib (10 ng ml1)
Sup+bevacizumab/sorafenib
Sup+bevacizumab/sunitinib
Sup+sorafenib/sunitinib

(pg ml1)

10 000
5000
200
100
0
IL-12

IL-10

IL-6

IL-8

Figure 5 Dendritic cell differentiation in the presence of RCC supernatants or sunitinib shows less IL-12 production. Cytokines were measured by CBA
assays in the supernatant of the indicated DC cultures treated for the length of their differentiation from monocytes as indicated. All the experiments were
performed with mature DC except when indicated as immature DC. (A) The effect of VEGF and drug inhibitors is shown, whereas the effect of RCC
supernatants, along with bevacizumab, sorafenib, sunitinib or its combinations, at the indicated concentrations is shown (B).

(Coppin et al, 2008). In addition, as mentioned earlier, VEGF


interferes with the development and function of DC (Gabrilovich
et al, 1996, 1998), and such an effect can be blocked by anti-VEGF
antibodies. Therefore, the study of potential interactions between
targeted agents currently used in the treatment of RCC and the
immune system is of interest.
Our data confirm that VEGF inhibits the functional differentiation of DC, and impairs their ability to induce allogenic T-cell
proliferation as shown by the decrease in the allogenic MLR
assay. Such an inhibition was not dependent on indoleamine
2,3-dioxygenase or on arginase activity factors, which have been
described as immunosuppressive in DC and other myeloid cells
(Munn et al, 2002; Ochoa et al, 2007). The addition of either
sorafenib or bevacizumab reverted the effects of VEGF on DC.
Sunitinib did not restore the ability of DC to induce a normal MLR,
& 2009 Cancer Research UK

despite the fact that the concentrations used were in the range as
those observed in cancer patients (31.8 65.9 ng ml1) (Faivre et al,
2006). The immune phenomena of these kinds in RCC patients
might be crucial.
To mimic in vivo conditions, we assessed the effects of
differentiating DC in the presence of VEGF-containing supernatants from RCC cell lines cultured under hypoxic conditions.
The supernatants induced a strong inhibition of DC differentiated
in such a manner that it was not reverted by the addition of any of
the anti-VEGF tested drugs. Moreover, combinations of the drugs
did not have any restoring effects on the MLR decrease induced by
the supernatants. Fractionation of those supernatants showed that
small molecule/s 43.5 kDa and o10 kDa suppressed the MLR
activity of DC, and that such molecules were thermostable. For this
reason, metabolic products, such as tryptophan metabolites, can
British Journal of Cancer (2009), 1 9

VEGF blockade and dendritic cell differentiation


C Alfaro et al

8
3H-Thymidine uptake (c.p.m.)

120 000
100 000
Control
RCC-10 Supernatant (Sup) 1:5

80 000

RCC-10 Sup 100C


RCC-10 >100 kDa

60 000

RCC-10 <100 kDa


RCC-10 <50 kDa
RCC-10 <30 kDa

40 000

RCC-10 (<10 kDa, >3.5 kDa)


PBL without allogenic DC

20 000
0
100

1000
No. of DC

10 000

Figure 6 The inhibitory activity of RCC-10 supernatants on the


differentiation of MLR-stimulating mature DC is mainly mediated by
thermostable molecules in the range of 3.5 10 kDa. RCC-10 was used as
crude or fractionated by filtration and/or dialysis in molecules of the
indicated molecular weight ranges. The inhibitory activity was present after
treating RCC-10 supernatants for 5 min at 1001C. Mature DC without
additives under identical conditions were used as positive controls for MLR
activity, and microcultures of allogenic lymphocytes without DC are plotted
as negative controls. The experiment shown is representative of the three
similarly performed.

also be excluded as being responsible for the suppression. These


results indicate that tumour cells exert their inhibitory function on
differentiating DC through soluble factors distinct from VEGF,
although VEGF could also be involved to a lesser extent.
Changes in DC differentiated in the presence of recombinant
VEGF or RCC supernatants included marked decreases in CD11c,
CD86 and HLA-DR expressions, which were completely reverted
by bevacizumab and sorafenib, whereas sunitinib only restored
normal expression of CD11c. The events in the transition
from monocyte to DC are important not only because they
represent a popular method of generating DC for immunotherapy
(Vulink et al, 2008) but also because they might resemble the
physiological differentiation of DC from myeloid precursors. The
effect of VEGF on monocytes is not surprising because these
leukocytes express abundant levels of the three identified receptors
for this cytokine (Supplementary Figure 5). Moreover, during
differentiation culture to DC, VEGFRs are downregulated.
It is interesting that, neither VEGF nor RCC supernatants were
able to inhibit MLR if they had been added only during DC
maturation/activation, rather than during the 7-day differentiation
culture from monocytes. This indicates that mature DC might be
more resistant to the effects of RCC supernatants or VEGF.
Alternatively, the good manufacturing practice-compliant maturation cocktail that we use may be of such a strength that is not
amenable to inhibition by these agents.
Exposure to VEGF during differentiation did not alter the
secretion of several cytokines, including that of IL-12, IL-10, IL-6
and IL-8. The addition of sunitinib during DC differentiation
induced a marked decrease in IL-12 concentrations and a less
relevant reduction in IL-10 secretion. Bevacizumab and sorafenib
did not reproduce these effects. These results suggest that the IL-12
inhibition induced by sunitinib is not mediated by the VEGF
pathway, and may have relevant implications when exploring
combinations of sunitinib with other immunotherapeutic
strategies. Indeed, sunitinib and sorafenib are far from being
specific inhibitors of a single tyrosine kinase and have been shown
to inhibit various kinases with different ICs50. These included
signalling pathways known to be critical in leukocyte biology
(Karaman et al, 2008). Therefore, this type of VEGF-R-independent
effects is not unexpected to see. Renal cell carcinoma culture
British Journal of Cancer (2009), 1 9

supernatants also inhibited IL-12 production, and neither of the


drugs tested reversed this effect, further supporting the fact that
IL-12 inhibition is dependent not on the VEGF pathway but on
other unidentified factors. Interleukin-12 is critical for therapeutic
immunity against cancer (Mazzolini et al, 2003). Artificial enhancement
of IL-12 production by DC strengthens elicited immunity and
therapeutic potential (Melero et al, 1999; Mazzolini et al, 2005).
Therefore, selective inhibition of IL-12 is likely to be suppressive
for cellular immune responses.
Other authors have also evaluated the effects of sorafenib and
sunitinib on DC (Hipp et al, 2008). They reported that sorafenib,
but not sunitinib, inhibited DC function, assessed through the
expression of cytokines and CD1a. The inhibitory effects were
mediated by the inhibition of phosphatidylinositide 3-kinase
production and mitogen-activated protein kinases, as well as by
nuclear factor-kB signalling. They treated C57BL/6 mice with both
drugs, and found that sorafenib significantly reduced the induction
of antigen-specific T cells, whereas sunitinib reduced the number
of regulatory T cells in peripheral blood. On the basis of these
findings, they concluded that sorafenib does not seem to be a good
candidate for combination with immunotherapeutic approaches,
whereas sunitinib does. The main difference between both studies
is that whereas Hipp et al (2008) investigated the effects on already
differentiated DC, we focused on the transition from myeloid
immature precursors to DC.
Our experimental observations join the literature on potential
mechanisms accounting for immune dysfunction in RCC. Direct
proapoptotic effects of RCC cells on human T cells have been
reported as being mediated by the FasL expression and gangliosides released into the surrounding media (Das et al, 2008).
Gangliosides are unlikely mediators of the effects on monocytes
because of lower molecular weights than that of the expected
factors according to Figure 6. In RCC patients, sunitinib treatment
improves type I responses by T lymphocytes with decreases in
regulatory T cells (Finke et al, 2008). Whether this is mediated
directly on T cells within the cancer-modified environment or by
indirect effects involving myeloid leukocytes, such as DC, remains
to be seen. The effects of sunitinib downmodulating the ability for
IL-12 production in our cultures are to be reconciled with the
improvements of type I responses seen in the patients and possibly
related to the complexity of the in vivo system (Finke et al, 2008).
In conclusion, our study shows that bevacizumab and sorafenib,
but not sunitinib, reverse the inhibitory effects of recombinant
VEGF on DC differentiation, but not of those mediated by RCC
culture supernatants, which are mainly mediated by other
unidentified substance(s). The changes induced by VEGF on
mature DC included a reduced expression of HLA-DR and CD86,
which was also reverted by bevacizumab and sorafenib. Finally,
RCC culture supernatants and, surprisingly, sunitinib decreased
IL-12 production by mature DC. Such inhibition was not restored
by any of the tested drugs, delivered either as single agents or in
combination. Moreover, our data indicate that soluble factors
from RCC exert effects on monocyte to DC differentiation,
which renders functionally impaired DC for T-cell stimulation.
Fortunately, such altered DC by tumour-soluble factors do not
interfere with the function of healthy DC differentiated apart from
the influence of tumour cells. Biochemical identification of the
inhibitory factors is the next step to progress in our understanding
of the new described phenomena.

ACKNOWLEDGEMENTS
This study was supported by the Spanish Society of Medical
Oncology (SEOM), Departamento de Salud del Gobierno de
Navarra (Beca Ortiz de Landazuri), Ministerio de Sanidad (Fondo
de Investigacion Sanitaria, PI060932), Ministerio de Sanidad
& 2009 Cancer Research UK

VEGF blockade and dendritic cell differentiation


C Alfaro et al

9
(Fondo de Investigacion Sanitaria, EC07/90133) and by Pfizer Inc.
We are indebted to Leyre Urruticoechea for providing sorafenib.

Supplementary Information accompanies the paper on British


Journal of Cancer website (http://www.nature.com/bjc)

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British Journal of Cancer (2009), 1 9

FIGURAS SUPLEMENTARIAS________________________
(Influence of bevacizumab, sorafenib and sunitinib as single agents or in combination on the
inhibitory effects of vegf on human dendritic cell differentiation from monocytes. C Alfaro, N
Surez et al. British Journal of Cancer, 2009 Abril 7; 100 (7): 1111-9)

109

Supplementary Figure 1

Supplementary Figure 1. VEGF added to DC differentiation cultures does not increase


IDO or arginase.
IDO was studied by kynurenine production normalized by [kynurenine]/[tryptophan] in the
supernatant of DC differentiated under the influence of the indicated compounds. Arginase was
measured in cell lysates by a colorimetric assay measuring urea production. Bevacizumab and
sorafenib, added at the indicated concentrations, normalized IDO activity when VEGF had been
added whereas sunitinib increased by itself arginase activity. For normalization, a relation index
(RI) was calculated as [kynurenine]/[tryptophan] in dendritic cells in the experimental
conditions divided by the same parameter in control dendritic cells without VEGF or RCC
supernatants during differentiation. DC were matured with TNF-, IFN- and poly I:C or IFN-
when indicated.

110

Supplementary Figure 2

Supplementary Figure 2.VEGF, RCC-10 supernatants during differentiation of monocytes


to DC change adherence and morphology under phase contrast microscopy.
As it can be seen RCC-10 but not VEGF changed the morphology towards a more spread and
adherent phenotype. Percentages of floating versus adherent cells are included for each
condition.

111

Supplementary Figure 3

Supplementary Figure 3.
DC differentiated with VEGF did not repress MLR stimulating activity when added at different
ratios to DC:T co-cultures.

Supplementary Figure 4

Supplementary Figure 4.
Effects on IL-12 production of lower concentrations of sunitinib, than those shown in figure 5
under identical conditions to promote maturation with TNF-, IFN- and poly I:C.

112

Supplemenatry Figure 5

Supplementary Figure 5.
FACS analysis by indirect immunofluorescencce with specific mAb of surface VEGFRs
expression on monocytes and DC derived from monocytes

113

Carcinoma-Derived Interleukin-8 Disorients Dendritic Cell Migration


without Impairing T-Cell Stimulation

C. Alfaro 1; N. Surez 1,2; I. Martnez-Forero 1 ; A. Palazn 1; A. Rouzaut 1; S. Solano1; E.


Feijoo 1; A. Grpide3 , E. Bolaos1,3; L. Erro 1; J. Dubrot 1; S .Hervas-Stubbs 1; A. Gonzalez 2;
J.L. Perez-Gracia 3; I. Melero 1,3

Gene Therapy and Hepatology Division, Centro de Investigacin Mdica Aplicada (CIMA),
Pamplona, Spain; 2 Biochemistry Department, Clnica Universidad de Navarra, Pamplona,
Spain; 3 Medical Oncology Department, Clnica Universidad de Navarra, Pamplona, Spain.

PloS ONE, 2011 Marzo 14; 6(3): e17922

115

Carcinoma-Derived Interleukin-8 Disorients Dendritic


Cell Migration Without Impairing T-Cell Stimulation
Carlos Alfaro1., Natalia Suarez1,2., Ivan Martnez-Forero1, Ass Palazon1, Ana Rouzaut1, Sarai Solano1,
Esperanza Feijoo1, Alfonso Gurpide3, Elixabet Bolanos1,3, Lorena Erro1, Juan Dubrot1, Sandra HervasStubbs1, Alvaro Gonzalez2, Jose Luis Perez-Gracia3", Ignacio Melero1,3*"
1 Gene Therapy and Hepatology Division, Centro de Investigacion Medica Aplicada (CIMA), Pamplona, Spain, 2 Biochemistry Department, Clnica Universidad de Navarra,
Pamplona, Spain, 3 Medical Oncology Department, Clnica Universidad de Navarra, Pamplona, Spain

Abstract
Background: Interleukin-8 (IL-8, CXCL8) is readily produced by human malignant cells. Dendritic cells (DC) both produce IL-8
and express the IL-8 functional receptors CXCR1 and CXCR2. Most human colon carcinomas produce IL-8. IL-8 importance in
malignancies has been ascribed to angiogeneis promotion.
Principal Findings: IL-8 effects on human monocyte-derived DC biology were explored upon DC exposure to recombinant
IL-8 and with the help of an IL-8 neutralizing mAb. In vivo experiments were performed in immunodeficient mice
xenografted with IL-8-producing human colon carcinomas and comparatively with cell lines that do not produce IL-8.
Allogenic T lymphocyte stimulation by DC was explored under the influence of IL-8. DC and neutrophil chemotaxis were
measured by transwell-migration assays. Sera from tumor-xenografted mice contained increasing concentrations of IL-8 as
the tumors progress. IL-8 production by carcinoma cells can be modulated by low doses of cyclophosphamide at the
transcription level. If human DC are injected into HT29 or CaCo2 xenografted tumors, DC are retained intratumorally in an IL8-dependent fashion. However, IL-8 did not modify the ability of DC to stimulate T cells. Interestingly, pre-exposure of DC to
IL-8 desensitizes such cells for IL-8-mediated in vitro or in vivo chemoattraction. Thereby DC become disoriented to
subsequently follow IL-8 chemotactic gradients towards malignant or inflamed tissue.
Conclusions: IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated
T-cell stimulation.
Citation: Alfaro C, Suarez N, Martnez-Forero I, Palazon A, Rouzaut A, et al. (2011) Carcinoma-Derived Interleukin-8 Disorients Dendritic Cell Migration Without
Impairing T-Cell Stimulation. PLoS ONE 6(3): e17922. doi:10.1371/journal.pone.0017922
Editor: R. Mosley, University of Nebraska Medical Center, United States of America
Received August 18, 2010; Accepted February 17, 2011; Published March 14, 2011
Copyright: 2011 Alfaro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Financial support was from MEC/MICINN (SAF2005-03131 and SAF2008-03294), Departamento de Educacion del Gobierno de Navarra, Departamento
de Salud del Gobierno de Navarra (Beca Ortiz de Landazuri), Redes tematicas de investigacion cooperativa RETIC (RD06/0020/0065), Fondo de investigacion
sanitaria (FIS PI060932), European commission 7th framework program (ENCITE) and SUDOE-IMMUNONET, Fundacion Mutua Madrilena, and UTE for project
FIMA. CA is supported by Fundacion Cientfica de la Asociacion Espanola Contra el Cancer (AECC). SH-S receives a Ramon y Cajal contract from Ministerio de
Educacion y Ciencia and AP a scholarship from FIS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: imelero@unav.es
. These authors contributed equally to this work.
" These authors also contributed equally to this work.

Chemokine receptors guide DC in physiology and in inflammation [7,8]. DC migration from inflamed/infected [9] or
malignant tissues [10,11] is important for the orchestration of
immune responses. Chemokine receptors do not only regulate
motility but also control other cellular functions such as activation
or survival in various cell types [11,12]. Therefore it would not be
a surprise if the chemokine microenvironment modified DC
functions other than migration [12].
Human tumor cells produce IL-8 in most cases [1,13] as a
biological dirty trick played by the malignant tissue to promote
angiogenesis [3,13,14,15] and possibly to support the type of
smoldering inflammation that promotes tumor progression and
metastasis [14,16,17]. Tumor growth in human patients statistically correlates with IL-8 serum concentrations [3,18]. Recently, a
role for IL-8 has been described in the resistance to antiangiogenic

Introduction
111

In a previous study [1] we showed that Indium-labeled DC


when injected into tumor lesions of patients suffering advanced
digestive carcinomas [2] tended to remain inside the injected
lesion. An explanation for such a retention was proposed in the
sense that the human tumors abundantly produce IL-8 [1,3] and
DC express CXCR1 and CXCR2 functional IL-8 receptors on
their plasma membrane [1,4,5]. However, no definitive proof was
provided for the role of IL-8 in intratumoral retention of DC [1].
An IL-8 homologue is absent from the mouse genome and these
precludes incisive definitive genetic experimentation on the role of
IL-8 in murine tumor models. However there are reports
suggesting that mouse CXCR1 is activated by human IL-8, hence
permitting to some extent experiments in xenografts [6].
PLoS ONE | www.plosone.org

March 2011 | Volume 6 | Issue 3 | e17922

Malignant Cell-Derived IL-8 Effects on DC

VEGF signal blockade with sunitinib [19]. Importantly escape


from sunitinib can be thwarted by co-treatment with neutralizing
anti-IL8 mAb [19].
IL-8 was originally discovered as a powerful attractor of
polymorphonuclear leukocytes (PMNs) [20,21] in acute inflammation [21], but may act on other leukocyte subtypes [1,22] and
on endothelial cells [15]. In turn, DC are both responsive to IL-8
[4,5], and produce IL-8 either when inactive or more overtly so,
when activated/matured [1]. Injecting DC inside tumors has been
intended to enhance antitumor activity for therapeutic purposes in
animal models [11,23] and in the clinic [2,24,25]. One of the
hurdles faced is that the tumor microenvironment is rich in
substances impairing DC functions [11,26]. DC migration into
lymph nodes is of critical importance in cancer immunotherapy
based on DC [27,28,29]. If retained intratumorally, DC would be
prey for tumor microenvironmental factors such as TGF-b for
longer periods of time [11], thereby causing damage to the
induction of anti-tumor immunity.
Here we show that xenografts of human tumors retain DC
inside the injected tumors by means of IL-8-mediated chemoattraction, that can also recruit DC to the malignancy when injected
in the subcutaneous connective tissue in the vicinity of the tumor.
However, the same functional recombinant IL-8 that attracts DC
and PMNs does not impair the abilities of DC to induce T-cell
activation and proliferation either in vitro or in vivo. Interestingly,
pre-exposure of DC to IL-8 restrains subsequent migration
towards IL-8 chemo-attractive gradients indicating desensitization
of the receptors.

in the 24 h supernatants from these subcultures by means of


ELISA (BD Biosciences, San Diego, CA). Cyclophosphamide
(Cytoxan) was purchased in our hospital pharmacy.

Semiquantitative RT-PCR for IL-8


Total cellular RNA was extracted with Trizol (Invitrogen,
Carlsbad, CA) according to the protocol provided by the
manufacturer. First-strand cDNA was synthesized from 1 mg total
cellular RNA using an RNA PCR kit (Takara Bio Inc., Otsu,
Japan) with random primers. Thereafter, cDNA was amplified
using 30, and 28 cycles for IL-8 and for b-actin, respectively. The
specific primers used were as follows: IL-8, forward primer 59ATGACTTCCAAGCTGGCCGTG -39 and reverse primer 59TTATGAATTCTCAGCCCTCTTCAAAAACTTCTC-39; and
for b-actin, forward primer 59-GTGGGGCGCCCCAGGCACCA-39 and reverse primer 59-CTCCTTAATGTCACGCACGATTTC-39. The product sizes were 300 bp for IL-8, and
548 bp for b-actin. The thermocycling conditions for the targets
were as follows: denaturing at 94uC for 30 s for IL-8, and b-actin,
annealing at 60uC for 30 s for IL-8 and b-actin, and extension at
72uC for 90 s for IL-8 and b-actin. The PCR products were
fractionated on 2% agarose gels and visualized by ethidium
bromide staining. The quantity of a band was measured by the
area under its intensity profile curve using BioRad Quantity One
1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA,
USA). b-actin was employed to normalize the amount of RNA
used in each reaction.

Mouse tumors

Methods

Nude mice, Rag2/2 or Rag2/2 IL-2Rc2/2 were obtained


from The Jackson Laboratory. Animal experiments were in
accordance to Spanish laws and approval was obtained from the
animal experimentation committee of the University of Navarra
(Study 03/007 approval). These mice were injected with the tumor
cell lines HT29 (56106 cells), CaCo2 (107 cells) or SW48 (56106)
to induce subcutaneous tumors. IL-8 in serum samples was
sequentially measured by ELISA (BD Biosciences). When
indicated 3 mg/mouse of cyclophosphamide were injected i.p.

Ethics statement
Animal studies have been performed in accordance with
Spanish legislation under specific approval from the institutional
ethics board by the Comite de Etica para la Experimentacion Animal of the
University of Navarra (Study 03/007 approval). Human cells are
obtained from Blood donors (public blood bank of Navarra) under
written informed consent for research.

Dendritic cell generation

In vivo migration

Dendritic cells were generated from filter buffy coats (FBC)derived monocytes donated by healthy donors [30] who explicitly
sign a written informed consent. To generate immature DCs from
monocytes, human peripheral blood was isolated by Ficoll-Paque
gradient centrifugation from FBC. Isolated mononuclear cells
from these sources were subjected to positive selection using antiCD14-conjugated paramagnetic beads and purified using the
AutoMacs system according to the manufacturers instructions
(Miltenyi Biotec, Bergisch Gladbach, Germany). Purified monocytes were cultured for 7 days in RPMI-1640 with 5% (v/v) heat
inactivated FCS. To differentiate dendritic cells from CD14+ cells,
culture medium was supplemented with GM-CSF (1000 U/mL;
Novartis, Basel, Switzerland) and IL-4 (500 U/mL; R&D Systems,
Minneapolis, MN). DCs were matured adding clinical grade TNFa (50 ng/mL; Boehringer Ingelheim, Ingelheim, Germany), IFN-a
(1,000 IU/mL; Schering-Plough, Kenilworth, NJ) and Poly I:C
(10 mg/mL; Ampligen, Bioclones, Tokai, South Africa) for 48 h.

Female nude mice, Rag2/2 or Rag2/2 IL-2Rc2/2 as


indicated, were subcutaneously injected with 56106 HT29
(n = 4), 106106 CaCo2 (n = 4) or 106 SW48 (n = 3) tumor cells.
When tumors reached about 1 cm diameter, 106 mature DCs
were labelled with 2.5 mM CFSE (Sigma, Barcelona, Spain) or
461026 M PKH26 (Sigma), washed and injected intratumorally.
Mouse IgG (100 mg, BD Pharmingen) or neutralizing anti-human
IL-8 mAb (100 mg, BD Pharmingen) were coinjected within the
same syringe into the tumors. In case of HT29 and SW48, cell
suspensions from the tumors were generated with the GentleMacs
dissociator device (Miltenyi Biotec). Cell suspensions were
analysed by FACS and fluorescent cells counted. In the case of
CaCo2 xenografts, three days later, tumors were mechanically
homogenized. Tissue homogenates were cleared from debris by
centrifugation and fluorescence was measured using a plate
fluorimeter (Polarstar Galaxy, BMG). Migration was calculated
as fluorescence in the tumor divided by total input fluorescence
injected (fluorescence was quantified in arbitrary units).

Cell lines and IL-8 concentrations in mouse serum and


culture supernatants

Cytokine production by maturing DC

HT29, CaCo2 and SW48 colon carcinoma cell lines were


obtained from American Type Culture Collection (Rockville,
MD). Cell lines were cloned by limiting dilution in 96-well plates
and subcultures (105 cells) were tested for the concentration of IL-8
PLoS ONE | www.plosone.org

For in vitro stimulations, 105 DC were cultured 48 h with


medium alone (control), LPS (1 mg/mL) purchased from Sigma,
R-848 imidazoquinoline (1 mM) purchased from Pharmatech
2

March 2011 | Volume 6 | Issue 3 | e17922

Malignant Cell-Derived IL-8 Effects on DC

PKH2-fluorescent DC per microscopic field (620) in the lower


chamber were quantitated in triplicate wells by a blinded observer.
Recombinant MIP3a was from R&D.

(Shangai, China) or sCD40L at 200 ng/mL purchased from


Abnova (Taipei, Taiwan). After culture for 48 h, supernatants
were collected and the cytokine concentration was determined by
immunoassay. Commercially available ELISA kits were used for
the detection of IL-12p70 and IL-10 (BD Bioscience).

Statistics

FACS analysis

Comparisons were made with paired students t tests. Values of


p are given in the corresponding experiments.

FITC and PE-labeled mAb specific for the DC maturation


markers: CD80, CD83, CD86 and HLA-DR (BD Bioscience) and
isotype-matched labeled controls were used to characterize cell
surface phenotypes by flow cytometry. Dendritic cells (105) were
washed in cold PBS and incubated 15 min at 4uC with specific
FITC or PE-labeled Abs. MAbs against IL-8 receptors (CXCR1
and CXCR2) were used by indirect fluorescence developed with a
rabbit anti-mouse antiserum tagged with FITC (Jackson ImmunoResearch Labs, West Grove, PA).

Results
HT29 and CaCo2 tumor cell lines xenografted into
immunodeficient mice generate tumors that produce
IL-8
A panel of human colon carcinomas was tested in order to
identify cultures that produce high amounts of IL-8 to the
supernatant [1]. All clonal subcultures of HT29 showed high
homogeneous outputs of IL-8 while the SW48 cell line did not
reach detectable levels in any experiment, and CaCo2 subcultures
showed around one half of the production when compared to the
levels attained by HT29 cells cultured at identical density for the
same period of time (Figure S1). Microenvironment conditions
and therapy may modify the ability of tumor cells to produce IL-8.
Figure 1A shows that the production of IL-8 secreted to culture
supernatants by viable HT29 cells was reduced in 24 h by
exposure to low concentrations cyclophosphamide, while cells still
preserved membrane integrity (.90% viability by trypan blue
exclusion). Interestingly, the low range of cyclophosphamide
concentrations was more effective at preventing IL-8 bioproduction and secretion to the supernatant. Gemcitabine and radiation
did not or more weakly affected IL-8 secretion (Figure 1A and data
not shown). Moreover, in a repeated set of experiments
semiquantitative RT-PCR for IL-8 in comparison with the house
keeping mRNA b-actin showed that cyclophosphamide inhibits
IL-8 production at the mRNA level in a dose-dependent manner
(Figure 1B), in such a way that low dose cyclophosphamide was
better at mediating this effect than higher concentrations.
These results open the possibility that IL-8 production can be
acutely reduced by cyclophosphamide for therapeutic purposes.
Indeed, metronomic cyclophosphamide is becoming an attractive
alternative for cancer management [31] and potentiation of a
variety of immunotherapies [32].
When HT29 was xenografted in athymic nude mice, it gave rise
to subcutaneous nodules that grew steadily over time (Figure 1C).
Sequential sera samples from such animals contained increasing
concentrations of IL-8 (Figure 1C) that correlated with tumor
progression as reported in human patients [3].
CaCo2 failed to graft as subcutaneous nodules in two thirds of
cases (data not shown), but grafted homogeneously as multiple
peritoneal nodules if injected intraperitoneally (Figure S2). CaCo2grafted animals also showed circulating IL-8 (Figure S2) but at
lower concentrations if compared to HT29-bearing mice, as
expected from the productions of IL-8 in the cell line cultures.
Apart from this quantitative difference, the tendency was similar in
tumors from both cell lines.
Importantly, treatment of mice with a single dose of 3 mg/mouse
of cyclophosphamide reduced the serum concentration of IL-8 in
the next 24 h in a range from 48 to 100% (Figure 1D), while those
concentrations rapidly rebound in 4872 h. It is of note that for this
experiment mice with 7-day palpable tumor xenografts were used,
so the concentrations of IL-8 in plasma were still low.
In conclusion, xenografted colon carcinomas retain the property
of producing high amounts of human IL-8, and our results
indicate that such a function could be modified by cyclophosphamide.

In vitro and in vivo MLR


In vitro MLR were performed as described [26]. Briefly, a total
of 26105 lymphocytes from a distinct donor were added on day 9
at different T cell:DC ratios (1280:1, 640:1, 320:1, 160:1, 80:1 and
40:1). After 3 days, the [methyl-3H]thymidine uptake was
determined by the addition of 1 mCi of [methyl-3H]thymidine.
Female Rag2/2 IL-2Rc2/2 were subcutaneously injected with
56106 HT29 cells. When tumors reached approximately 1 cm in
diameter, these mice and tumor-free mice were intraperitoneally
injected with 16106 DC and 56106 PKH2-labeled PBLs. After 4
days, cells were obtained by intraperitoneal lavages and samples
were analysed using a FACSCalibur Flow Cytometer (Becton
Dickinson). The number of T cell divisions is proportional to the
dilution of PKH2 intensity and was found to be negligible in the
absence of DC (data not shown). For FACS analysis lymphocytes
were gated based on FSC/SSC features.

PMN purification and fluorescence labelling


In vitro neutrophil and DC migration was measured in Transwell
Chambers (5 mm; Corning Costar, Corning, NY). PMN cells were
enriched by sedimentation of peripheral blood mixed in a dextran
(6% v/v) solution. After sedimentation, floating fractions were
collected. Red cells in the resuspended pellets were osmotically
lysed. The remaining cell suspensions were layered onto FicollPaque gradients and pellets were collected and washed after
centrifugation. Neutrophil purity was .95% (CD15bright neutrophils)

In vitro chemotaxis assay


In vitro neutrophil and DC migration was measured in Transwell
Chambers (5 mm; Corning Costar, Corning, NY). Both PKH26DCs (105) and PKH2-labeled neutrophils (105) or only PKH2labeled neutrophils were added to the upper chamber and
migration stimuli were placed in the lower chamber. In this
experiment IL-8 (R&D Systems) was used at 20 ng/mL as positive
control. In other cases PKH26-DCs with or without IL-8
neutralizing mAb or IgG as control (BD Pharmigen) at 20 mg/
mL was placed in the lower chamber as indicated. Transmigrated
cells in the lower chamber were quantified using a FACSCalibur
flow cytometer (BD Biosciences) or fluorescence microscopy
imaging of the lower chamber. In some cases the lower chamber
contained a subconfluent monolayer of HT29 cells. The
chemotactic index was calculated as the number of migrated cells
in the experimental conditions divided by number of migrated
cells in the negative control, which is complete culture medium. In
the experiments with HT29 cell in the lower chamber number of
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Figure 1. HT29 cells when xenografted secrete IL-8 to the plasma of the mice. ELISA determination of IL-8 in the supernatant of HT29
confluent cultures in the presence of indicated concentrations of cyclophosphamide (1000 to 0.001 mg/mL), or gemcitabine (1000 to 0.1 mg/mL)
during the 24 h prior to supernatant collection. When indicated the solution vehicle of both drugs was added at the highest concentration. Results
represent mean6SEM from three experiments. (B) Separate set of experiments as in A but in this case HT29 were collected and IL-8 mRNA was
quantified by RT-PCR. PCR bands are shown in the upper panel and densitometry quantitative data given in the lower panel representing relative
expression of IL-8 mRNA in comparison with b-actin mRNA. (C) Subcutaneously xenografted HT29 cells in Rag2/2 IL-2Rc2/2 mice gave rise to
progressing tumors (left axis depicting mean tumor diameter) and increasing serial serum concentrations of human IL-8 (right axis). Results represent
mean6SEM of three independent experiments with 6 mice per experiment. Similar results were observed with CaCo2 tumors xenografted in the
peritoneal cavity (Figure S1). (D) IL-8 serum concentrations of three individual Rag2/2 IL-2Rc2/2 mice bearing HT29 tumors for seven days before a
single intraperitoneal injection of 3 mg of cyclophosphamide and 24 and 48 h after treatment. The percentages of reduction at 24 h are indicated.
Experiments were repeated at least three times with the exception of (D) that was performed with three individual animals.
doi:10.1371/journal.pone.0017922.g001

analysis representing two cases are shown in figure 2B as an example.


These results were confirmed in athymic nude mice bearing
subcutaneous CaCo-2 tumors (Figure S3) indicating that the
phenomenon was not exclusive of HT29-derived tumors.
SW48 cells, that failed to produce IL-8 as shown in figure S1,
were xenografted in Rag2/2 mice. In this case, the tumors could
not retain DC labeled with the fluorescent dye PKH26. Figure 2C
shows representative dot plots from three mice 72 h post
intratumoral injection along with the fluorescence of input DC
(left dot-plot of figure 2C). These results on the colon cancer cell
line that does not produce IL-8 further indicate that this
chemokine was important for the retention of DC inside the
tumor upon intratumoral release.

Exogenously injected human DC inside xenografted


HT29 tumors are retained by IL-8 in the tumor
microenvironment
In order to study whether IL-8-producing tumors would retain
intratumorally DC injected inside the lesion, we first chose the
HT29 xenografts because of their higher bioproduction of IL-8.
Human DC were derived from CD14+ monocytes in the presence
of GM-CSF and IL-4 and labeled with PKH26. Fluorescent DC
were injected into HT29 tumor nodules subcutaneously implanted
into Rag2/2 IL-2Rc2/2 mice. In some of the mice, DC were
injected with control polyclonal mouse IgG antibody, while in other
cases were resuspended with 100 mg/mL of an anti-human IL-8
neutralizing mAb. 72 h after DC injection, tumors were removed
and a single cell suspension was generated. The number of fluorescent
human CD11c+ cells versus total cells was quantified. As can be seen
in figure 2A, IL-8 indeed retained DC intratumorally since the
neutralizing anti-IL-8 mAb decreased the number of cells that
remained inside the tumor by more than one half (Figure 2A). FACS
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Lack of IL-8 effects on DC-mediated T-cell stimulation


Functional response to IL-8 involves signalling pathways that
might alter DC functions, since these cells are known to express
CXCR1 and CXCR2 [1]. We explored this question in detail
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Figure 2. IL-8 produced by tumor cells in vivo retains DC inside xenografted tumor nodules. (A) HT29 was xenografted into Rag2/2 IL2Rc2/2 double KO mice. Tumor nodules, 812 mm in diameter, were injected with 56106 PKH26-labeled monocyte-derived DC. When indicated, the
100 mL DC suspensions contained 100 mg/mL of mouse IgG (control antibody) or anti-IL-8 neutralizing mAb. The figure shows the proportion of
PKH26+ events with respect to total tumor cells upon FACS analysis, three days after DC injection. Four mice with two bilateral xenografted tumors
each per condition were used. Of note, DC cultured in the presence of the anti-IL-8 mAb at 100 mg/mL did not show loss of viability at least in 72 h
(data not shown). (B) Representative FACS dot plots from A in two tumor nodules from two mice are shown as an example. Similar data were
obtained with xenografts of the CaCo2 cell line (Figure S2). (C) Absence of PKH26-labeled DC in 3 out of 3 SW48 xenografts processed as in A,
following injection of fluorescence labeled human DC. In the left dot-plot, the fluorescence intensity of injected PKH26-labeled DC is shown for
reference. Dot plots are from representative experiment of two actually performed with three animals per group each.
doi:10.1371/journal.pone.0017922.g002

of IL-8. No change was observed again in the ability of DC to


induce proliferation of allogeneic T lymphocytes (Figure 3C). To
rule out alterations of the recombinant protein used, in every case,
IL-8 was controlled for functionality since it readily attracted
human neutrophils, as shown in chemotaxis assays (Figure S4).
We had previously shown that DC expressed CXCR1 and
CXCR2 [1]. We observed that the exposure to the ligand for two
hours induced the modulation/internalization of both receptors
(Figure 3D) in the very same DC used to set up the T-cell
allostimulation experiments. Therefore, the pathways guiding IL8-directed migration and those governing the capabilities for Tcell stimulation seem to be fairly independent in the DC.
The absence of effects on T cell:DC co-cultures suggested that
the molecular factors employed by the DC for T cell activation
were not affected by IL-8.

using Mixed Lymphocyte Reaction (MLR) assays in which DC


were co-cultured in decreasing amounts with fully allogeneic
Peripheral Blood Mononuclear Cells (PBMC) containing alloreactive T-cells. When added during the MLR reaction, IL-8 did
not change the proliferation of T cells (Figure 3A) or the ensuing
production of INF-c to the supernatant (data not shown).
DC were derived from CD14+ monocytes in the presence of
GM-CSF and IL-4 and during this process we had observed that
tumor-derived compounds impair differentiation [26]. However,
in the case of IL-8 as a recombinant protein, resulting DC
stimulated allogenic MLRs as strongly as those DC derived in the
absence of the IL-8 chemokine (Figure 3B).
Alternatively, IL-8 could alter the maturation/activation of DC
[29]. To induce maturation, DC were incubated for 48 h with a
mixture of INF-a, TNF-a and Poly I:C in the presence or absence
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Figure 3. IL-8 does not impair T cell stimulation by DC that express functional CXCR1 and CXCR2. (A) Human monocyte-derived DC and
T-cells were seeded in the indicated proportions. Functional recombinant IL-8 was added at different concentrations as indicated in the graph
legends. T-cell proliferation was measured by 3H-thymidine incorporation 3 days later. A representative case out of three independently performed
experiments with cells from different combinations of donors is shown. (B) Experiments as in A, but in this case DC were incubated with the indicated
amounts of IL-8 added to monocytes during the 7-day differentiation culture in the presence of GM-CSF+IL-4. A representative experiment out of at
least three is shown. (C) Similar experiments as in B but in this case IL-8 at the indicated concentrations was added during the maturation 48 h culture
onto differentiated DC matured for 48 h with IFN-a, TNF-a and poly I:C. A representative experiment out of at least three performed is shown. As a
control every IL-8 batch was shown to readily attract human PMNs in chemotaxis assays (Figure S3). (D) Mature DC as those used for the MLRs were
tested by indirect immunofluorescence for the expression of CXCR1 and CXCR2. Incubation with 100 ng/mL of IL-8 during 2 h resulted in a loss of
fluorescence intensity upon immunostaining of the surface receptors indicating receptor internalization. The mean fluorescence intensity (MFI) of
each histogram is provided. FACS Experiments were repeated in two occasions with similar results.
doi:10.1371/journal.pone.0017922.g003

migration of DC in 16 h that was abolished by anti-IL8


neutralizing mAb. Interestingly, if DC had been pre-exposed for
24 h to recombinant IL-8, migration was also abolished indicating
that desensitized DC could not migrate towards the IL-8producing carcinoma cells. Data are quantified in figure 5B,
which also shows that monolayers of the SW48 (the cell line that
does not produce IL-8) also fail to attract DC. Likewise, pretreatment of the HT29 cells with low concentrations of
cyclophosphamide decreased the ability of HT29 to attract DC
because of reducing IL-8 secretion (Figure 5C).
Therefore while IL-8 seems to leave T cell stimulation by DC
unimpaired, chronic exposure to IL-8 may profoundly affect the
migration capabilities of DC towards IL-8 gradients and possibly
of other leukocyte subsets as well.
In a previous study we reported that DC produce IL-8 [1].
Figure S7A confirms that DC produce IL-8 at the protein and
mRNA level. It was conceivable the autocrine IL-8 may
downregulate CXCR1 and CXCR2 expression, as seen in
figure 3D with exogenously added IL-8. Indeed, we observed
brighter immunofluorescence specific for CXCR1 and CXCR2
when IL-8 was neutralized with a specific mAb (Figures S7B and
C) and when DC were cultured at very low cell densities upon
agitation to dilute the secreted IL-8 (Figures S7B, C and D).
Therefore autocrine IL-8 determines the level of receptor surface
expression in DC providing an interesting mode of regulation.

Indeed, table S1 shows that IL-8 at various concentrations does


not alter the level of expression the maturation markers CD80,
CD83, CD86 and MHC class II on mature and immature DC.
Moreover, IL-8 did not alter the production of IL-12 and IL-10
upon maturation as induced by lipopolysaccharide (LPS) plus the
R-848 imidazoquinoline or recombinant CD40L (Figure S5).
Furthermore, we explored the issue of whether immunodeficient
animals bearing IL-8-producing tumors would impair MLR alloreactions of human leukocytes seeded inside their peritoneal cavities. For
this purpose, we grafted HT29 tumors for 57 weeks and co-injected
PKH2-labeled PBL and allogeneic DC inside the peritoneal cavity. As
can be seen in figure 4, T cells readily proliferated in tumor-free mice
within 4 days and such proliferative responses were clearly downsized
by the presence of subcutaneous tumors.
If neutralizing anti-IL-8 mAb was co-injected with the PBL and
the allogenic DC, no recovery of proliferation was observed. This
is interpreted in the sense that factors other than IL-8 downregulate T-cell proliferation. This is in agreement with the lack of
IL-8 effects on DC-mediated T-cell stimulation in the in vitro
alloreactive co-cultures.
In addition, we performed experiments (shown in figure 4B) that
demonstrate that treatment of the HT29-xenografted mice with
cyclophosphamide did not improve the alloreactive T-cell
stimulation. Moreover SW48 xenografts, that do not produce
IL-8, also inhibit the intraperitoneal alloreactive response to an
extent comparable to that observed with HT29 (Figure 4B).
Regardless the fact that there is no IL-8 homologue gene in the
mouse genome, human IL-8 exerts at least some agonist activity
on mouse CXCR1 as described [6]. Indeed, we were able to
observe IL-8 chemotactic activity on mouse bone marrow-derived
DC (Figure S6A). Therefore we set up experiments in which we
activated CD4+ TCR-transgenic OT-2 T cells responding to
ovalbumin in the peritoneal cavity of Rag2/2 IL-2Rc2/2 mice.
DC were pulsed with the cognate peptide and the mice were
bearing or not established HT29 subcutaneous tumors. As shown
in figures S6B and C, HT29 tumors also suppressed proliferation
of the mouse T cells in this setting, although such an inhibition was
not affected again by IL-8 neutralizing antibodies. Our data
further indicate the existence of immunosuppressive factors in the
tumor bearing mice which are different from IL-8.

IL-8 produced by DC retains and attracts neutrophils


A function of IL-8 could be to favor a rendezvous between
polymorphonuclear (PMN) cells and DC by co-attracting both
subsets of leukocytes. In our hands, both immature and mature
DC produce abundant IL-8, although mature DC produce about
four-five-fold more quantity on a per cell basis (Figure S7).
Classical chemotaxis assays were set up to determine if IL8
could regulate DC and PMN migration in a concerted fashion. As
can be seen in figure 6A and figure S4, neutrophils are attracted by
recombinant IL-8. However, if neutrophils are seeded together
with DC (at 1:1 ratio), neutrophil migration as induced by IL-8
was abolished. These data might indicate that DC have a means to
attract and/or retain PMNs that otherwise would migrate away.
If the assays were set up with PMNs in the upper and DC in the
lower chamber, neutrophils were attracted by DC seeded into the
lower chamber. Importantly, addition of neutralizing anti-IL-8
mAb eliminated most of the attraction of fluorescence-labeled
neutrophils by the DC seeded in the lower chamber (Figure 6B)
while control antibody exerted no effect.
Our results as a whole indicate that although IL-8, abundantly
produced by tumors, would not damage DC-mediated stimulation
of T-cells. However, tumor-derived IL-8 would alter migration
and interactions with other leukocyte subtypes such as neutrophils.
For instance, if DC are prevented from migrating towards MIP3a
gradients by HT29 supernatants they would reach the inflamma-

Pre-exposure of DC to IL-8 disorient DC to subsequently


follow IL-8-guided migration
DC in tumor bearing subjects would be chronically exposed to
IL-8. As we have shown in figure 3D, exposure of DC to IL-8
determines receptor downregulation. Therefore we hypothesized
that pre-incubation of DC with IL-8 could inhibit subsequent
responses to IL-8 gradients.
In figure 5, chemotactic experiments were set up plating IL-8producing HT29 cells in the lower chamber and PKH2-labelled
DC in the upper chamber. The colon carcinoma cell line induced
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Figure 4. Impairment of DC-induced human T-cell proliferation inside the peritoneum of HT29 xenografted mice. (A) Rag2/2 IL-2cR2/2
mice (3 per group) were xenografted with HT29 cells or remained tumor-free. Four weeks later mice received intraperitoneal injections of human PKH2labeled PBLs and fully allogenic mature DC (ratio 5:1 to a total of 66106 cells). Proliferation was monitored four days later by dye dilution on FACS-gated
lymphocytes from peritoneal lavages by dilution of the fluorescent dye. In the group of indicated mice an intraperitoneal injection of 100 mg of neutralizing
anti-IL-8 mAb was provided immediately following the injection of PBLs and DC. Percentages of dividing lymphocytes in each log cursor interval are shown
in the histograms. Fluorescence intensity in the input undivided PBL was over 95% above the third log interval (upper histogram). (B) Similar experiments as
in A, quantifying PKH2-dilution as the percentage of cells that reach the 3rd and 4th log scales of the flow cytometry histograms. Log regions are depicted in
the upper histogram of A. Data from animals bearing subcutaneous SW48 xenografts, that do not produce IL-8, have been included. Data represent
mean6SD.
doi:10.1371/journal.pone.0017922.g004

tory focus in lesser numbers and as a result would stimulate T cells


less efficiently (Figure S8). IL-8-disoriented migration could
thereby contribute to weaken immune responses to cancer.

should be attracted to the tumor nodule. Indeed, fluorescencelabelled DC were recovered from the tumor tissue and such
migratory behaviour was inhibited if DC were co-injected with the
neutralizing anti-IL-8 mAb (Figure 7B). More importantly, preexposure of the DC cultures to IL-8 for 24 h prior to injection also
greatly impaired the migration towards the tumor. Therefore
tumors can attract DC by means of IL-8 but chronic exposure to
IL-8 desensitizes DC for this in vivo migration. Owing to these
effects, IL-8 produced at malignant lesions profoundly impairs the
migratory orientation of DC.

IL-8 produced by tumor xenografts attracts DC to the


tumor tissue unless they have been desensitized by preexposure to IL-8
As seen in figure 1, HT29 xenografts are sites of intense IL-8
production. Therefore we reasoned that DC injected in the
subcutaneous tissue 5 mm away from the tumor (Figure 7A)
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Figure 5. DC pre-exposed to IL-8 become desensitized to respond to carcinoma-derived IL-8 as a chemoattractant. (A) Chemotaxis
assays were set up with HT29 confluent monolayers in the lower chamber and fluorescent DC in the upper chamber. Phase contrast microscopy
images and the corresponding UV fluorescence microscopic fields of the lower chamber are shown. When indicated the lower chamber contained
neutralizing anti-IL-8 mAb (20 mg/mL) or the DC had been pre-exposed for 24 h to recombinant IL-8 (1 mg/mL). (B) In the upper panel representation
of data from three independent experiments similarly performed to those in A with HT29 cells represented as mean6SD in which four random fields
were counted for each triplicate well. In the lower panel experiments performed as in A, but in this case the confluent monolayers in the lower
chamber were formed by SW48 cells that do not produce IL-8. (C) Experiments as in A, but in this case HT29 cells had been pretreated with various
cyclophosphamide concentrations for 24 h as indicated in the figure. Results represent mean6SD.
doi:10.1371/journal.pone.0017922.g005

the serum concentration of the chemokine in circulating blood.


The rapid decrease and recovery of serum concentrations indicate
the rapid turnover in blood of a polypeptide below the renal
filtration threshold and with a short half-life [34]. We are
exploring the therapeutic implications of low-dose cyclophosphamide effects on IL-8 patho-phisiology. In fact, this effects on IL-8
output can be a factor behind the beneficial effects of the described
metronomic dosing of the drug [31,32]. In addition acute
reductions in IL-8 output as induced by cyclophosphamide might
be exploited to support intratumoral injections of DC in order to
favor migration to lymph nodes.
In a clinical trial IL-8 was suspected to mediate intratumoral
retention of DC artificially delivered in such locations by imageguided procedures of injection [1]. However, the role of IL-8 at in

Discussion
By producing IL-8, tumors may profoundly alter the migrationguiding gradients of this important chemokine in the tissues of
tumor-bearing hosts [33]. Indeed, the chemokine network is well
known to modify cancer biology in multiple ways from metastasis
and angiogenesis to the attraction of a nurturing leukocyte
infiltrates [33]. In this study, we demonstrate that IL-8 as
produced by human tumor cells is capable of attracting (or
retaining) human DC in vivo, but that IL-8 does not functionally
affect the ability of DC to stimulate alloreactive T cells.
We found that at least in vitro, IL-8 production by tumor cells
can be decreased by low dose cyclophosphamide at the protein
and mRNA level. In vivo this is reflected by transient decreases in
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Figure 6. DCs retain neutrophils in migration assays towards IL-8 and attract neutrophils in an IL-8-dependent fashion. (A) Transwell
chemotaxis assays were set up with PKH2-labeled neutrophils. Neutrophils migrated to recombinant IL-8 added to the lower chamber. However, if
neutrophils are seeded in the upper chamber together with DC (1:1) migration is totally impaired. Of note there is IL-8 by the DC as shown in figure
S6A. This experiment is representative of two independently performed in triplicate wells with migration lasting for 120 min. (B) Percentage of
migration of PKH2-labeled neutrophils that were seeded into the upper chamber of transwell migration assays towards recombinant IL-8 or DC
placed in the lower chamber. When indicated, IL-8-neutralizing mAb (20 mg/mL) was added to the lower chamber along with the DC. Migration was
analyzed at 120 minutes. Similar experiments analyzed as early as 60 minutes rendered similar results (data not shown). Results are representative of
two separate triplicate experiments independently performed. Asterisks indicate statistical significance p,0.01 in students t tests.
doi:10.1371/journal.pone.0017922.g006

by mouse factors or random migration. What we document is that


IL-8 mediates the retention inside the tumor microenvironment.
This is not a surprise since IL-8 receptors, CXCR1 and CXCR2,
are expressed on DC and are functional in classical chemotaxis
assays [1]. The production of IL-8 by DC themselves could be also
operating in an autocrine or paracrine fashion in this intratumoral
setting. Nonetheless, we clearly observe that HT29 tumor
xenografts are capable to chemoattract DC when injected in the
connective tissue that surrounds the malignancy.

vivo retention could not be experimentally documented. In this


study, we observe that DC are retained inside xenografted colon
carcinomas by IL-8. The evidence was generated by means of a
neutralizing anti-IL-8 mAb injected alongside the DC. These
findings further support our interpretations with regard to the
apparent retention of intratumorally injected DC in patients
according to scintigraphic scans [1,2].
In our mouse xenograft system, we do not yet understand
whether migration out of tumors is the result of chemotaxis driven

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Figure 7. HT29 xenografts attract human DC injected in the subcutaneous tissue which surrounds the tumor. (A) Mice bearing HT29
established xenografts as the one shown in the pictures were injected approximately 5 mm away from the tumor lesion with 56106 human
immature DC labelled with PKH2 that were resuspended in 50 ml of saline buffer to form a small subcutaneous bump which disappeared in less than
2 hours. (B) 24 hours later tumors were surgically removed and a cell suspension was obtained in which the number of fluorescent DC were
enumerated by flow cytometry and normalized as the percentage of injected DC that were recovered from the tumor. When indicated DC were pretreated for 24 hours in culture with 1 mg/ml of rIL-8 or DC were co-injected with 100 mg of neutralizing anti-IL8 mAb.
doi:10.1371/journal.pone.0017922.g007

The IL-8 receptors, when ligated, turn on various signaling


pathways [35] and rearrange the cytoskeleton [13]. IL-8 could
thereby potentially alter the functional performance of DC.
Therefore we hypothesized that DC under the influence of IL-8
in the tumor, would be poorer T cell stimulators. However, fully

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functional IL-8 (as checked in migration assays) was completely


incapable of decreasing T-cell allostimulation as mediated by DC.
This is in agreement with the fact that IL-8 does not affect the
expression of costimulatory receptors or T-cell stimulating
cytokines. It remains to be seen if IL-8 alters the antigen

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presenting machinery or other biological activities of DC that are


not required for alloreactive stimulation. Although not formally
ruled out, this possibility is considered unlikely.
Nonetheless, if DC were retained in the tumor milieu by IL-8,
those DC would remain under the concentrated influence of
tumor-derived factors that repress DC functions [26,36,37,38].
Evidence for this phenomenon also comes out from our
alloreactive reactions of human lymphocytes inside the peritoneum of immunodeficient mice bearing HT29 and SW48 xenografts
[39]. Some of the malignant tissue immunorepressor molecules
include TGF-b [40,41], VEGF [42,43], interleukin-13 [44],
prostaglandins, kynurerines [40] and most likely other unknown
polypeptide moieties [26], as well as certain lipids [45].
Collectively, our data can be interpreted in the sense that IL-8
retains DC in the precise location where such antigen presenting
cells are most efficiently damaged in their function by tumorderived biomolecules. Our results regarding the in vivo inhibition of
T-cell allostimulation in animals xenografted with human tumors
offer a useful experimental system to dissect tumor-dependent
mechanisms of inhibition that are operating distantly from the
tumor implant. This experimental tool is employed in our
laboratory [39].
DC intratumoral retention has been described as an evasion
strategy in breast cancers [46]. We can observe in in vitro
chemotaxis that if HT29 cells prevent DC from migrating to
MIP3a mimicking an inflammatory focus then fewer DC reach
the location to stimulate T cells and the immune response is
accordingly less intense.
These phenomena certainly pose an obstacle for the intratumoral route of DC administration in human immunotherapy [11],
an approach that has been described to be very successful in a
number mouse models [47,48,49,50]. From the therapeutic point
of view, low doses of cyclophosphamide can be useful to reduce IL8 output by viable tumor cells. Neutralization of IL-8 with mAb
could be also therapeutically feasible. This has been recently
demonstrated in a situation in which tumor xenografts escape
from sunitinib-induced anti-angiogenesis by means of an IL-8dependent mechanism [19].
IL-8 attracts neutrophils and possibly immature forms such as
myeloid derived suppressor cells [37,51]. Interestingly, DC secrete
IL-8 that may act in an autocrine fashion [1]. Apart from these
largely unexplored autocrine effects that modulate CXCR1 and
CXCR2 surface levels; we show that DC are capable of attracting
or retaining neutrophils in an IL-8-dependent fashion. The
physiological consequences of DC-neutrophil interactions [52] in
the tumor context are currently being actively explored in our
laboratory, with emphasis on the implications for cross-presentation of tumor antigens [53,54].
What seems also plausible is that under high circulating levels of
IL-8, as occurs in HT29-xenografted mice or patients with bulky
disease, migration-driving gradients of IL-8 would be disrupted
and thereby DC-production of IL-8 might be overwhelmed in its
ability to attract neutrophils and other leukocytes. Indeed, we have
obtained evidence in the sense that overwhelming pre-exposure to
IL-8 results in desensitization of the DC to the chemotactic effects
of IL-8. In other words, this could mean that DC chronically
exposed to IL-8 in the context of tumor-bearing hosts would
become disoriented and thus unable to follow migration cues set
up by IL-8 concentration gradients. In vivo evidence of desensitization for migration further supports this notion. Such immune
disorientation as caused by the abundantly and ectopically
expressed chemokine may result in disordered immune responses
and ought to have relevant prognostic consequences for patients.
PLoS ONE | www.plosone.org

Supporting Information
Figure S1 Colon carcinoma cell lines HT29 and CaCo2
produce high levels of IL-8 in a clonal stable fashion
while SW48 does not produce IL-8. Clonal limiting dilution
subcultures (four for each cell line) of the colon cancer-derived cell
lines HT29, CaCo2 and SW48 were tested for the production of
IL-8 as measured in 24 h culture supernatants by ELISA.
(TIF)
Figure S2 CaCo2 carcinoma cells xenografted in immunodeficient mice develop progressive intraperitoneal
tumors that correlate with raising serum concentrations
of IL-8. Intraperitoneally xenografted CaCo2 cells developed
progressive peritoneal colon carcinomas in athymic nude mice
(measured as weight increase in the left axis) and accumulated
increasing concentrations of serum IL-8 (right axis).
(TIF)
Figure S3 DC are retained inside CaCo2 tumors in a IL8-dependent fashion. CaCo2 cells were xenografted in athymic
nude mice. Only 1/3 of such animals successfully xenografted
tumor lesions. Tumor nodules, 812 mm in diameter, were
injected with CFSE-labeled human DC derived from monocytes,
as in A. DC were injected in 100 mL of saline buffer with control
antibody or neutralizing anti-IL-8 mAb. In these cases, tumors
were homogenated and cleared of debris by centrifugation.
Fluorescence in the lysate was measured in a fluorimeter. The
amount of fluorescence remaining in the tumor compared to that
present in the lysate from an identical number of DC before being
injected in the tumor was quantitated. Data are presented as the
percentage of fluorescence lost from the tumor. Experiments were
performed with four mice bearing a single tumor nodule. The inset
shows a correlation of fluorescence (arbitrary units) and number of
DC in lysates containing increasing amounts of CFSE-labeled DC.
(TIF)
Figure S4 Recombinant IL-8 rendering negative results
at modifying DC functionality is capable of attracting
PMNs. Migration transwell assays with purified neutrophils in the
upper chamber and different concentrations of the recombinant
IL-8 in the lower chamber to prove that IL-8 used in figure 3A, B,
C and D was fully functional.
(TIF)
Figure S5 IL-8 does not modify IL-12 nor IL-10 secretion
by DC matured in the presence of LPS+R848 or
trimerized CD40L. IL-12 and IL-10 were quantified in the
supernatant of DC cultures treated with the indicated concentrations of IL-8 during the 48 h maturation culture. Concentrations
(mean6SD) represent triplicate wells from a single experiment.
(TIF)
Figure S6 Activation of antigen specific murine CD4 T
cells by DC in mice bearing HT29 tumors is suppressed
by factors distinct from IL-8. (A) Mouse BM-derived DC
[50,55] were subjected to classical transwell chemotaxis assays
towards culture medium (control), 105 heat-inactivated E. coli
bacteria used as a positive control, or recombinant IL-8 as
indicated. Data show a modest but reproducible attraction of
mouse DC by human recombinant IL-8. (B) Schematic representation of experiments in which HT29-bearing Rag2/2 IL-2Rc2/2
mice were injected in the peritoneal cavity with 56106 CFSElabelled CD4 OT-2 cells [56] and 106 syngeneic DC pulsed with
the OVA323-339 synthetic peptide. (C) Assessment of OT-2 T-cell
proliferation by dilution of CFSE as in figure 4. Experiments were
performed in mice bearing or not HT29 tumors with or without
12

March 2011 | Volume 6 | Issue 3 | e17922

Malignant Cell-Derived IL-8 Effects on DC

cognate peptide stimulation by the DC (n = 3 mice per group).


When indicated 100 mg of anti-IL8 mAb were co-injected into the
peritoneal cavity.
(TIF)

seeded in the upper chamber with or without conditioned medium


of HT29 cells or SW48 cells as indicated. When indicated an IL-8
neutralising antibody was added. In the right panel DC recovered
from the lower chamber were used to stimulate allogenic PBL, and
T-cell proliferation was recorded three days later as c.p.m. in
3
H-Thy incorporation assays. Data represent three independent
replicates.
(TIF)

Figure S7 DC produce IL-8 and such autocrine IL-8


modulates in part the surface expression of CXCR1 and
CXCR2. (A) Left axis: IL-8 concentration in the supernatant of
mature (mDC) and immature DC (iDC); Right axis: mRNA
encoding IL-8 in the corresponding DC cultures assessed by semi
quantitative RT-PCR. (B and C) IL-8 concentration in the
supernatant (left axes) and CXCR1 (B) and CXCR2 (C) surface
expression as mean fluorescence intensity (MFI) analyzed by
FACS (right axes). In B and C a mAb neutralising IL-8 (20 mg/ml)
was added when indicated, or the DC were cultured under gentle
agitation at the cellular densities given. Results are presented as
mean6SD from triplicate experiments. IL-8 neutralisation or
lower IL8 concentrations in the supernatants correlate with higher
MFIs for CXCR1 and CXCR2 on the DC. (D) Shows
representative FACS histograms from B and C.
(TIF)

Table S1 Lack of IL-8 effect on surface expression of

dendritic cell maturation markers. Mean fluorescence


intensity of the indicated surface markers of DC upon FACS
analyses in human DC (mean6SD from three different experiments) using either immature (iDC) or LPS+R848 matured DC
(mDC), that were cultured in the absence or the presence of
increasing concentrations of IL-8 as indicated in the columns.
Experiments are representative of three similarly performed with
different donors.
(TIF)

Acknowledgments

IL-8 in conditioned supernatants from HT29


cells impedes DC from migrating to MIP3a gradients. As
a consequence fewer DC reaching the lower chamber results in
less T-cell allostimulatory activity at this location. In order to
model whether DC-disoriented migration would give rise to less T
cell stimulation, we set up in the left panel chemotaxis assays in
which DC migrated towards recombinant MIP3a (100 mg/ml).
Data are presented as mean6SD of the chemotactic index
normalized with culture medium without MIP3a (Neg). DC were
Figure S8

Elena Ciordia and Eneko Elizalde are acknowledged for excellent animal
facility management, as well as technical help by Arantza Azpilikueta,
Manuela Gonzalez-Aparicio and Ana Larraga.

Author Contributions
Conceived and designed the experiments: IM JLP-G AG CA NS AR.
Performed the experiments: CA NS AP LE SS EB JD EF. Analyzed the
data: IM SH-S JLP-G IM-F AR AG. Contributed reagents/materials/
analysis tools: IM EF AR. Wrote the paper: IM JLP-G CA NS.

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FIGURAS SUPLEMENTARIAS________________________
(Carcinoma-Derived Interleukin-8 Disorients Dendritic Cell Migration without Impairing TCell Stimulation. C. Alfaro; N. Surez et al. PloSONE, 2011; 6(3)).

131

Supplementary Figure 1

Supplementary Figure 1. Colon carcinoma cell lines HT29 and CaCo2 produce high levels
of IL-8 in a clonal stable fashion while SW48 does not produce IL-8.
Clonal limiting dilution subcultures (four for each cell line) of the colon cancer-derived cell
lines HT29, CaCo2 and SW48 were tested for the production of IL-8 as measured in 24 h
culture supernatants by ELISA.

132

Supplementary Figure 2

Supplementary Figure 2. CaCo2 carcinoma cells xenografted in immunodeficient mice


develop progressive intraperitoneal tumors that correlate with raising serum
concentrations of IL-8.
Intraperitoneally xenografted CaCo2 cells developed progressive peritoneal colon carcinomas in
athymic nude mice (measured as weight increase in the left axis) and accumulated increasing
concentrations of serum IL-8 (right axis).

133

Supplementary Figure 3

Supplementary Figure 3. DC are retained inside CaCo2 tumors in a IL-8-dependent


fashion.
CaCo2 cells were xenografted in athymic nude mice. Only 1/3 of such animals successfully
xenografted tumor lesions. Tumor nodules, 812 mm in diameter, were injected with CFSElabeled human DC derived from monocytes, as in A. DC were injected in 100 L of saline
buffer with control antibody or neutralizing anti-IL-8 mAb. In these cases, tumors were
homogenated and cleared of debris by centrifugation. Fluorescence in the lysate was measured
in a fluorimeter. The amount of fluorescence remaining in the tumor compared to that present in
the lysate from an identical number of DC before being injected in the tumor was quantitated.
Data are presented as the percentage of fluorescence lost from the tumor. Experiments were
performed with four mice bearing a single tumor nodule. The inset shows a correlation of
fluorescence (arbitrary units) and number of DC in lysates containing increasing amounts of
CFSE-labeled DC.

134

Supplementary Figure 4

Supplementary Figure 4. Recombinant IL-8 rendering negative results at modifying DC


functionality is capable of attracting PMNs.
Migration transwell assays with purified neutrophils in the upper chamber and different
concentrations of the recombinant IL-8 in the lower chamber to prove that IL-8 used in figure
3A, B, C and D was fully functional.

135

Supplementary Figure 5

Supplementary Figure 5. IL-8 does not modify IL-12 nor IL-10 secretion by DC matured
in the presence of LPS+R848 or trimerized CD40L.
IL-12 and IL-10 were quantified in the supernatant of DC cultures treated with the indicated
concentrations of IL-8 during the 48 h maturation culture. Concentrations (meanSD) represent
triplicate wells from a single experiment.

136

Supplementary Figure 6

137

Supplementary Figure 6. Activation of antigen specific murine CD4 T cells by DC in mice


bearing HT29 tumors is suppressed by factors distinct from IL-8.
(A) Mouse BM-derived DC [50], [55] were subjected to classical transwell chemotaxis assays
towards culture medium (control), 105 heat-inactivated E. coli bacteria used as a positive
control, or recombinant IL-8 as indicated. Data show a modest but reproducible attraction of
mouse DC by human recombinant IL-8.
(B) Schematic representation of experiments in which HT29-bearing Rag/ IL-2R/ mice
were injected in the peritoneal cavity with 5106 CFSE-labelled CD4 OT-2 cells [56] and 106
syngeneic DC pulsed with the OVA323-339 synthetic peptide.
(C) Assessment of OT-2 T-cell proliferation by dilution of CFSE as in figure 4. Experiments
were performed in mice bearing or not HT29 tumors with or without cognate peptide
stimulation by the DC (n=3 mice per group). When indicated 100 g of anti-IL8 mAb were coinjected into the peritoneal cavity.

138

Supplementary Figure 7

139

Supplementary Figure 7. DC produce IL-8 and such autocrine IL-8 modulates in part the
surface expression of CXCR1 and CXCR2.
(A) Left axis: IL-8 concentration in the supernatant of mature (mDC) and immature DC (iDC);
Right axis: mRNA encoding IL-8 in the corresponding DC cultures assessed by semi
quantitative RT-PCR.
(B and C) IL-8 concentration in the supernatant (left axes) and CXCR1 (B) and CXCR2 (C)
surface expression as mean fluorescence intensity (MFI) analyzed by FACS (right axes). In B
and C a mAb neutralising IL-8 (20 g/ml) was added when indicated, or the DC were cultured
under gentle agitation at the cellular densities given. Results are presented as meanSD from
triplicate experiments. IL-8 neutralisation or lower IL8 concentrations in the supernatants
correlate with higher MFIs for CXCR1 and CXCR2 on the DC.
(D) Shows representative FACS histograms from B and C.

140

Supplementary Figure 8

Supplementary Figure 8. IL-8 in conditioned supernatants from HT29 cells impedes DC


from migrating to MIP3 gradients.
As a consequence fewer DC reaching the lower chamber results in less T-cell allostimulatory
activity at this location. In order to model whether DC-disoriented migration would give rise to
less T cell stimulation, we set up in the left panel chemotaxis assays in which DC migrated
towards recombinant MIP3 (100 g/ml). Data are presented as meanSD of the chemotactic
index normalized with culture medium without MIP3 (Neg). DC were seeded in the upper
chamber with or without conditioned medium of HT29 cells or SW48 cells as indicated. When
indicated an IL-8 neutralising antibody was added. In the right panel DC recovered from the
lower chamber were used to stimulate allogenic PBL, and T-cell proliferation was recorded
three days later as c.p.m. in 3H-Thy incorporation assays. Data represent three independent
replicates.

141

Supplementary Table 1

Supplementary Table 1. Lack of IL-8 effect on surface expression of dendritic cell


maturation markers.
Mean fluorescence intensity of the indicated surface markers of DC upon FACS analyses in
human DC (meanSD from three different experiments) using either immature (iDC) or
LPS+R848 matured DC (mDC), that were cultured in the absence or the presence of increasing
concentrations of IL-8 as indicated in the columns. Experiments are representative of three
similarly performed with different donors.

142

Synergistic effects of CTLA-4 blockade with tremelimumab and


elimination of regulatory T lymphocytes in vitro and in vivo

N. Surez 1; C. Alfaro 2; J. Dubrot 2; A. Palazn 2; E. Bolaos 2; L. Erro 3; S. Hervas-Stubbs 2; I.


Martnez-Forero 2; A. Morales-Kastresana 2; S. Martn-Algarra 3; B. Sangro 4; F. Lecanda 2; JL..
Prez-Gracia 3; A. Gonzlez 1 ; and I. Melero 2,3.

Biochemistry Department, Clnica Universidad de Navarra, Pamplona, Spain; 2 Gene Therapy


and Hepatology Division, CIMA, Pamplona, Spain; 3 Medical Oncology Department, Clnica
Universidad de Navarra, Pamplona, Spain; 4 Hepatology Department, Clnica Universidad de
Navarra, Pamplona, Spain.

International Journal of Cancer, 2010 Septiembre; 129: 374-386

143

IJC
International Journal of Cancer

Synergistic effects of CTLA-4 blockade with tremelimumab and


elimination of regulatory T lymphocytes in vitro and in vivo
Natalia Suarez1*, Carlos Alfaro2*, Juan Dubrot2, Asis Palazon2, Elixabet Bolanos2, Lorena Erro3,
Sandra Hervas-Stubbs2, Ivan Martinez-Forero2, Aizea Morales-Kastresana2, Salvador Martin-Algarra3, Bruno Sangro4,
Fernando Lecanda2, Jose L. Perez-Gracia3, Alvaro Gonzalez1 and Ignacio Melero2,3
1

Biochemistry Department, Clnica Universidad de Navarra, Pamplona, Spain


Gene Therapy and Hepatology Division, CIMA, Pamplona, Spain
3
Medical Oncology Department, Clnica Universidad de Navarra, Pamplona, Spain
4
Hepatology Department, Clnica Universidad de Navarra, Pamplona, Spain
2

Tumor Immunology

Anti-CTLA-4 monoclonal antibodies (mAb) that block the interaction of CTLA-4 with CD80 and CD86 such as tremelimumab and
ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de-repress tumor-specific
cellular immunity. Addition of the fully human anti-CTLA-4 (tremelimumab) to cultures of human T cells with allogenic
dendritic cells (DCs) did not increase proliferation. Magnetic bead-mediated elimination of CD41 CD251 regulatory T cells
(Treg) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast,
predepletion of CD41 CD251 Treg and culture in the presence of tremelimumab synergistically resulted in increased
proliferation and DC:T-cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy
mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of Treg elimination, thereby offering more
targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer
patients) were coinjected in the peritoneum of Rag22/2 IL-2Rc2/2 mice. In these conditions, tremelimumab injected
intravenously did not significantly enhance alloreactive proliferation unless Treg cells had been predepleted. Synergistic effects
in vivo were again largely restricted to the CD4 T-cell compartment. In addition, Treg depletion and CTLA-4 blockade
synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together,
these experiments indicate that tremelimumab therapy may benefit from previous or concomitant Treg depletion.

T cell depends much on CD28 stimulation by CD80 and


CD86 at immune synapses formed between T cells and
dendritic cells (DC).1,2 This costimulatory pathway acts in
a complementary fashion to the signals that stream from
the TCR upon antigen recognition.2 This costimulatory activity is regulated by a CD28 homolog that receives the
name of CTLA-4 (CD152).1,2 This molecule has several

hundred fold more avidity than CD28 for the CD80 and
CD86 countereceptors.1 CTLA-4 has been demonstrated to
be an inhibitory (or co-inhibitory) molecule for T cells in
culture.3 It is expressed on the surface of regulatory T cells
(Treg), and becomes intracellularly expressed in activated
effector T cells. Indeed, its absence from the genome in
mice determines a severe autoimmunity phenotype that

Key words: CTLA-4, T cell, regulatory T cells, dendritic cell, tremelimumab


Additional Supporting Information may be found in the online version of this article
Grant sponsors: MEC/MICINN; Grant numbers: SAF2005-03131, SAF2008-03294; Grant sponsor: Redes tematicas de investigacion
cooperativa RETIC; Grant number: RD06/0020/0065; Grant sponsor: Fondo de investigacion sanitaria; Grant number: FIS PI060932; Grant
sponsors: Departamento de Educacion del Gobierno de Navarra, Departamento de Salud del Gobierno de Navarra (Beca Ortiz de
Landazuri), European Commission VII Framework Program (ENCITE), Immunonet SUDOE, Fundacion Mutua Madrilena, UTE for Project
FIMA and Fundacion Cientca de la Asociacion Espanola Conta el Cancer (AECC)
Conicts of interest: I.M. and S.H.-S. receive support from DIGNA-BIOTECH. Tremelimumab and a research grant for related studies were
provided by Pzer inc. I.M. has received consulting honoraria from Pzer and Bristol Myers-Squibb
*N.S. and C.A. contributed equally to this work
DOI: 10.1002/ijc.25681
History: Received 6 Jul 2010; Accepted 3 Sep 2010; Online 17 Sep 2010
Correspondence to: Carlos Alfaro, Gene Therapy and Hepatology Division (CIMA), Clnica Universidad de Navarra and Medical School,
University of Navarra, Avda. Pio XII, 55, 31008, Pamplona, Spain, E-mail: calfale@unav.es; or Ignacio Melero, Gene Therapy and Hepatology
Division (CIMA), Clnica Universidad de Navarra and Medical School, University of Navarra, Avda. Pio XII, 55, 31008, Pamplona, Spain,
E-mail: imelero@unav.es

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mental evidence for this synergy has been reported in mice


with a demonstration of improvements in immune response
and therapeutic antitumor effects.31,32
This study demonstrates that T-cell alloreactivity in culture
is enhanced by anti- CTLA-4 mAb only when Treg cells are
depleted from the starting culture population. Indeed, Treg predepletion before the alloreactive culture results in enhanced
expression of CTLA-4 on the effector T cells thus offering more
target molecules for the blocking anti-CTLA-4 mAb. Combined
transfer of T cells and DC from unrelated donors to immunodecient mice resulted in alloreactive proliferation that was signicatively enhanced by intravenous anti-CTLA-4 mAb treatment again only when Treg had been removed.

Material and Methods


DC differentiation and maturation

Dendritic cells were generated from lter-buffy coats (FBC).33


The protocol and informed consent for obtaining the FBC were
approved by both the Ethics Committee of our Institution and
the local Government of Navarra. Isolated mononuclear cells
were subjected to positive selection using anti-CD14-conjugated
paramagnetic beads and puried using the AutoMACS system
(Miltenyi Biotec, Bergisch Gladbach, Germany). Puried monocytes were cultured for 7 days in RPMI medium supplemented
with granulocyte-macrophage colony stimulating factor (GMCSF) (1000 U/mL; Leukine, Berlex Richmond, CS) and interleukin-4 (IL-4) (500 U/mL; R&D Systems, Minneapolis, MN). Dendritic cells were matured with tumour necrosis factor-a (TNF-a;
50 ng/mL; Boehringer Ingelheim, Ingelheim, Germany), IFN-a
(1000 IU/mL; Schering-Plough, Kenilworth, NJ) and poly I:C
(20 lg/mL; Ampligen, Bioclones, Tokai, South Africa) for 48
hr.33 When indicated, T cells and dendritic cells were derived
from peripheral blood mononuclear cells from leukoapheresis
samples of advanced cancer patients (two cases of melanoma
and three cases of colorectal cancer) enrolled in a dendritic cell
immunotherapy clinical trial (NCT00610389) under informed
consent for the use of these cell samples.
Isolation of T cells

Peripheral Blood mononuclear cells (PBMC) from FBC were isolated by Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ) gradient centrifugation. To obtain CD8 T cells, PBMC were subjected
to positive selection using anti-CD8-conjugated paramagnetic
beads (Miltenyi Biotec). To obtain CD4 T cells and CD4
CD25 T cells, PBMC were isolated using a CD4 CD25 Regulatory T Cell Isolation Kit, according to the manufacturers manual (Miltenyi Biotec). T cells were routinely analyzed by ow
cytometry and the purity of selections were >98%.
Cell culture and reagents

DC and T cells were maintained in complete RPMI medium


composed of RPMI 1640 with Glutamax-I (Gibco, Invitrogen,
Carlsbad, CA) containing 10% heat-inactivated FBS (SigmaAldrich, Dorset, UK), 100 IU/mL penicillin and 100 mg/mL
streptomycin (Biowhittaker, Walkersville, MD).

Tumor Immunology

results in the death of the animals within 4-8 weeks


of age.46
The cellular immune response is kept under control by a
specialized population of CD4 lymphocytes termed Treg
cells7,8 that are characterized by the expression of the FoxP3
transcription factor and express CD25 at steady state.7 These
cells suppress the functions of effector CD4 and CD8 T cells
as well as those of DC.8
Interestingly, these Treg lymphocytes express CTLA-4 on
the plasma membrane in mice6 and humans under baseline
conditions.9 This contrasts with the fact that CTLA-4 is not
expressed in the rest of T cells, unless they undergo antigendriven activation.1,2 Even in activated effector T cells, CTLA4 is mainly retained in the cell interior and only emerges to
the surface at the precise area of an immune synapse.1012
The suppressive mechanism of action of CTLA-4 could be
multifarious. It can operate on the activated effector cell outcompeting CD28 ligation13,14 or signaling through phosphatases recruited by its cytoplasmic tail15,16 that in turn crosstalk to the T-cell receptor.17
In addition, CTLA-4 ligation can operate regulating the
functions of regulatory T cells.6,18 Elegant mouse data from
the groups of Sakaguchi and Chambers. demonstrated that
lack of CTLA-4 expression on Treg cells seriously impaired
these cells from restraining effector T-cell-mediated lethal
autoimmunity.6,19
Because of the role of CTLA-4 as a cellular immunity brake
system, the group of Allison pioneered work in which in vivo
blockade of CTLA-4 with mAbs resulted in rejection of experimentally transplanted tumors in mice.20,21 This effect was
clearly enhanced when combined with tumor vaccines.22,23
As a result of this work, two human immunostimulatory
mAbs blocking CTLA-4 (tremelimumab and ipilimumab) are
under phase II/III clinical testing. In melanoma, an encouraging rate of 520% of long-lasting clinical responses has been
reported.2428 An interesting recently published clinical trial
shows survival benet in patients with stage IV melanoma.29
However, very little immune correlates of activity have been
described with the exception of ICOS and HLA-DR expression on a fraction of CD4 T cells,9,30 and the ratio of regulatory/effector cells in post-treatment tumor biopsies.23 More
importantly, no biomarker parameter seems to accurately
predict which patients will respond to treatment.
The involvement of Treg cells in the therapeutic effects of
anti-CTLA-4 mAbs as observed in humans is unlikely. The
group of Rosenberg has reported that treatment of cancer
patients with the anti-CTLA-4 mAb ipilimumab did not
change either the number or the function of Treg cells despite
CTLA-4 expression in this lymphocyte subset.9 Our preliminary results in a clinical trial for hepatocellular carcinoma
with tremelimumab (NCT01008358) agree with these ndings. If anti-CTLA-4 mAb and Treg depletion overlap incompletely or not at all in their mechanism of action, additive or
even synergist effects may be feasible, i.e.: depleting or inactivating Treg followed anti-CTLA-4 mAb treatment. Experi-

375

376

Dendritic cells were cultured in 96-well plates in RPMI 1640


complete medium. A total of 2  105 lymphocytes from a distinct donor were added on day 9, at different T cell/DC ratios.
Proliferation was assessed by [methyl-3H] thymidine nuclear
uptake. IFN-c production at 72 hr with an enzyme-linked
immunoabsorbent assay (ELISA) kit (R&D Systems).
When indicated, mAb anti-CTLA4 (tremelimumab)24,34 was
added to culture medium at different concentrations. Human
GMP-manufactured IgG (Beriglobina P, ZLB Behring, Barcelona,
Spain) was used as control at the same quantities or concentrations of anti-CTLA4 mAb tremelimumab in each experiment.

Anti-CTLA-4 mAb and Treg Depletion

specic lysis at the indicated effector to target cell ratios was analyzed using lymphocytes from day 7 of cocultures as effector
cells. Cytotoxicity performed in triplicate U-bottomed wells and
calculations were performed as published previously.36
Statistical analysis

All data are presented as the mean 6 standard error of the


mean (SEM). For comparisons, unpaired Students t-tests or
MannWhitney U tests were used [Prism software (Graph
Pad Software)].

Tumor Immunology

Results
In vitro mixed lymphocyte reactions
Human peripheral blood lymphocyte (PBL) or PBL without
Treg were stained with 2.5 lM of CFSE (Invitrogen). To have
a reference of CFSE intensity, a sample of PBL was xed
with paraformaldehyde 10% the rst day of experiment.
A total of 106 DC and 107 PBL or PBL without Treg (1:10
ratio) were injected into the peritoneum of Rag2/ IL-2Rc/
mice (on the basis of protocol approval by the animal experimentation committee of the University of Navarra). AntiCTLA-4 mAb (tremelimumab, 10 mg/kg) were administrated
intravenously to the indicated the mice. As a control antibody, other mice were treated with human IgG (Beriglobina
P). After 3 days, intraperitoneal lavages were obtained, cells
were stained with anti-human CD4 and anti-human CD8
and xed with paraformaldehyde 10% to be analyzed by ow
cytometry using a FACSCalibur Flow Cytometer (Benton
Dickinson). The number of T-cell divisions is proportional to
dilution of CFSE intensity.
FACS and enumeration of DC:T-cell aggregates

Immunouorescence and FACS was performed as described33


and BD perm/Fix Buffer was used prior to intracellular
immunostainings with anti-FoxP-3 and anti-CTLA-4 PEtagged mAbs (Becton Dickinson).
T cells (either total PBL or magnetically puried CD4 and
CD8 cells) were co-cultured with allogenic DC as in MLR
(1:5 ratio DC:T cell) in 24-well plates. Before culture, DC
were labelled with PKH26 red (Sigma-Aldrich, Saint Louis,
MO) and T cells with PKH2 green (Sigma-Aldrich) at 2 
106 M. Washed cells were co-cultured for the indicated
time and resuspended gently by pipetting up and down. Cells
were analyzed by ow cytometry analyzing the fraction of T
lymphocytes (green) attached to DC (red).
In vitro stimulation with EBV-transformed
cells and cytotoxicity
EBV cell lines from two healthy donors were established 5 years
ago following standard procedures.35 These donors were still
available for blood donation. PBL were seeded in co-culture
with irradiated (500 cGy) autologous cell lines (5:1). On day 7,
lymphocytes were counted with trypan-blue exclusion and cytotoxicity monitored against the autologous EBV cell lines and the
allogenic colon carcinoma cell line HT29 (ATCC). Percentage of

Anti-CTLA-4 blocking mAb (tremelimumab) enhances


proliferation in alloreactive PBL:DC co-cultures
only when Treg are removed

As shown in Figure 1a, with four representative cases, combination of alloreactive co-cultures of PBL and mature DC
from different donors undergo an intense degree of DNA
replication in the lymphocyte compartment. In none of the
cases (all of 8), did addition of 15 lg/mL of the human antiCTLA-4 mAb (tremelimumab) increase proliferation over
baseline at the different DC to PBL ratios. When magnetic
beads were used to eliminate CD25 cells prior to culture, a
slight increase was observed in some, but not all cases, in spite
of complete elimination of FoxP3 T cells, as routinely
checked by ow cytometry (Supporting Information Figure 1).
In contrast, pre-elimination of CD4 CD25 T cells and
addition of 15 lg/mL of tremelimumab to the cultures,
resulted in clear increases in proliferation in all of 8 experiments using leukocyte combinations from various healthy
donors (Fig. 1a). This increase was not seen when polyclonal
human IgG was used as control antibody (data not shown).
Such an increase was also seen but less clearly when immature DCs were used (Supporting Information Figure 2 shows
a representative experiment).
A dose response of tremelimumab in these MLR cultures was
performed in several experiments as the one shown Figure 1b.
Concentrations of tremelimumab as low as 15 lg/mL were already able to mediate the maximal effect on alloreactive proliferation enhancement. Again such enhancement only takes place
when Treg have been predepleted. Similar serial concentrations
of clinical grade polyclonal human IgG (Beriglobina P) used as a
control did not modify the proliferation (data not shown).
Next, we examined the concentration of IFN-c in the
supernatants of these alloreactive mixed leukocyte cultures
(MLR). In this case, tremelimumab did not increase the production of IFN-c in the 3-day co-cultures, while Treg elimination induced a slight increase as shown in Figure 1c.
CTLA-4 is expressed on Treg and in a fraction of activated
T cells in the alloreactive cultures and Treg predepletion
enhances the levels of CTLA-4 expressed by
effector T cells

The main target of tremelimumab in the alloreactive co-cultures was questioned by experiments in Figure 1. It was clear
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Tumor Immunology

Suarez et al.

Figure 1. Treg depletion and anti-CTLA-4 mAb enhance proliferation of T cells in response to mature allogenic DC. (a) PBLs from healthy
donors were mixed at the indicated ratios with fully allogenic mature DC and proliferation of the cultures was assessed by [methyl-3H]
thymidine nuclear uptake. When indicated, Treg (CD25 cells) were magnetically depleted and/or anti-CTLA-4 mAb (15 lg/mL) was added to
the cultures. Data from four representative co-cultures out of eight are presented in different panels indicating the donors of T cells and
DC. (b) Experiment as those in (a) checking the effects of a range of different concentrations of tremelimumab or control immunoglobulin
up to 250 lg/mL. A representative dose-response experiment out of four similarly performed is presented. (c) IFN-c concentration in tissue
culture supernatants of a representative MLR set up as in (a).

Tumor Immunology

378

that CTLA-4 was expressed on Treg cells (FoxP3) but not in


FoxP3 CD4 and in CD8 T cells prior to the alloreactive
stimulation (Fig. 2a). Inmunostaining for CTLA-4 prior to
permeabilization (necessary to analyze the expression of FoxP3
transcription factor), indicated that CTLA-4 is expressed on
the plasma membrane of Treg (Fig. 2a), but not on the surface
of effector T cells in agreement with multiple previous
reports.2,12,26 After 5 days in the alloreactive co-cultures, some
CD4 and CD8 effector cells acquired some intracellular expression of CTLA-4 but very dimly on the cell surface (Fig. 2b).
This result is of interest because in spite of the more
intense expression of CTLA-4 on Treg cells, the effects of
CTLA-4 blockade on the intensity of the alloreactive proliferation was observed in the case of cultures that had been predepleted from Treg.
Importantly, it was observed as a result of Treg predepletion that both CD4 and CD8 effector T cells expressed intracellular CTLA-4 much more intensely than the same cells
in cultures set up without Treg depletion, upon analysis on
gated FoxP3-negative lymphocytes (Fig. 2c). Nonetheless, surface expression of CTLA-4 could not be detected on the
effector T cells (data not shown). However, it must be taken
into account that CTLA-4 is thought to operate by selective
emergence at the signaling immunological synapses. Thereby,
minor changes in surface expression levels have major effects
on the outcome of T-cell activation.12
Together, these observations help to understand the synergistic effects, since Treg depletion results in the expression of
higher numbers of inhibitory CTLA-4 molecules, whose function needs to be inhibited by tremelimumab.
Tremelimumab increases the proliferation of alloreactive
CD4 T cells but not CD8 T cells

Setting up the primary alloreactive T cell:DC cultures with


magnetically sorted CD4 and CD8 T cells showed interesting
differences with regard to the effect of tremelimumab and
Treg depletion. In the case of CD4 cells, proliferation
increased only when Treg depletion was combined with the
addition of 15 lg/mL of tremelimumab to the co-culture
(Supporting Information Figure 3A). In contrast, CD8 T cells
that readily proliferated in the presence of allogenic mature
DC did not further respond to tremelimumab (Supporting
Information Figure 3B). This was true even when CD8 T
cells were puried from PBL that had been predepleted from
CD4 CD25 T cells to follow the same processing as in the
case of CD4 cells (this condition is named [CD8-Treg] in the
Supporting Information Figure).
Treg depletion and tremelimumab enhance T-cell
aggregation with allogenic DC

Productive T-cell activation takes place in molecularly structured interactions across the plasma membranes of T lymphocytes and DC (immunological synapses). Duration and
strength of the synapses have been reported to involve
CTLA-4 function11 and Treg.37

Anti-CTLA-4 mAb and Treg Depletion

We observe in the T cell:DC alloreactive co-cultures an


early formation of T cell:DC aggregates. Interestingly, Treg
predepletion and CTLA-4 blockade with tremelimumab
enhanced the percentage of T cells forming aggregates with
DC as early as in 24 and 48 hr after culture onset (Fig. 3a).
The effect was readily observed with puried CD4 cells
when this subset was deprived of Treg (Fig. 3b), whereas the
effect could not be detected in the case of puried CD8 T
cells (Fig. 3c). We conrmed that aggregates showed more
intense forward scattering of the laser light in FACS analysis
further indicating that they were larger events in comparison
with single cells in the same suspension (data not shown).
In Supporting Information Figure 4A, a series of representative FACS dot plots show the fractions of the two-color
aggregates of distinctly uorescence labeled DC and T cells
under the different experimental conditions. These data are
reminiscent of the proliferation outcomes of these cultures.
Differences in proliferation are also observed by PKH2 dilution upon cell division of these cultures (Supporting Information Figure 4B).
Alloreactive proliferation of CD4 T cells is increased by
tremelimumab treatment in Rag22/2 IL-2Rc2/2 mice
transferred with human PBL and allogenic mature
DC when Treg are depleted from PBL

To conrm if the in vitro ndings were sustained in vivo settings, immunodecient mice were adoptively transferred intraperitoneally with PBL and allogenic mature DC (at 10:1
ratio) (Fig. 4a). PBLs were labeled with CFSE so they could
be followed and the dilution of CFSE was used as a correlate
of the experienced proliferation. In some cases, CD4 CD25
Treg cells were magnetically removed from the PBL. When
indicated 10 mg/kg of tremelimumab were injected intravenously immediately following adoptive transfer of the human
cells. Human leukocytes were recovered 3 days later by peritoneal lavages and proliferation was assessed by ow cytometry
(Fig. 4a). Prior to transfer, PBL did not show any proliferation
(Fig. 4b). However, even if injected without DC a number of
T cells undergo a cycle of proliferation (Fig. 4b). When allogenic mature DC are cotransfered into the peritoneal cavity
along with the PBL, lymphocytes readily proliferated. In these
experiments, 10 mg/kg of human IgGs were injected intravenously as a control with no evidence of any enhancing effect.
Intravenous administration of anti-CTLA-4 mAb increased
proliferation with a slight effect that was observed both on
CD4 and CD8-gated lymphocytes (Fig. 4c). Treg predepletion before injection of the PBL also increased proliferation to
some extent in the CD4 and CD8 compartments (Fig. 4c).
It is important to note that FoxP3 events were gated out in
the analysis of the CD4 lymphocyte population.
Strikingly, simultaneous predepletion of Treg and intravenous administration of tremelimumab resulted in a very
intense proliferation during the 72-hr period, which resulted
in marked dilution of CFSE. This marked increase in proliferation was seen only in the gated CD4 T cells, whereas
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Tumor Immunology

Suarez et al.

Figure 2. CTLA-4 is expressed on the surface of FoxP3 cells and in activated effector alloreactive T cells mainly intracellularly. Treg
predepletion leads to higher levels of CTLA-4 expression on CD4 and CD8 effector T lymphocytes. (a) Multicolor FACS staining of
permeabilized cells. FoxP3, CD4 and CD8 co-stainings were used for gating the indicated subpopulations. Experiments were performed on
resting PBL and on MLR cultures on day 5. (b) Similar stainings in cells that were not permeabilized prior to anti-CTLA-4 staining so only
surface expression of this molecule is detected at the same time points analyzed in (a). (c) Intracellular staining of CTLA-4 in FoxP3negative lymphocytes on day 3 of MLRs set up with T cells predepleted or not from Treg cells.

380

Anti-CTLA-4 mAb and Treg Depletion

A series of two independent experiments with four mice


per group each, as those shown in Figure 4, was carried out
and summarized as pooled data in Figure 5a. As it can be
seen, a marked difference in the level of proliferation induced
by combined Treg depletion and tremelimumab iv treatment
was found consistently 72 hr postintraperitoneal cell transferences. Differences in proliferation were also substantiated in
longer periods of in vivo alloreactive activation up to 56
days (data not shown).
Furthermore, independent experiments were set up with T
cells and DC from leukoapheresis drawn from metastatic
cancer patients (two melanoma cases and three colorectal
carcinoma cases). In these experiments shown in Figure 5b a
similar behavior was observed, albeit T cells from patients
tended to proliferate less in every condition if compared to
cells from healthy volunteers. In addition, parallel experiments showed that Treg predepletion resulted in higher levels
of CTLA-4 expression on effector CD4 and CD8 T lymphocytes, thereby offering more molecular targets to the CTLA-4
antibody (Supporting Information Figure 5).

Tumor Immunology

Treg depletion and CTLA-4 blockade synergize to enhance


the immune response against autologous
EBV transformed cells

Figure 3. Treg elimination and tremelimumab enhance early T


cell:DC aggregate formation in culture. (a) Prestained PBL and
allogenic DC with uorescent dyes were co-cultured for 24, 48 and
96 hr. When indicated, 15 lg/mL of tremelimumab or CD25mediated Treg predepletion was applied. Data represent triplicate
percentages of lymphocytes forming aggregates with DC as
detected by FACS double staining. Similar experiments were
performed with (b) puried CD4 cells (with or without Treg) and

To address if combinations encompassing Treg elimination


and CTLA-4 blockade with tremelimumab could result in
more effective antitumor cytotoxicity in an autologous setting, we used EBV-transformed cell lines. EBV-transformed
cell lines were available from two healthy donors. Irradiated
EBV-transformed cell lines were co-cultured for 7 days with
autologous peripheral blood lymphocytes predepleted or not
from Treg cells and in the presence or absence of tremelimumab (Fig. 6a). Cytotoxicity in the resulting cultures against
the autologous EBV-B cell lines and HT29 colon carcinoma
cells (as a specicity negative control) were monitored in 5hr chromium release assays. As shown in Figure 6b, clear
synergistic increases in cytotoxicity were detected in the two
donors tested in three independent experiments each,
although with a more prominent effect in donor 1. Percentage of specic lysis of HT29 cells was below 10% in every
case and E:T ratio (data not shown). Figure 6c shows the recovery of viable lymphocytes which was also monitored
under each culture condition and was found to be increased
in the case Treg depletion CTLA-4 blockade. These results
as a whole indicate that Treg predepletion and CTLA-4 blockade act in synergy to strongly enhance cytotoxicity against
autologous transformed cells.

(c) CD8 T cells. Experiments in the gure are representative of


three similarly performed (**p < 0.01 and *p < 0.05 according to
Students t-tests) and representative dot plots are presented as
Supporting Information Figure 3.

CD8 T cells proliferated as much as those cases in which


only Treg had been predepleted without tremelimumab treatment (Fig. 4c).

Discussion
Our study highlights that the immunotherapeutic effects of
Treg depletion/inactivation in humans may not overlap with
the effects of CTLA-4 blockade with antibodies, thereby
opening up the opportunity for combination treatments. This
is in spite of bright CTLA-4 surface expression on human
and mouse Treg cells that would offer a site of action for
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Figure 4. Depletion of Treg and administration of tremelimumab synergize to enhance CD4 T-cell alloreactive proliferation in vivo. (a) Rag2/ IL2Rc/ mice were injected intraperitoneally with 107 PBL and 106 mature DC from unrelated donors. PBL were stained with CFSE and cells were
harvested from peritoneal lavages 48 hr later as schematically indicated in (a). In some conditions, Treg were depleted from PBL. When indicated,
anti-CTLA-4 mAb or control antibody was intravenously injected into the mice. (b) Control of CFSE labelling intensity depicting spontaneous
proliferation in 72 hr when PBL were injected without DC and when coinjected with DC and/or human IgG intravenously as a control. (c) CFSE
intensity of expression in total CFSE cells, CD4 FoxP3 gated cells and CD8 gated cells from peritoneal lavages drained from mice
intraperitoneally injected with PBL and mature allogenic DC. When indicated, intravenous treatment with anti-CTLA-4 mAb or control was provided.
When indicated, Treg cells were magnetically predepleted from the input PBL population. Data represent one experiment with two animals per group.

Anti-CTLA-4 mAb and Treg Depletion

Tumor Immunology

382

Figure 5. Synergy of Treg depletion and treatment with anti-CTLA-4 mAb for the in vivo enhancement of alloreactive proliferation of T cells
from healthy donors and cancer patients. (a) Two experiments as those shown in Figure 5 were performed with four mice per group and
results from individual mice are pooled in this gure. CFSE uorescence was monitored by FACS in CD11c-negative cells eluted from
peritoneal lavages as in Figure 5. Data are presented as the relative number of events in the indicated arbitrary uorescence intensity FL-1
regions (regions 4 and a sum of regions 4 and 3). Such regions as in Figure 5 (depicted in the upper diagram) were set in such a way that
in the case of undivided PBL (those injected without allogenic DC) >95% of events were in region 1. Data on total and gated CD4 and CD8
PBL are individually provided along with the mean represented by a line. (b) Similar experiments as in (a) but with combinations of PBL
and mature DC obtained from advanced metastatic cancer patients (two melanoma cases and three colorectal carcinoma cases). *Indicates
p < 0.01 according to MannWhitney U tests.

CTLA-4 blocking drugs. Paradoxically, elegant genetic evidence in mice pinpoint a critical role for CTLA-4 expression
for the function of Treg cells,6,18 since Treg lacking CTLA-4
molecules fail to control effector T cells and Treg-selective
deciency of CTLA-4 results in lethal autoimmunity.19 However, Allison et al. have elegantly reported using Rag/
reconstituted mice, whose mouse Treg cells express human
CTLA-4 while effector T cells express the murine version,18
that most of the effect of anti-CTLA-4 mAb to enhance antitumor immunity in mice is exerted on effector T cells with a
modest therapeutic effect when only Treg CTLA-4 is blocked
with mAb. Therefore we do not contradict the compelling
genetic evidence that CTLA-4 is a key functional moiety for
Treg. However, our results clearly indicate that blockade of
CTLA-4 with mAb tampers insufciently with Treg functions
while satisfactorily relieving effector T cells from CTLA-4mediated restrain.
We made our observations employing a relatively simple
method to stimulate human T lymphocytes as a result of
allogenic recognition of foreign HLAs on mature DC. This
system allows us to look at the effect of CTLA-4 blockade at
productive T-cell activation events leading to clonal expansion. Although articial, these experiments may represent
events pertaining to activation by other types of antigens
such as tumor or viral antigens presented by DC. The
advantage of the system is that a high fraction CD4 and
CD8 T cells are antigen reactive because of the bias in the
TCR repertoire to recognize foreign HLA molecules. Our
data using these human alloreactive lymphocytes are in
agreement both with those by Rosenberg et al. on the treatment of cancer patients with ipilimumab in whom no
numeric or functional changes of Treg were observed9 and
with our own preliminary results in a clinical trial with tremelimumab. Furthermore, our data are in agreement with the
mentioned weak functional effects exerted by CTLA-4 mAb
blocking antibodies on mouse Treg cells.18
The molecular/cellular mechanism behind the synergy of
Treg depletion and CTLA-4 blockade is interpreted in the
sense that Treg depletion before stimulation of effector T cells
results in enhanced levels of CTLA-4 expression on the effector-to-be lymphocytes. Enhanced CTLA-4 expression on
effector T cells, as we observe in vitro and in vivo, would
more efciently counteract activation, unless blocked with the
anti-CTLA-4 neutralizing mAb. This series of effects provides
a simple and plausible explanation to the synergy between
Treg depletion and CTLA-4 blockade. Indeed, quantitative
changes in the CTLA-4/CD28 competition for their counterreceptors have been described to be functionally important.38
The combined effects of Treg depletion and CTLA-4
blockade with mAb seems to be largely restricted to CD4 T
cells as it is not observed in CD8 counterparts either in
vitro or in vivo. The unexplained molecular reasons behind
these differences between lymphocyte subsets are being currently addressed and do not seem to involve differences in
the levels of CTLA-4 expression on effector cells following
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383

Treg predepletion. Our results are in line with observations in


mice from the group Allison who has also reported that
CTLA-4 blockade dramatically enhance therapeutic effects
upon adoptive transfer of minute amounts anti-tumor CD4 T
cells.39 Intriguingly, we observe that Treg elimination and
CTLA-4 blockade synergize for in vitro reactivation of a CTL
response against EBV-transformed cell lines. This could
reect the fact that in the case of EBV, we are re-stimulating
a response from EBV-seropositive individuals.
Experimentation in mice offers the opportunity to work
with autologous/syngeneic tumors. In humans these experiments are very cumbersome, if possible at all. Nonetheless, we
have managed to perform experiments in an autologous setting
with transformed B cells to show that Treg depletion and
CTLA-4 blockade synergize in culture to induce specic cytotoxicity. Most of such response is likely to go against viral
rather than true tumor antigens, but still is clinically relevant
as extensively demonstrated by the group of Brenner.40,41
It was informative to observe more frequent T cell:DC
aggregates when Treg were removed and tremelimumab
added to the cultures. Longer and stronger T cell:DC interactions are a likely cause for enhanced T-cell responses to antigens.
Ongoing studies will deal with molecular changes at the immunological synapses possibly controlled by Treg and CTLA-4. The
control of CTLA-4 on intercellular adhesion at the CD4 T-cell
synapse has been the subject of an interesting article that
describes that CTLA-4 functions to induce less durable interactions between mouse T cells and antigen presenting cells.42
Accordingly, blockade of CTLA-4 with tremelimumab conceivably results in more durable DC:T-cell contacts.
Several corollaries arise from our observations on the
alloreactive reactions taking place inside short term xenografted mice:
1. Blocking CTLA-4 and Treg depletion with agents such
as cyclophosphamide or ONTAK43 may result in
enhanced antitumor immunity. Clinical trials with this
kind of combinations should be designed. Caution
must be stressed because undesired autoimmunity may
be enhanced as well by Treg elimination or inactivation.
2. Baseline pretreatment Treg may be a predictor of clinical response to CTLA-4 blockade. This will be examined retrospectively and/or prospectively in series of
tremelimumab and ipilimumab treated patients.
3. CTLA-4 blockade on Treg-depleted lymphocytes seems
to be much more active on CD4 than on CD8 T cells.
This is important for therapy because human and
mouse CD4 T cells are known to mediate antitumor
effects by themselves27,39,44 and because the augmentation of antitumor CTL responses may be indirectly dependent on de-repression of T helper cells. Activation
of T helper cells is involved in the inammatory
adverse events of anti-CTLA-4 mAb in humans by
releasing them to cooperate in cellular and humoral
immunity towards self and shared tumor antigens.45,46

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384

Anti-CTLA-4 mAb and Treg Depletion

Figure 6. CTLA-4 blockade and Treg predepletion enhance cytotoxicity raised in culture against autologous EBV-transformed cells lines.
(a) Two EBV transformed cell lines from independent donors were co-cultured with autologous PBL for 7 days in the presence or
absence of 15 lg/mL of tremelimumab or following CD25 Treg depletion. (b) Cytotoxicity assessed by 5 hr51 Chromium-release assays as
percentage of specic lysis against autologous EBV-transformed B cells is presented at a series of effector to target ratios. HT29 colon
carcinoma cell targets which were used as a specicity control remained under 10% of specic lysis for every condition and E:T ratio
(data not shown). (c) Viable lymphocyte cell recovery (absolute number) upon 7 day co-cultures. Results show a representative of
three independent experiments performed with the two donors.

4. Considering recognition of alloantigens, these phenomena could be also important for graft versus host and
graft versus leukemia effects in bone-marrow transplantation, as suggested by experiments in mice.47
Overall, our results are in agreement with preclinical evidence for such a Treg depletion plus anti-CTLA-4 mAb synergy as reported by the group of Melief in mouse B16 melanoma.31 Importantly, our in vivo observations on this
synergy also take place when using T cells and DC from
advanced cancer patients. The enhancing effect is also
observed against autologous transformed cells at least
in vitro. Moreover, clinical response to anti-CTLA-4 mAb

treatment correlates with high ratios of effector to regulatory cells in the tumor microenvironment further suggesting the appropriateness of approaches tilting this delicate
balance.23 As a consequence, combined approaches consisting of CTLA-4 blockade and depletion/inactivation of Treg
are going to be applied either sequentially or simultaneously to cancer patients in planned clinical trials at our
institution and elsewhere.

Acknowledgements
Enrique Grande-Pulido (Pzer) is acknowledged for the supply of tremelimumab and scientic support. Collaborations and scientic discussions pertaining CTLA-4-related research with Drs. Jesus Prieto, Jose I. Riezu, Pablo
Sarobe and Juan J. Lasarte are also acknowledged including the kind gift

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Int. J. Cancer: 129, 374386 (2011) V

Suarez et al.

385

of EBV-transformed cells. We are grateful to the excellent animal facility


support provided by Eneko Elizalde and Elena Ciordia and to technical help
by Arantza Azpilikueta and Sarai Solano. S.H.-S. receives a Ramon y Cajal

contract from Ministerio de Educacion y Ciencia and A.P. a scholarship


from FIS. C.A. receives salary support from Fundacion Cientca de la Asociacion Espanola Contra el Cancer (AECC).

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FIGURAS SUPLEMENTARIAS________________________
(Synergistic effects of CTLA-4 blockade with tremelimumab and elimination of regulatory T
lymphocytes in vitro and in vivo. N. Surez ; C. Alfaro et al.International J of Cancer, 2010;
129: 374-386).

159

Supporting Information Figure 1

Supporting Information Figure 1. Efficiency of Treg depletion from PBL evaluated by


FACS staining prior to MLR setting.
Multicolor immunostaining showing the efficiency of magnetic elimination of FoxP3+ cells.

160

Supporting Information Figure 2

Supporting Information Figure 2. CTLA-4 blockade and Treg depletion also enhance
alloreactive proliferation induced by immature DC.
Representative experiment of those in figure 1 but performed with immature monocyte-derived
DC (iDC) that were not exposed to maturation agents prior to setting up the MLR culture.

161

Supporting Information Figure 3

162

Supporting Information Figure 3.


CTLA-4 and Treg depletion increase the alloreactive proliferation of CD4 T cells but not of CD8
T cells. CD4+ and CD8+ cells were purified and tested by MLR in the presence of mature
allogenic DC. When indicated CD25+ Treg were pre-depleted and/or CTLA-4 mAb
(tremelimimab) added to the MLR cultures at 15 g/mL. Proliferation was assessed at the
indicated T:DC ratios. Two representative cases out of four are presented for CD4 (A) and CD8
(B) purified T cells. In the case of CD8 cells positive immunoselection was performed when
indicated from samples pre-depleted of CD25+ cells (a condition termed [CD8-Treg]).

163

Supporting Information Figure 4

Supporting Information Figure 4. Series of representative FACS dot plots show the
fractions of the two-colour fluorescent aggregates.
(A) Representative dot plots from experiments presented in figure 4A to assess T cell:DC
aggregates in MLR cultures.
(B) PKH2 fluorescence (FL-1) represented in histograms assessing the dilution of the dye
staining in T lymphocytes due to proliferation.

164

Supporting Information Figure 5

Supporting Information Figure 5. Treg pre-depletion resulted in higher levels of CTLA-4


expression on effector CD4 and CD8 T lymphocytes.
Representative histograms of CTLA-4 expression in CD4 and CD8 cells in experiments with
human PBL or PBL without Treg obtaining from peritoneal lavages of in vivo experiments as
those shown in figure 6.

165

Dendritic cells take up and present antigens from viable and apoptotic
polymorphonuclear leukocytes.

N. Surez *, C. Alfaro*, JL. Perez-Gracia, I. Martinez-Forero*, S. Hervs-Stubbs*, I.


Rodriguez*, C. Oate*, E. Bolaos*, A. Palazn*, A. Morales-Kastresana*, A. Gonzalez, and I.
Melero*,.

Gene Therapy Unit. CIMA. Universidad de Navarra. Pamplona, Spain. Biochemistry


Department. Clnica Universitaria de Navarra. Universidad de Navarra. Pamplona, 31008 Spain.

Medical Oncology Department. Clnica Universitaria de Navarra. Universidad de Navarra.


Pamplona, 31008 Spain.

Manuscrito en preparacin

167

Dendritic cells take up and present antigens from viable and apoptotic
polymorphonuclear leukocytes.

N. Surez *, C. Alfaro*, JL. Prez-Gracia, I. Martnez-Forero*, S. Hervs-Stubbs*, I.


Rodrguez*, C. Oate*, E. Bolaos*, A. Palazn*, A. Morales-Kastresana*, A. Gonzlez, and I.
Melero*,.

Gene Therapy Unit. CIMA. Universidad de Navarra. Pamplona, Spain.


Biochemistry Department. Clnica Universitaria de Navarra. Universidad de Navarra. Pamplona, 31008
Spain.

Medical Oncology Department. Clnica Universitaria de Navarra. Universidad de Navarra. Pamplona,


31008 Spain.

ABSTRACT
Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types
to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of
being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected
tissue. Human and mouse DC can readily internalize viable or UV-irradiated PMNs. Such
internalization was abrogated at 4 C and partly inhibited by anti-CD18 mAb. In mice, DC
which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to
cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in
humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be
subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if
human neutrophils had endocytosed bacteria, they were able to trigger the maturation program
of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot
pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph
nodes. Using CT26 (H-2d) mouse tumor cells, it was observed that if tumor cells are
intracellularly loaded with OVA protein, and UV-irradiated, they become phagocytic prey of H2d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically
re-isolated and phagocytosed by H-2b DC, such DC productively cross-present OVA antigen
determinants to OT-1 T cells. In summary, our results indicate that antigens phagocytosed by
short-lived PMNs can be in turn internalized and cross-presented by DC.

169

INTRODUCTION
Neutrophils are the first defense barrier against bacteria or fungi that have penetrated the body
surfaces. Many features equip these cells to perform such a critical function (Borregaard 2010).
Originating in the bone marrow, neutrophils have a short 4h half life in circulation and readily
extravasate upon infection or inflammation in any infected tissue (Ley et al. 2007; Summers et
al. 2010). Neutrophils sense and migrate in response to chemotactic gradients at inflammatory
sites (Ley et al. 2007). Interleukin-8 (Mukaida 2000; Waugh y Wilson 2008), bacterial formyl
peptides and complement factors are the main chemotactic stimuli, while extravasation is
guided by LFA-1 integrin interactions with ICAM-1 (Ley et al. 2007(Ley et al. 2007;
Vestweber 2007). Once in contact with the invading microorganisms, leukocytes degranulate
microbiocide substances, perform phagocytosis and oxidative killing of microorganisms (ElBenna et al. 2009; Borregaard 2010), take up tissular debris, and entrap bacteria by means of
forming trapping nets with their own DNA (Brinkmann et al. 2004; Borregaard 2010). Pus as it
appears in acute inflamed foci is a collection of exudates rich in the remains of neutrophils,
microorganisms, and tissular debris.
Adaptive immune responses rely on antigen presentation to T-cells as performed by
professional dendritic cells (DC) (Steinman 2007). At least some of the DC subsets present in
the body can take-up antigen from third party cells and present them to lymphocytes
(Villadangos y Schnorrer 2007). This process is referred to as antigen cross-presentation
(Carbone y Heath 2010) and can be performed both through the MHC class II and I pathways.
The paucity of cross-presenting DC in human peripheral blood or mouse lymphoid organs has
precluded direct experimentation and therefore most experiments have been performed with DC
differentiated in vitro from their precursors. The ability of DC to stimulate a T-cell response is
not constitutive (Steinman et al. 2003). On the contrary, DC depend on detection of microbial
molecular patterns (Granucci et al. 2005), tissue damage denoting molecules (Klune et al. 2008)
and proinflamatory cytokines to experience their maturation program. Maturation means
migration towards lymphoid organs triggered by CCR7 ligands (MartIn-Fontecha et al. 2003),
brighter surface expression of co-stimulatory molecules for T cells and production of T-cell
stimulating cytokines(Granucci et al. 2005).
An interesting article indicated that DC and neutrophils may interact in the body in sites of
acute inflammation such as a phlegmonous appendicitis (van Gisbergen et al. 2005).
Importantly the Van Kooyk group demonstrated that a molecular interaction dependent on DCSIGN on the DC side and siayil Lewis-Y sugars anchored on the CD11b integrin mediated the
heterotypic adhesion of human PMN to DC (van Gisbergen et al. 2005; van Gisbergen et al.
2005). Other authors have shown that neutrophils enhance the expression of co-stimulatory

170

molecules on DC (van Gisbergen et al. 2005; Yang et al. 2009). There have been also reports of
the ability of neutrophils to directly present antigen to T cells (Beauvillain et al. 2007).
From our point of view, it makes physiological and economic sense that scanty DC would be
able to interact with abundant neutrophils that bear signs of having internalized relevant
microbiological antigens. Such stressed PMN leukocytes would be thereby a concentrated
source of microbiological antigens. Even under sterile conditions neutrophil granule proteins
have been observed to elicit maturation of DC (Soehnlein 2009; Yang et al. 2009; Tewary et al.
2010). However, it is clear that if DC internalized infected neutrophils this would not only
constitute a rich source of microbial antigens, but also a concentrated source of microbial
molecular patterns to elicit DC maturation. If this hypothesis is correct, the ability of PMNs to
ferry antigenic material for DC efficient cross-presentation might be useful in cancer
immunotherapy.
We found that human and mouse DC internalize viable and dead PMNs. Interestingly, if such
PMN had been pre-exposed to dying tumor cells, the tumor-related material ends up being taken
up by DC. We demonstrate in mice that antigens internalized by PMNs can be cross-presented
by DC. Furthermore, intracellular antigens from dead tumor cells can be ferried by PMNs to be
readily up-taken by DC and subsequently cross-presented to CD8+ T cells. The importance of
second and third hand antigen presentation might have been overlooked, and PMN leukocytes
may play a critical role as a source of concentrated antigen and in providing maturation stimuli
to DC.

MATERIALS AND METHODS


Purification of Human neutrophils
10 ml of peripheral blood drawn in heparin containing tubes were mixed with 10mL of cold
PBS and 10 ml of 6% Dextran/0.9% NaCl solution. After being inverted 18-20 times to ensure
adequate mixing, the mixture was pipetted into a 50 ml tube and the tube was placed upright at
room temperature for 1-1.5 h, or until separation was completed. The yellowish supernatant was
recovered into a 50 ml tube and spun at 1150 rpm for 12 minutes at 4C with low brake. The
supernatant was then discarded and the pellet re-suspended in 5 ml ACK buffer, 5 min at room
temperature. After that incubation, the cells were washed with 45 ml of cold PBS. The
supernatant was discarded and the pellet was resuspended in 2.5 ml of PBS. The cell suspension
was laid over 3 ml of Ficoll-Hypaque gradients in a 15ml tube and spun at 1500 rpm for 30
minutes at 4C. The pellet was resuspended in 2 ml cold PBS and stored at 4 C. To ensure the
correct purification the pellet was immunostained with CD15-PE and a Giemsa staining of

171

cytospins was performed. Neutrophil purity was >95% (CD15bright neutrophils) (Alfaro et al.
2011).
Purification of mouse neutrophils
BALB/c mice neutrophils were obtained from bone marrow as follows. Hind limbs were
collected in ethanol and bones were cleaned with a scalpel (peeling off as much flesh as
possible). When all bones were cleaned, their ends were cut and a 27G1/2 needle placed in the
central cavity of the long bones to flush them with mouse complete medium [RPMI 10% FBS
(Gibco) + penicillin/ streptomycin (Gibco) + -mercaptoethanol (Gibco)]. An immunomagnetic
selection was made with Ly6G AutoMACS microbeads (Miltenyi Biotec, Bergisch Gladbach,
Germany) obtaining the positive and the negative fractions to monitor cell population
enrichment. Anti-Ly6C-biotine plus Streptavidine-PE was used for monitoring enrichment by
flow cytometry using a FACSCalibur Flow Cytometer (Benton Dickinson).
Human DC generation and maturation
DC were generated from filter buffy coat (FBC)-derived monocytes donated by healthy human
donors (Mazzolini et al. 2005). The protocol for obtaining FBC was approved by the Ethics
Committee of our institution and donors gave informed consent. Isolated mononuclear cells
were subjected to positive selection using anti-CD14-conjugated paramagnetic beads and
purified using the AutoMACSs system according to manufacturers instructions (Miltenyi
Biotec, Bergisch Gladbach, Germany). Purified monocytes were cultured for 7 days in complete
medium (RPMI 10% FBS + penicilin/streptomycin + -mercaptoethanol) supplemented with
GM-CSF (1000 U/ml; Leukine) and IL-4 (500 U/ml; R&D Systems, Minneapolis, MN).
Cytokines were added every other day. DC were matured with clinical grade TNF- (50 ng/ml;
Boehringer Ingelheim, Ingelheim, Germany), IFN- (1,000 IU/mL; Schering-Plough,
Kenilworth, NJ) and Poly I:C (20 g/ml; Ampligen, Bioclones, South Africa) during 48 h
(Alfaro et al. 2009).
Murine DC generation
Mice DC precursors were obtained from bone marrow. Cells were put in culture [RPMI 10%
FBS (Gibco) + penicillin/streptomycin (Gibco) + -mercaptoethanol (Gibco)] at a density of 2 x
106 cells/mL in 10 ml per petri dish in the presence of 20 ng/ml of rmGM-CSF (R&D) (Tirapu
et al. 2004). After 72 h, recombinant mouse GM-CSF (rmGM-CSF) at 10ng/ml was added in 10
ml in RPMI complete medium per dish. On 6th day of culture, 10 ml of medium were
withdrawn per dish. On 9th culture day, non adherent cells were collected by thoroughly
pipetting with RPMI medium. Adherent cells were collected by adding 2ml of accutase (L11007 PAA laboratories) per dish and incubated 15 minutes at 37 C.

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Interaction between DC and neutrophils in vitro


DC were stained with PKH26 and neutrophils with PHK2 membrane marker (Sigma- Aldrich)
as reported (Alfaro et al. 2011). Half of the neutrophils were killed with UV light (1,232 J/cm2).
After that, live or dead neutrophils were co-cultured with DC for 4 and 24 h at 4 C or 37 C.
The neutralizing antibodies anti-CD11b, anti-CD18 (kindly provided by Francisco Sanchez
Madrid. Hospital de la Princesa. Madrid) and anti-DC-SIGN (kindly provided by Angel Corbi.
CIB Madrid) were added to the culture medium at 10/ml or at a 1/5 dilution of the
concentrated Hybridoma supernatant. After 2, 4 and 24 h, co-culture cells were collected to
analyse double positive cells using a FACSCalibur Flow Cytometer or examined by confocal
microscopy.
In vitro chemotaxis assay
In vitro neutrophil and DC migration was measured in Transwell Chambers (5 m; Corning
Costar, Corning, NY). Both PKH26-DCs (105) and PKH2-labeled neutrophils (105) were added
to the upper chamber and migration stimuli (subconfluent monolayer of HT29 cells) were
placed in the lower chamber. DC and neutrophils were added to the upper chamber in complete
medium, complete medium plus 20 g/ ml antiIL-8 mAb (BD Pharmigen), or complete medium
plus mIgG2b as control. Transmigrated cells in the lower chamber were quantified using
fluorescence microscopy imaging of the lower chamber. The number of PKH26-fluorescent DC
or PKH2-labeled neutrophils per microscopic field (x20) in the lower chamber was quantitated
in triplicate wells by a blinded observer.
Flow cytometry analyses
FITC and PE-labeled mAb specific for the DC maturation markers CD80, CD83, CD86 and
HLA-DR (BD Bioscience) and isotype-matched labeled controls were used to characterize cell
surface phenotypes by flow cytometry. PE-labeled mAb specific for CD15 and Ly6C-biotin +
SAV-PE (BD Bioscience) were used to analyse purity in human and mice leukocyte samples.
Splenocytes were obtained from OT-1 and OT-2 TCR transgenic mice (Jackson Laboratories.
Bar Harbor, Maine, USA) (Murillo et al. 2009). Lymphocyte division was analysed by serial
dilution of carboxifluorescein succinimidyl ester (CFSE) in CD4+ and CD8+ cells (BD
Bioscience). Cells (105) were washed in cold PBS and incubated 15 min at 4 C with specific
APC, FITC or PE-labeled Abs to electronically gate CD4 and CD8 cells upon FACS analyses.
Immunofluorescence using confocal microscopy
Confocal fluorescent images were obtained using a LSM 510 Zeiss confocal scan head mounted
on a Zeiss Axiovert 200M on an inverted-based microscope using a 40x or 63x oil immersion

173

objective. Sequential excitation at 488 nm and 543 nm was provided by argon and helium-neon
gas lasers, respectively. Emission filters BP500-550 and LP560 were used for collecting green
(PKH2) and red (PKH26) in channels one and two, respectively. After sequential excitation,
green and red fluorescent images of the same cell were saved with Laser Sharp software. Images
were analyzed by Zeiss software. The term colocalization refers to the coincidence of green and
red fluorescence, as measured by the confocal microscope.
Protein quantification
After culturing the neutrophils in complete medium supplemented with 1mg/ml of OVA protein
(Calbiochem) for 2h, the supernatants of serial centrifugations were collected to verify the
complete elimination of extracellular OVA. Protein concentration was measured in a Nanodrop
Spectrometer ND-1000 at 280nm. Stock solution was used as control.
Chemokine production
Supernatants were collected in the indicated culture time points, and the cytokine concentration
was determined by immunoassay. Commercially available ELISA kits were used for the
detection of human IL-12p70 and mouse IFN- (BD Biosciences Pharmingen. San Diego. CA.).
Tumor cell lines
HT29 and CT26 tumor cell lines were obtained from American Type Culture Collection
(Rockville, MD).
Elicitation of DC maturation by E coli associated to neutrophils
2x105 human neutrophils were incubated with heat-inactivated E. coli bacteria in various
numbers ranging from 105-102 for 60 min. The neutrophils were then washed eight times and the
pellets and the supernatants collected. Washed PMN and the supernatants were added to human
immature DC (iDC) cultures and 48h later IL-12 concentrations in the supernatants were
monitored. Subsequently, such DC (105) were washed in cold PBS and incubated 15 min at 4
C with specific FITC or PE-labeled mAbs to measure the mean fluorescence intensity for CD80,
CD83 and CD86 surface immunostaining as assessed by flow cytometry. Mature DC (mDC) in
the presence of poly I:C and TNF- and IFN- were used as a positive control.
OVA-specific T cell response analysis
Splenocytes from OT-1 and OT-2 transgenic mice were stained with CFSE solution 2.5uM.
After 24 h co-culture DC plus live or UV-irradiated neutrophils (cultured during 2 h with or
without OVA at 1mg/ml), OT-1 or OT-2 splenocytes were added to the co-culture. The capacity
of DC to cross-present OVA from neutrophils was measured as the dilution of membrane CFSE

174

from CD4+ (OT-2 mice) or CD8+ (OT-1mice) splenocytes that were gated by immunostaining
with CD8 or CD4 specific mAb in a FACS Scalibur.
OVA protein electroporation in the tumor cell line CT26
For electroporation, CT26 (3.5x106 cells/ 500 l RPMI without serum and antibiotics) were
incubated with 500 l OVA at 2mg/mL for 10 minutes at room temperature. Electroporation
was carried out using Gene Pulser II Electroporation System (BioRad): two pulses 200V/cm
and 300 F of capacitance. Immediately after electroporation, CT26 cells were cultured in
RPMI complete medium during at least 1 h. To ensure the internalisation of OVA in CT26 cells,
a control fluorochrome-labelled protein (Streptavidine-PE) was electroporated in the same
cuvette with OVA, and its internalisation was assessed by flow cytometry after extensive
washing of non internalized protein.
Co-injection of DC and PMN into the mouse foot pad
Mouse PMNs were co-cultured for 2h with 105 heat-inactivated E. Coli bacteria. After 8
extensive washes, 5x106 PMN-PKH26 and 107 mouse DC-PKH2 were injected into the foot
pads of mice in separate syringes with 30 l PBS for each injection. 24 h later, popliteal and
inguinal lymph nodes were surgically excised and were disaggregated to give single cell
suspensions. Cells were immunomagnetically selected to enrich CD11c+ cells. Immunoselected
cells were processed for subsequent analysis using flow cytometry and confocal microscopy.
Statistics
All data are presented as the mean SD. For comparisons, unpaired Students t-tests or Mann
Whitney U tests were used [Prism software (GraphPad Software)].

RESULTS
DC and PMN are co-attracted by IL-8 derived from tumor cells.
We have previously shown that human carcinoma cells produce functional IL-8 (Feijoo et al.
2005). One of the corollaries is that such IL-8 might attract human PMNs and monocyte-derived
DC (Alfaro et al. 2011). Indeed, in classical transwell chemotaxis assays towards IL-8producing monolayers of HT-29 human colon carcinoma cells, both PKH26-labelled DC and
PKH2-labelled PMNs are actively co-attracted to the lower chamber containing a confluent
monolayer of HT29 cells (Figure 1 A and B). The effect was mediated by IL-8 as demonstrated

175

by the abrogation of the migration by adding a neutralizing anti-IL8 mAb to the lower chamber
(Figure 1A and B).
DC take up live and UV-irradiated PMN leukocytes that might carry microbial
maturation signals.
Human DC and PMN were florescence-labelled and as can be seen in supplementary figure 1,
fluorescence was readily observed by flow cytometry and confocal microscopy. In figure 2
confocal microscopy pictures of short 4h co-cultures of PMN and DC show that fluorescencent
material from human PMNs was internalized by human DC (Figure 2). This phenomenon
occured both when the PMN had been irradiated with UV-light or when live untouched PMNs
were used. Evidence for internalization in confocal planes is shown at different magnifications
and summarized in a video reconstruction of multiple confocal planes in the Z axis
(Supplementary Video 1).
In figure 3A a two color FACS dot plot shows the single stained cultures and the double
positive events corresponding to DC that have internalized green-fluorescent material from
PMNs. Using this technique, we looked at the requirements for internalization. We observed
that neither an anti-DC-SIGN antibody not and anti-CD11b antibody were able to decrease
internalization (Figure 3B). It is of note that such adhesion molecules have been described as
the main mediators of the DC-neutrophil heterotypic aggregation (van Gisbergen et al. 2005). In
contrast, anti-CD18 integrin common chain mAb clearly decreased double staining. When the
experiment was performed at 4 C to repress metabolism, internalization was drastically reduced
(Figure 3B).
If PMN have internalized bacteria they are likely to contain microbiological biomolecules that
activate/ mature DC (i.e: LPS or bacterial DNA). In fact, we exposed PMNs to E. coli bacteria
which were rapidly internalized. We removed bacteria from cells by extensive washing and
centrifugation (up to 8 washing cycles with 50 ml of culture medium each). PMN that had been
exposed to E. coli were able to mature DC in terms of IL-12 production and enhancement of
CD80, CD83 and CD86 surface expression. The maturation activity was only present in the
cellular pellets since the supernatant of the eighth wash was not able to trigger DC maturation in
any case (Figure 4). These findings are important since extravasated PMN that would capture
gram negative bacteria would concentrate LPS and bacterial DNA for a subsequently interacting
DC.
Internalization of mouse bone marrow PMN by mouse DC.
To study the consequences of PMN internalization by DC, it was of interest to move on to
mouse experimental systems. Consequently, we purified Ly6G cells from bone marrow cells by
immunomagnetic selection (Supplementary Figure 2A) and DC were derived from bone marrow

176

in the presence of GM-CSF for 9 days. Supplementary figure 2 shows immunostainings for
Ly6C and Giemsa stainings of cytospins from the enriched PMN population. Mouse DC and
PMNs were also differentially labelled with fluorescent dyes. Co-cultures of DC and PMN for 4
h showed evidence of internalization by flow cytometry and confocal microscopy
(Supplementary Figure 2B and Figure 5A at 24 h of co-culture). Pictures in figure 5B show
confocal microscopy evidence in the sense that internalization of PMN by DC is inhibited at 4
C both in 4 h and 24 h co-cultures. Summarized data are presented in figure 5C. Therefore the
phenomena observed with human DC and PMN seems to be similar in both humans and mice.
Dead carcinoma cells are internalized by PMN leukocytes that if subsequently
phagocytosed by DC ferry carcinoma cell material.
Human DC can take up fluorescent material from apoptotic tumor cells (Hoffmann et al. 2000).
We confirmed this point in figure 6A that shows that red-labelled DC can take up green
fluorescent apoptotic bodies from UV-irradiated HT29 colon carcinoma cells (Figure 6A).
Apoptotic tumor cells can be prey of PMN phagocytes as well. We reasoned that if PMN
leukocytes take up carcinoma cell debris, this material will end up inside DC, in the case that
such DC eventually internalized a PMN with HT29 material inside. To test this idea we labelled
HT29 colon carcinoma cells with PKH2 and following UV-irradiation fed them to PMN
leukocytes. After 2 h of co-culture, CD15+ cells were immunomagnetically selected to re-isolate
the PMNs. Subsequently CD15+ PMNs were co-cultured with PKH26-labelled DC for 24 hours.
The experimental approach is summarized in figure 6B. As can be seen in figure 6C, green
fluorescent material from HT29 cells is found inside red-labelled DC. Furthermore, quantitative
data from FACS analysis indicated that the internalization phenomenon is quite efficient with
more than half of the DC taking up green fluorescent material derived from the carcinoma cells
(Figure 6). Such internalizing activity was abolished if the co-incubation of PMN and DC took
place at 4 C.
Accordingly it could be envisaged that PMN exposed to debris from tumor cells could transfer
this debris to third party antigen-presenting DC. However, to explore the role in antigen crosspresentation we needed to use mouse models.
Antigen carried by PMN can be cross-presented by DC in vitro.
In order to demonstrate that DC could cross-present antigen material from internalized PMNs,
we performed experiments with bone-marrow neutrophils derived from the bone marrow of
BALB/c mice (H-2d) from which PMN were isolated as shown in supplementary figure 2. Such
PMN were given OVA protein for two hours in culture followed by extensive washes (x 5
times). These PMNs were then co-cultured overnight with DC from C57Bl/6 mice (H-2b) and
exposed to OT-1 or OT-2 TCR-transgenic lymphocytes. Proliferation of OT-1 or OT-2 T cells

177

was monitored by CFSE dilution 72 h later. An schematic representation of the approach based
on the fact that H-2d class I and II antigen presenting molecules cannot present to OT-1 or OT-2
T lymphocytes is presented in figure 7A. In figure 7B a representative case shows the dilution
of CFSE that was triggered by the cognate peptides pulsed on DC as a positive control and by
DC that had been incubated with PMNs preloaded with OVA. T cell division did not take place
in the rest of control conditions including PMN that had not been exposed to OVA fed to DC
(Figure 7B). Analysis of IFN- concentrations released to the tissue culture supernatants showed
a comparable result with both CD8 OT-1 and CD4 OT-2 lymphocytes (Supplementary Figure
3).
Carcinoma cells loaded with OVA transfer antigen to PMNs, then PMNs to DC and such
DC cross-present antigen to T lymphocytes.
In order to study antigen transfer to DC via a PMN intermediate, experiments were set up as
summarized in figure 8A. CT26 colon carcinoma cells of BALB/c origin (H-2d) were
electroporated in the presence of soluble OVA protein. Electroporation in the presence of a
soluble protein leads to internalization of protein as checked with a mixture of OVA with
streptavidin-PE. Supplementary figure 4 shows internalization of PE fluorescence by CT-26
contingent on the electrical shock. Once CT26 had been electroporated with or without OVA,
they were extensively washed, UV-irradiated and fed to PMN of BALB/c origin. Such PMN
were recovered 2 h later from the cultures by Ly6G magnetic immunoselection (taking
advantage of the magnetic beads still bound to the cells). Immunoselected PMN were then cocultured overnight with DC of C57Bl/6 (H-2b) origin. Subsequently these DC were used to
stimulate CFSE-loaded OT-1 T lymphocytes. As can be seen in a representative experiment
shown in figure 8B, dilution of OT-1 CFSE only took place when the CT26 tumor cells had
been electroporated with OVA, but not in the remaining control co-cultures. As a positive
control DC pulsed with the cognate peptide were used and elicited strong proliferation of OT-1
lymphocytes. A summary of replicate experiments is provided in supplementary figure 5. This
correlated with IFN- release to the co-culture supernatant as shown in Supplementary figure 6.
These data indicate that it is possible that tumor derived antigens can end up productively
presented by DC to T cells after being transiently ferried by PMNs that are internalized by the
DC.
DC co-injected in the foot pad with PMN that have internalized bacteria reach draining
Lymph nodes with PMN-derived material.
To present antigen to T cells DC need to reach lymph nodes through afferent lymphatic vessels
(Rouzaut et al. 2010). This is a function tightly controlled upon maturation of the DC by the
induction of CCR7 expression (MartIn-Fontecha et al. 2003; Granucci et al. 2005). If DC and
PMN are injected in the foot pad they should be able to meet thus mimicking inflamed tissue. In

178

the experimental approach shown in figure 9A, differentially fluorescence labelled bone marrow
derived DC and PMN preloaded with E. coli bacteria were injected with separate syringes into
the foot pad. 24 h later draining lymph nodes were surgically excised and CD11c cells enriched
by immunomagnetic selection. Double immunofluorescence events analyzed by flow cytometry
and summarized in figure 9B indicate that about 2% of the injected DC were recovered from the
draining LN carrying fluorescent material from the PMNs while about 5% of the injected DC
were recovered from the lymph nodes with no evidence of carrying PMN-derived fluorescent
material. These data indicate that is possible for DC which internalize PMN material to reach T
cell compartments. It is of note that if PMN had not been exposed to bacteria very few CD11c+
fluorescent events were recovered from lymph nodes and virtually no double positive cells were
found (data not shown). Confocal microscopy images of cytospins made with CD11c+ selected
cells from draining lymph nodes indicated co-localization of fluorescent dyes in the same cells.

DISCUSSION
In the defence of the organism against infection it makes sense that the early innate response
would cue the adaptive immune responses. In the bridge between innate and adaptive immune
responses dendritic cells play a pivotal role (Janeway y Medzhitov 2002). We reasoned that
pyocytes (neutrophils forming pus) would be an excellent source of concentrated microbial
antigens and microbial molecular pattern signals, if eventually internalized by DC.
Therefore we first found that both human and mouse DC avidly internalize PMNs in co-culture.
Both apoptotic and viable PMN are internalized although with more efficiency in the case of
apoptotic PMN. It is postulated that under steady state conditions this phenomenom should feed
DC with innocuous self antigens that if presented by the immature DC should lead to tolerance
(Steinman et al. 2003).
The nature of the surface molecules playing a role in the internalization is unclear. We found
partial inhibition by blocking the CD18 integrin chain but not upon blockade of DC-SIGN and
CD11b in many attempts. This is in disagreement with a paper from the group of Van Kooyk
whose findings indicated a role for DC-SIGN on DC and sialyl Lewis X decorating CD11b in
the heterotypic cellular interaction (van Gisbergen et al. 2005; van Gisbergen et al. 2005). Since
internalization rather than aggregation is our read out, reasons for this difference could come
from various sources. Internalization requires active metabolism since it was nearly abolished at
low temperatures.
Under acute inflammation, extravasated PMN would be full of phagocytosed microbes and
microbial material. An important aspect is that such pyocytes, if entering into contact with and
become internalized by DC, would transmit to DC both the presence of the biomolecules

179

denoting a microbial pathogen and microbial antigens. In our proof of concept experiments, E.
coli bacteria actively loaded into PMN was able to intensely mature the subsequently
encountered DC. This makes sense because ingestion of PMN by DC or their tissular precursors
will launch the proper maturation program in DC. In mice these events also elicit migration of
DC to draining lymph nodes. It remains to be seen which molecular patterns of bacteria are
detected, but most likely these involve LPS detection by TLR4 and bacterial DNA detection by
TLR9 in our experiments.
DC have been in the limelight of tumor immunology and immunotherapy (Steinman y
Banchereau 2007). In solid tumors multiple myeloid cells nest in the stroma including cells
resembling PMN. Such PMN have the opportunity to phagocytose apoptotic or necrotic tumor
cells. We thought that it was interesting to determine if PMN could take up antigen from
carcinoma cells and then transfer it to DC, in such a way that the relevant antigens would end up
cross-presented to T cells. To start exploring these possibilities we moved to mouse systems in
which we can probe for antigen presentation with TCR-transgenic T cells. Our data in humans
strongly argued in favour of this possibility because HT29 fluorescent carcinoma cell debris
taken up by PMNs is internalized by DC, if those PMN are subsequently phagocytosed.
However, it could be argued that the PMN-associated fluorescent material could be degraded
and is thereby become not suitable for antigen presentation. However, in the mouse system we
found that PMN which had internalized OVA protein from soluble sources or OVAelectroporated tumor cells efficiently cross-presented antigens to either OT-1 or OT-2
lymphocytes. Direct presentation by PMN or tumor cells was ruled out because of incompatible
antigen presenting molecules although in other circumstances this can take place according to
published reports (Beauvillain et al. 2007; Culshaw et al. 2008).
The study of the intercellular crosstalk between DC and PMN leukocytes is in its infancy. A
recent report indicates that PMN could deactivate DC upon arrival to lymph nodes in aseptic
conditions (Yang et al. 2010). This makes sense for immature DC but it remains to be seen if it
is the case in vivo under septic conditions that ought to be more relevant for the defence of the
body against infection. Our data showing the ability of DC loaded with material derived from
PMN with internalized bacteria to reach the lymph nodes argue in favour of DC-maturing role
of PMNs under acute infection. Our current research is addressing if the DC which reach lymph
nodes ferrying PMN material can be operational in terms of antigen presentation of PMNassociated antigens to T cells in vivo.
Our current research is focused on unravelling the implications of these PMN-DC interactions
by carrying out in vivo experiments in mouse models. In humans these events are much more
difficult to dissect in vivo. However, there is published evidence of DC-PMN interactions in
appendicitis surgical samples (van Gisbergen et al. 2005; van Gisbergen et al. 2005) and we

180

have found that DC and PMN can be co-attracted to solid malignancies by means of the IL-8
chemokine.
All in all, our experiments point to an entangled and complex relationship between PMN and
DC. PMN greatly outnumber DC and therefore could act as concentrators of antigen and
microbial molecules for DC. If PMN leukocytes become accessible to DC they can be
internalized and then modulate DC functions while transferring the antigens that PMN may
carry. However, exactly how relevant are these functions for the overall physiology of the
immune system still remains to be seen.

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FIGURAS____________________________________________
(Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear
leukocytes. N. Surez; C. Alfaro et al. Manuscrito en preparacin)

185

Figure 1

Figure 1. DC and PMNs are co-attracted by IL-8 secreted by HT29 colon cancer cells.
(A) Chemotactic migration of DC (labeled with PKH26) and PMN (labeled with PKH2) from
the upper chamber towards confluent monolayers of HT29 cells in the lower chamber. When
indicated, control IgG mAb or anti IL-8 mAb were added to the lower chamber. Microscopy
photographs represent visible light and UV illumination fields with filters for red and green
fluorescence showing the lower chamber when taken 24h later.
(B) Quantitative data based on experiments as in A, representing three independent triplicate
experiments counting four microscopic fields per well (meanSD).

186

Figure 2

DC + UV-irradiated PMNs

DC + Alive PMNs

x100

x200

x200

x200

x400

x200

x200

x400

Figure 2. Human DC internalize UV-irradiated and viable PMNs.


Confocal microscopy images at the indicated magnifications of 4h co-cultures of human DC
(membrane-red labeled with PKH26) and either (A) peripheral blood viable PMNs (green
labeled with PKH2) or identical PMNs lethally irradiated with UV (B).

187

Figure 3

Figure 3. Human DC internalize PMNs in a temperature and CD18 dependent fashion.


(A) FACS dot plots showing PKH26 staining of Human DC and PKH2 staining of PMNs and
37 h co-cultures of DC and PMN showing double positive events in a representative experiment
of at least five similarly performed.
(B) Co-culture experiments as in A, depicting the percentage of double positive events at 4h of
co-culture. When indicated blocking mAb to DC-SIGN, CD11b and CD18 were added or the
co-cultures were performed at 4C.

188

Figure 4

189

Figure 4. PMNs carrying E. coli bacteria trigger the maturation of DC.


PMN from human peripheral blood were exposed for 1h to E. coli bacteria for 60 min in various
numbers ranging from 105-102 and then were washed eight times and the pellet and the
supernatant collected. Washed PMN and the indicated washing supernatants were added to
human immature DC (iDC) cultures and 48h later IL-12 concentration in the DC culture
supernatants was monitored as well as the mean fluorescence intensity for CD80, CD83 and
CD86 surface immunostainings on the DC. Mature DC (mDC) in the presence of poly I:C and
TNF- and IFN- were used as a positive control.

190

Figure 5

191

Figure 5. Mouse DC take up mouse PMN leukocytes.


(A) Confocal microscopy images of 24 h co-cultures of viable or UV-irradiated PMNs labeled
with PKH2 and bone-marrow derived DC labeled with PKH26.
(B) Similar experiments as in A but with some co-cultures performed at 4C as indicated.
(C) Summary of data from five microscopic fields per condition in which internalization was
identified by yellow staining.

192

Figure 6

Figure 6. Fluorescent material from human carcinoma cell debris can be taken up by
PMN and transferred to DC.
(A) PKH2-labeled HT29 cells were UV-Irradiated and co-cultured for 24 h with human PKH26labelled DC and confocal images were taken indicating internalization by DC. (B) Summary of
the experimental approach of co-cultures consisting of HT29 carcinoma cells previously labeled
with PKH2 and UV-irradiated with PMNs which were subsequently retrieved 2 h later by CD15
immunomagnetic selection. Then PMN were co-cultured for 24 h with PKH26-labeled DC.
(C) Confocal microscopy images of the indicated co-culture conditions.
(D) FACS dot plots showing each cell type labeled with the corresponding fluorescent dye and
co-cultures performed at 37 C and 4 C.

193

Figure 7

Figure 7. Ovalbumin taken up by mouse PMNs can be transferred to DC that in turn


cross-present antigen.
(A) Schematic representation of the experimental conditions in which OVA was added to bonemarrow-derived PMNs from BALB/c mice which were extensively washed 2 h later and
subsequently subjected or not to UV-irradiation. Then PMNs were co-cultured overnight with
DC derived from the bone marrow of C57Bl/6 mice. The next morning CFSE-loaded OT-1 or
OT-2 TCR-transgenic T cells were added to the cultures.
(B) FACS histograms showing CFSE dilution at 72 h of culture in the indicated conditions. DC
pulsed with the cognate OVA peptides recognized by OT-1 and OT-2 T lymphocytes were used
as positive controls. The experiment shown is representative of at least three similarly
performed.

194

Figure 8

Figure 8. Internal antigens can be ferried from tumor cells to DC via PMNs and then be
subsequently cross-presented to T lymphocytes.
(A) Schematic representation of the experiments in which CT26 tumor cells were electroporated
in the presence or not of soluble OVA protein, extensively washed 30 min later and then UVirradiated. Such apoptotic tumor cells were co-cultured with Ly6G-immunoselected PMN from
the bone marrow of BALB/c mice (H-2d) during 2 h. These PMN that kept labeled with the
immunomagnetic beads were re-isolated in columns under a magnetic field and subsequently
exposed to DC from C57BL/6 mice, whose H-2Kb antigen presenting molecules can present
antigen to OT-1 T cells. Following overnight DC culture with the PMNs, CFSE OT-1
lymphocytes were added to the cultures and CFSE dilution in CD8 cells was monitored 72 h
later.
(B) Representative histograms of CFSE dilution in the indicated conditions including DC pulsed
with the cognate peptide of OT-1 T cells as a positive control.
(C) Summary (meanSD) of three experiments similarly performed.

195

Figure 9

Figure 9. Co-injection of PMN with endocytosed bacteria and DC in subcutaneous tissue


gives rise to the detection of DC carrying PMN-derived material in the draining lymph
node.
(A) Schematic representation of experiments in which bone marrow-derived DC and PMN that
had been pre-exposed to E. coli and washed were injected in separate syringes into the foot pads
of mice. Both leukocyte types were differentially labelled with fluorescence dyes (PKH26 for
PMN and PKH2 for DC).
(B).Calculations of the percentages of injected DC recovered as CD11c+ immunomagnetically
selected cells from popliteal lymph nodes measuring double or single positive fluorescent
events as indicated by flow cytometry.
(C) Representative confocal images of cytospins made with the CD11c-immunoselected cells
from popliteal lymph nodes. Experiments are representative from at least two similarly
performed with three mice per group each.

196

Supplementary Figure 1

Supplementary Figure 1. Labelling of human PMN and DC with the indicated fluorescent
dyes visualized by FACS and by confocal microscopy.

197

Supplementary figure 2
A

198

Supplementary Figure 2.
(A) Immunomagnetic selection of Ly6G cells from single cell suspensions from mouse femur
and tibia bone-marrow before and after immunomagnetic selection (Automacs). Purity was
assessed by cells showing bright Ly6C immunestaining and upon May-Grnwald Giemsa
staining of cytospins.
(B) FACS dot plot analysis of mouse DC and PMN labeled with PKH2 and PKH26 as a single
cell type or in co-culture as indicated. 4 h co-cultures were performed at 37C or 4C as
indicated. Representative confocal images of co-cultures set up with the PMN and DC at the
indicated temperature conditions.

199

Supplementary Figure 3
A

Supplementary Figure 3.
(A) INF- concentrations released in 72 and 96 h to the supernatant of cultures by OT-1 and
OT-2 lymphocytes in conditions identical to those in figure 7 as indicated in the figure.
(B) Shows the extent of CFSE dilution in OT-1 and OT-2 cells in the same experiment in which
INF- concentrations in the supernatant were monitored in A.

200

Supplementary Figure 4

Supplementary Figure 4.
Dot plots FSC/SSC and red fluorescence (FL2) of CT26 tumor cells set in the presence of a
mixture of OVA and Streptavidin-PE and given or nor electroporation as indicated. Cells were
analyzed by flow cytometry 2 h following electroporation or mock electroporation

201

4- DISCUSIN GENERAL E IMPLICACIONES FUTURAS

203

4- Discusin general e implicaciones futuras


Las CD ocupan un importantsimo lugar en la investigacin inmunolgica tanto bsica
como aplicada, con 2239 publicaciones registradas en Medline a lo largo del ao 2010, que
contienen el trmino dendritic cells solamente en el ttulo del trabajo.
No podemos dudar del papel central de las CD tanto en inmunidad, como en tolerancia
inmunolgica. Recientemente se han generado modelos transgnicos de ratn en los que es
posible la deplecin selectiva de estas poblaciones (Hochweller et al. 2008; Bar-On y Jung
2010) y de la experimentacin con estas cepas se obtendrn conclusiones definitivas sobre la
funcionalidad de las clulas dendrticas tanto en situaciones de salud como de enfermedad.
Las clulas dendrticas se caracterizan por establecer muchas interacciones funcionales
con otros tipos celulares. La evidencia de su interrelacin con leucocitos polimorfonucleares
abre un campo de investigacin de gran inters.No sabemos si tendr realmente importancia en
inmunologa del cncer pero claramente s en la respuesta vrente a microorganismos.
El aspecto ms atrayente es el convencimiento de la comunidad cientfica sobre la
utilidad teraputica de las CD en protocolos de vacunacin frente al cncer, frente a agentes
infecciosos e incluso en la etiopatogenia y tratamiento de la autoinmunidad. As, en el
tratamiento de enfermedades autoinmunes se intentan vacunaciones con CD presentadoras de
auto-antgenos tratadas en condiciones de cultivo que favorecen un efecto tolerognico (MarinGallen et al. 2010). Su utilidad todava no se ha demostrado clnicamente.
En enfermedades infecciosas se han realizado ensayos para inducir inmunidad teraputica
en pacientes infectados por VIH (Garcia et al. 2011) basados en datos interesantes sobre su
eficacia en modelos de simios no humanos (Lu et al. 2003). Est claro que la vacunacin con
CD maduras, pulsadas con virus autlogo inactivado tienen la capacidad de aumentar la
respuesta inmunitaria frente al virus.
En nuestra institucin se va a comenzar un ensayo clnico para vacunar a pacientes con
hepatitis crnica tipo C (HCV) con CD transfectadas con un adenovirus que codifica antgenos
de la proteasa del HCV (Zabaleta et al. 2008). Las interacciones con leucocitos
polimorfonucleares indican que las CD pueden tener un papel muy relevante en infecciones
pigenas. En este sentido es posible que el pus constituya un entorno idneo para la carga
antignica de lsa CD.

205

En este momento existen en el mundo una enorme cantidad de ensayos clnicos basados
en inmunizaciones con CD para pacientes oncolgicos. Los datos de eficacia ms prometedores
se observaron en glioblastoma multifocal (Okada et al. 2011) donde varios estudios muestran
evidencia de efectos teraputicos, en ausencia de ensayos aleatorizados publicados hasta el
momento (Liau et al. 2005; Wheeler 2010; Prins et al. 2011).
Tras aos de desarrollo sigue an en debate cual es la mejor ruta de administracin del
tratamiento, cual es el mejor cctel de maduracin de las CD y si es necesario transfectar ciertos
genes a las clulas dendrticas. En inmunoterapia se percibe cada vez ms claramente la
necesidad de terapias combinadas con diferentes agentes. La vacunacin con CD es sinrgica en
varios modelos preclnicos con anticuerpos anti-CTLA-4 (Hodi y Dranoff 2006; Pedersen y
Ronchese 2007; Saha y Chatterjee 2010), anti-CD137 (Lynch 2008) y anti-PD1 (Alexandrescu
et al. 2010; Weber 2010; Wolchok et al. 2010). Estas combinaciones tienen indudable inters
traslacional.
Es importante resaltar que la eliminacin quirrgica de carga tumoral es muy conveniente
para eliminar mecanismos inmunosupresores dependientes del tumor y puede dar lugar a un
nuevo paradigma segn el cual las vacunaciones antitumorales se apliquen a pacientes en
estadios de enfermedad mnima residual.
El grupo de Ralph Steinman ha sido pionero en la dianizacin de antgenos in vivo a
clulas dendrticas mediante anticuerpos y protenas quimricas, para luego promover la
maduracin sistmica de las CD con agonistas de receptores TLR o con anticuerpos anti-CD40.
Estas ideas pueden tener especial inters tras el descubrimiento de las clulas BDCA-3+ cCLEC9+ como las principales inductoras de linfocitos T citotxicos (Jongbloed et al. 2010). Esto se
deduce por analoga con las funciones de las clulas CD11chighCD8+ de ratn.
No es fcil aventurar cul ser la estrategia de vacunacin que muestre mayor eficacia en
cncer. El beneficio clnico con Provenge (vacunaciones repetidas con clulas mononucleares
cultivadas con una protena de fusin GM-CSF-PSLA) (Madan y Gulley 2011) en cncer de
prstata resistente a bloqueo hormonal, ha motivado una gran expectacin. Sin embargo, su
coste es prohibitivo para conseguir un alargamiento en la mediana de supervivencia de tan slo
cuatro meses. El conocimiento de los mecanismos celulares y moleculares de las CD, que
incluya informacin fehaciente sobre los mecanismos de migracin y de inmunosupresin
tumoral, ser un elemento importante para la toma de decisiones en el diseo de los ensayos
clnicos.

206

La situacin en el momento presente se resume en la realizacin concomitante de ensayos


clnicos de mltiples estrategias de tratamiento inmunolgico a menudo combinadas entre s o
con terapias convencionales. Sus correlatos inmunolgicos proporcionan datos de extraordinaria
relevancia para entender los mecanismos y mejorar progresivamente la eficacia. En las
combinaciones

teraputicas

est

claro

que

es

crucial

interferir

con

mecanismos

inmunosupresores y quiz los mejores mtodos de carga antignica para CD estn an por
desarrollarse. En nuestra institucin se est apostando por cuatro aspectos: (i) tratamiento en
estadios de enfermedad mnima residual; (ii) inflamacin del tejido tumoral con anlogos de
sustancias microbianas; (iii) combinacin con anticuerpos monoclonales inmunoestimulantes;
iv) uso de CD equivalentes a las clulas CD11c highCD8+ de ratn.
El futuro dir si estas aproximaciones son las acertadas.

207

5- CONCLUSIONES

209

5- Conclusiones

1. El tratamiento combinado basado en la vacunacin con clulas dendrticas de tipo 1 y la


administracin del agente quimioterpico ciclofosfamida, reduce la cantidad de
linfocitos T reguladores circulantes en pacientes con cncer avanzado, as como las
clulas endoteliales circulantes y la concentracin de clulas tumorales circulantes en
algunos pacientes.

2. Las clulas dendrticas de tipo 1 producen elevadas cantidades de interleuquina-12 y


expresan abundantes molculas de coestmulo.

3. La administracin intraganglionar de clulas dendrticas alcanza eficientemente cadenas


ganglionares profundas en aproximadamente 2/3 de los pacientes, sin embrago las dosis
administradas de forma intradrmica alcanzan en una parte minoritaria el ganglio
linftico de drenaje.

4. Los sobrenadantes de cultivo celular de lneas de carcinoma renal contienen abundante


VEGF, cuya concentracin aumenta en condiciones de hipoxia.

5. La diferenciacin a clulas dendrticas, a partir de monocitos, se ve alterada si se aade


al medio de cultivo sobrenadante condicionado de lneas de carcinoma renal o VEGF
recombinante. Como resultado se obtienen clulas dendrticas con menor actividad
estimulante en ensayos MLR y una expresin menos intensa de CD80, CD83, CD86 y
HLA-DR. El efecto inmunosupresor del VEGF se puede revertir en presencia de
Bevacizumab y Sorafenib, pero no as en presencia de Sunitinib.

6. La administracin subcutnea de clulas de lneas tumorales de cncer de colon (HT29


y CaCo2) a ratones inmunodeficientes da lugar a tumores productores de elevadas
cantidades de interleuquina-8 que se acumula en su suero. Debido a la actividad

211

quimiotctica de la interleuquina-8, las clulas dendrticas inyectadas en el tumor


quedan retenidas en el microambiente tumoral.

7. La interleuquina-8 no modifica la actividad estimuladora de las clulas dendrticas para


los linfocitos T.

8. En ratones IL2R-/-Rag-/- con poblaciones de linfocitos T humanos y clulas dendrticas


alognicas en su cavidad peritoneal, la inyeccin intravenosa del anticuerpo anti-CTLA4 Tremelimumab incrementa intensamente la proliferacin linfocitaria tan slo cuando
los linfocitos T reguladores han sido previamente eliminados.

9. La eliminacin previa de linfocitos T reguladores combinada a la incubacin en


presencia de Tremelimumab, incrementa la respuesta inmunitaria especfica de
linfocitos T cocultivados con linfocitos B autlogos transformados por el virus de
Epstein-Barr.

10. Las clulas dendrticas internalizan leucocitos polimorfonucleares.

11. Las clulas dendrticas y los neutrfilos pueden ser quimiotcticamente coatrados por
las clulas tumorales de un modo dependiente de interleuquina-8.

12. Los leucocitos polimorfonucleares que internalizan bacterias o detritus de clulas


tumorales, son capaces de transferirlos a las clulas dendrticas que los han fagocitado y
les trasfieren tanto seales madurativas como antgenos reconocibles por los linfocitos
T.

212

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7.- LISTA DE ABREVIATURAS________________________

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7.1- Lista de abreviaturas


ADN: cido Desoxirribonucleico
ADNc: ADN complementario
ARN: cido Ribonucleico
ARNm: ARN mensajero
BAFF: B-cell Activating Factor
BDCA: Blood Dendritic Cell Antigen
bEGF: Factor de Crecimiento Fibroblstico bsico
CCR: Carcinoma Renal de Clulas Claras
CEA: Antigeno Carcinoembrionario
CEACAM1: Carcinoembrionic Antigen-Related Cell Adhesion Molecule 1
CD: Clula Dendrtica
CD-: Cluster of Differentiation
CMH: Complejo Mayor de Histocompatibilidad
CPA: Clula Presentadora de Antgenos
CRC: Cncer de Colon y Recto
CRTAM: Molcula Asociada a linfocitos T restringida a Clase I
CTLA-4: Antgeno Asociado al Linfocito T Citotxicos -4
DC-SIGN: Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Non-Integrin
EGF: Factor de Crecimiento Epidrmico
FDA: Food and Drug Administration
FoxP3: Forkhead box P3
GM-CSF: Factor Estimulador de colonias Granulocitos-Macrofgicos
GMP: Good Manufactuting Practice
HCV: Hepatitis Crnica C
HIF: Factor Inducible por Hipoxia
HMGB1: High Mobility Group Box1 Protein
HRE: Elementos de Respuesta a Hipoxia
ICAM: Molculas de Adhesin Intercelular
IDO: Indolamina 2,3 Dioxigenasa
Ig: Inmunoglobulina
IgV: Regin variable de las Inmunoglobulinas
IL: Interleuquina
IFN: Interfern

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LPS: Lipopolisacrido
LTC: Linfocitos T Citotxicos
LYNAP: Lymphocyte Derived Neutrophil Activating Peptide
MAC-1: Antgeno de macrfagos-1 (tambin CD11b/CD18)
MAP: Protenas Activadas por Mitgenos
MCP-1: Protena Quimiotctica de Monocitos-1
MDSD: Clulas Supresoras inmaduras Gr1+ de origen Mieloide
MGL: Macrophage Galactose-Type Lectin
MLR: Reaccin Mixta Linfocitaria
MTOC: Centro Organizador de Microtbulos
MUC: Mucina
NK: Natural Killer
NOS: xido Ntrico Sintasa
PGE2: Prostaglandina E2
PDGF: Factor de Crecimiento Derivado de las Plaquetas
PDL1: Programmed cell Death Ligand 1 (tambin B7-H1)
PGF: Factor de Crecimiento de Placenta
PI3K: Fosfatidil inositol 3 Quinasa
PKC: Protena Quinasa C
PMAP: Patrones Moleculares Asociados a Patgenos
PMN: Leucocitos Polimorfonucleares
Poly I:C: cido Poliinosnico-Policitidinico
RECIST: Response Evaluation Criteria In Solid Tumors
RMN: Resonancia Magnetica Nuclear
TCR: Receptor de Clulas T
TGF: Factor de Crecimiento Trasformante
TIL: Linfocitos Infiltrantes de Tumor
TLR: Receptores Tipo Toll
TNF: Factor de Necrosis Tumoral
VEGF: Factor de Crecimiento Endotelio Vascular
VHL: Von Hippel-Lindau
VIH: Virus de la Inmunodeficiencia Humana

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