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1.

Introduction
Central tolerance

Peripheral
tolerance

(thymic epithelial cells


and
hematopoietic myeloid
cells)

liminate self-reactive T cells


Lymphocytes with low affinity to self antigens

Foxp3+ Tregs
Clonal anergy or clonal deletion

Plays an essential role in the


development and function of D4+CD25+
Regulatory T cells.
FOXP3 act as a transcriptional
repressor of cytokine promotors.

I
P
E
X

The function of Foxp3 is highly


conserved among mammal
counter- species.

one mechanism
regulating
recessive
tolerance
is
costimulation.

OX40 and CD30


Disease
pathogenesis
Autoimmune
conditions

mmunosuppressive therapy

one marrow transplantation

Foxp3
mutant
scurfy
mice

For studying novel


immunosuppressive
therapies
with
relevance to the
human
IPEX
syndrome and other
autoimmune

These signaling pathways include


upregulating genes that control T cell
division and survival, and promoting
transcription of cytokine genes, as
well as expression of cytokine
receptors.

OX40

OX40 activates
PI3k/PKB, NFB1, and NFAT
pathways.

central memory cells (Tcm)


effector memory cells (Tem)

Suppressive events

CD30/TNF
RSF8

Is a cell membrane protein


TNF-R family
Expressed by activated T and B cells.
Interact with TRAF2 and TRAF5 (NFkappa-B)
Play a role in the regulation of cellular

2. Material and methods


B6.CgFoxp3sf/J (sf)
sf Thy1.1 mice
Rag1/ mice
Foxp3DTR mice

X-linked recessive inheritance


Lethality at 21-25 days
Wasting syndrome
Exfoliative dermatitis
Small, thickened ears

Wild type
C57BL/6

Wild type

Scurfy

Scurfy

B6.Cg-Foxp3sf/J (sf)

Severe autoimmune disorder


Hepatosplenomegaly
Enlarged lymph nodes
Multi-organ lymphocytic infiltrates
Elevated cytokines
(GM-CSF, IL-2, -4, -5,-6,-7,-10, IFN-, TNF)

2.2. Rescue and therapy


experiments

Anti-CD4
antibody
[YTS177.9].
Reported to block
MHC class II T cell
responses in vivo
and in vitro and
induces tolerance.

Express a
simian
diphtheria
toxin. DT
administration
results in
ablation of
Foxp3+CD4+T
reg cells.

2.3. Adoptive T cell transfer

strain

hymocytes are CD8-CD4LB IgM or IgD -/-

mutation produce no
mature T cells or B cells
have no CD3+or T cell
receptor (TCR)

T lymphocyte
specific Thy1a

costimulatory
signaling
through
CD28 in mouse T cells
(as a substitute
activating signal for

2.4. Flow cytometry


Labeled:
ethidium
monoazide
photolysis
propidium
iodide
DAPI.
CD3
CD90
CD8

CD30
Foxp3
CD44
OX40

CD4
CD62L Antibodies
CD69

Fc receptors were
blocked using
CD16/32

2.5. qPCR

On
negatively
isolated

Tnfrsf4
5-gcttggagttgactgtgttcc-3
5- gggtctgctttccagataagg-3
Tnfrsf8
5-tggagaggaggttgtcaagtc-3
5-gaggaaggcagctcacagat-3

2.6. Histological analysis and


scoring

The severity of
autoimmunity:
Inflammatory
cell
Infiltration
Necrosis

Analysed
using the
program
AutMess

Liver
Lung
Pancraetic
Skin
Sirius Red staining

collagen
fibres in
liver
sections

2.7. ELISA

antimouse IgG

OptEIA TMB

AMA titers
antimitochondrial
antibodies
Optical density
450 nm vs 570
nm.

Autoantibodie
90%
of patients
s
with
autoimmune
primary
cirrhosis

biliary
develop

2.8. Cytometric bead assay


(CBA)

Mouse Th1/Th2 Multiplex Kit


BMS FlowCytomix Software

Serum levels of:


IL-5
IFN-

Fig. 1. Anti-CD4 treatment rescues sf


mice devoid of functional Foxp3+
Tregs.

Developed
typical
autoimmune
disease
(exfoliative dermatitis of
the tail, ears, growth
retardation and lethargy)
2- 3 w

Rescue at 21 days
of age
Lacked
signs
autoimmune desease.

To examine whether anti-CD4 treatment also reduced the autoimmune


damage of these organs,

Inflammation scores
of liver, lung and
pancreas were all
clearly reduced in
YTS177.9-treated

A single injection of anti-CD4 inhibits systemic autoimmunity independent of

Fig. 2. CD4 blockade tolerizes autoreactive sf


CD4+ T cells. Isotype- and YTS177.9- treated
mice were sacrificed at 8-9 days of age.

For explored if
anti-CD4induced
tolerance on a
cellular level.

polyclonal
CD4 T cell
activation
(62L/69)

Analyzed AMA as an
indirect activity of
autoantigen-specifc CD4
T cells.

PDC-E2-specifc CD4 T cells


were tolerized by the nondepleting
anti-CD4

Sf

IFN- and IL-5

Tx

IFN- and IL-5

CD44+CD62Llow T cells

Foxp3DTR
DT 0 day
Isotype 0,28 d

Peripheral blood

Fig. 3. Anti-CD4 treatment is required concurrent to


regulatory T cell depletion in order to prevent
autoreactive T cell activation and autoimmunity.

anti-CD4 antibody
28
d
anti-CD4
antibody on
day 0.
Efficient depletion of
Foxp3 Tregs in all DTtreated

Reduced CD4 T cell


activation
7
days
after the first DT

augmented the
Survival
no inhibition of T cell
activation

median life expectancy of foxp3DTR


mice in contrast to Prior treatment
and isotype treatment

anti-CD4 antibody can only


induce
tolerance
if
autoreactive
T
cells
are
stimulated simultaneously

Fig. 4. YTS177.9 directly tolerizes


scurfy CD4+ T cells in a recessive and
non-infectious fashion
The

adoptive
transfer of sf
CD4 T cells into
Rag1-/- mice is
sufficient
to
transfer
fulminant
autoimmunity

The diseasefree survival


improved

investigated the
characteristics
of
anti-CD4induced Foxp3independent
tolerance
in
detail.

RAG -/sf(YTS177.9)

To distinguish
between recessive
T
cell-intrinsic
tolerization
and
classical dominant /
infectious tolerance

sf(Iso)

develope
d disease

Isotype / sf CD4 T
YTS177.9 / CD4 T
cells
cells
+
+
untx
sf
Th1.1 CD4
untx sf Th1.1 CD4

Mixtures of sf
CD4 T cells were
analyzed prior to
injection

Thy1.1 vs.
CD4
expression
of
CD4+CD3+ T
cells

55 days after
mixed
T cell transfer

the frequencies
of sf CD4 T cells
among
the
mixture had not
changed
sf(YTS177.9)
have no survivalor
proliferation
defect compared
to sf(Iso)

Cx.
YTS177.9induced tolerance is
transient, T cellintrinsic and non
infectious
in
the
absence
of
Foxp3

Fig. 5. Anti-CD4 therapy ameliorates


established autoimmunity in sf mice

anti-CD4 treatment could


1ameliorate
?
pre-established
disease

autoimmune

every 4 days

TX. were more mobile


but they had to be
sacrificed
due
to
progressing
skin
inflammation (ears)

REST was not altered by anti-CD4 therapy

liver
pathology
was clearly
reduced
following
anti-CD4
therapy

serum

(AMA)
associated
with
autoimmune liver diseases
were reduced.

Fig. 6. Reduced liver damage in sf


mice following YTS177.9 therapy.

Chronic liver
inflammation
resulting
in
fibrosis
and
eventually
liver
cirrhosis

Evaluated

collagen
deposition in liver
sections from sf
mice

CX.
anti-CD4
therapy not only
reduced
inflammation but
also
secondary fibrosis
and
tissue

fbrosis

reduced Sirius
Red staining

Tx

OX40was
strongly
expressed
3-4
weeks
old

CD4 T cells

splenic CD4 T cells

Fig. 7. CD4 blockade impairs CD30


and OX40 expression by scurfy CD4+ T
cells.
No Tx

Hypothesized:
1
anti-CD4
could
attenuate
signals
from one or both
receptors.

anti-CD4
treatment reduces
OX40 and CD30
expression in vivo

expression

Sf YTS177.9 of both OX40and CD30


mRNA

YTS177.9 reduced OX40 protein


expression on CD4 T cells when compared
to isotype-treated sf mice

Cx. anti-CD4-mediated tolerance


acts in part via dampening
pathogenic OX40 and CD30
signals

Anti-CD4 antibodies can inhibit autoimmunity in mice


completely devoid of functional Foxp3+ Tregs (therapy could
not control all manifestations of autoimmunity).
investigate whether the combination of anti-CD4 therapy with
additional immunosuppressive therapies can result in enhanced
control of autoimmunity.
Represent a novel therapeutic option for IPEX patients or to