Potential of Nasopharynx-associated Lymphoid Tissue

for Vaccine Responses in the Airways

Per Brandtzaeg1,2
1

Laboratory for Immunohistochemistry and Immunopathology, Centre for Immune Regulation, University of Oslo, Olso, Norway; and 2Department
of Pathology, Oslo University Hospital, Rikshospitalet, Oslo, Norway

Nasopharynx-associated lymphoid tissue (NALT), constituting Waldeyers ring in humans, is a unique inductive site for B-cell responses
and plasma cell generation. This makes the nasal route of vaccine
administration interesting for induction of mucosal and systemic
antibodies. The unpaired nasopharyngeal tonsil (adenoids) and the
paired palatine tonsils are prominent NALT structures, functionally
similar to the paired rodent NALT structures located dorsal to the
cartilaginous soft palate. Human NALT is more strategically located,
however, because its elements are exposed to both airborne and
alimentary antigens and have antigen-retaining crypts. It also shows
similarities with lymph nodes and participates both in systemic- and
secretory-type mucosal immunity. Primary follicles occur at 16 weeks
of gestation, which is similar to Peyers patches but different from
rodent NALT whose organogenesis begins at birth. The formation
of germinal centers reflecting B-cell activation does not take place
until shortly after birth, and terminal differentiation of plasma cell
can be seen about 2 weeks postnatally. Germinal centers arise in T
celldependent B-cell responses and are associated with somatic
hypermutation of Ig V-region genes. Downstream switching to various Ig isotypes also takes place, with or without concurrent expression of the J-chain gene. The J chain is a crucial part of dimeric IgA and
pentameric IgM, making these Ig polymers able to interact with the
epithelial polymeric Ig receptor. This interaction is central in the
formation of secretory IgA and secretory IgM. Accumulating evidence suggests a major role for NALT in antibody immunity of the
respiratory tract and associated glands.
Keywords: mucosa-associated lymphoid tissue; B-cell homing; mucosal immunity; secretory IgA; IgD

The unpaired nasopharyngeal tonsil (also called the adenoids)

and the paired palatine and lingual tonsils constitute the major
part of Waldeyers ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components (1). These organs seem
to be functionally comparable with the nasopharynx-associated
lymphoid tissue (NALT) in rodents, which is composed of two
paired lymphoepithelial structures besides the nasopharyngeal
duct, dorsal to the cartilaginous soft palate (2, 3). Of note,
rodents do not have tonsils, and Waldeyers ring is more strategically situated than their NALT to generate mucosal immunity
because its elements are exposed to both airborne and alimentary antigens. Human NALT may therefore play an important
immune-inductive role as part of mucosa-associated lymphoid
tissue (MALT). It also shows similarities with lymph nodes and
may, in addition, contribute as an effector site of local systemictype and mucosal secretory-type of adaptive humoral immunity.
Tonsils contain four specialized tissue compartments contributing to immune functions (4, 5): (1) the reticular crypt

(Received in original form November 4, 2010; accepted in final form March 18, 2011)
Correspondence and requests for reprints should be addressed to Per Brandtzaeg,
D.D.S., M.Sc., Ph.D., Department of Pathology, Rikshospitalet, P.O. Box 4950
Nydalen, N-0424 Oslo, Norway. E-mail: per.brandtzaeg@medisin.uio.no
Am J Respir Crit Care Med Vol 183. pp 15951604, 2011
Originally Published in Press as DOI: 10.1164/rccm.201011-1783OC on March 18, 2011
Internet address: www.atsjournals.org

epithelium, (2) the extrafollicular area, (3) the mantle zones of

lymphoid follicles, and (4) the follicular germinal centers (GCs).
Primary follicles are present in human tonsils as early as 16 weeks
of gestation (4), which is similar to Peyers patches of gutassociated lymphoid tissue (GALT) but different from rodent
NALT, whose organogenesis begins at birth (3). Nevertheless,
the formation of tonsillar GCs that reflects B-cell activation induced by exogenous antigens does not take place until shortly
after birth; and terminal differentiation of effector B cells to
extrafollicular plasma cells (PCs) can first be seen approximately
2 weeks postnatally (4).
The GCs characteristically arise in T celldependent B-cell
responses and are associated with (1) clonal expansion of B cells;
(2) somatic hypermutation in B-cell immunoglobulin (Ig) variable
region genes (Ig V-region genes); (3) positive selection of B cells
that are able to receive antigen-specific signals by high affinity; (4)
downstream switching of Ig heavy chain constant region genes (CH
genes) to various isotypes; (5) differentiation to memory and effector B cells and PCs of the expressed Ig isotypes; and (6) concurrent
induction of the J-chain gene in a variable subset of these cells. This
gene encodes a 15-kD peptide, the joining (J) chain, which is a crucial structural part of dimers and trimers of IgA (collectively called
polymeric IgA [pIgA]) and pentameric IgM (6). Without the incorporation of J chain, pIgs cannot bind to the epithelial pIg receptor (pIgR), also called membrane secretory component (SC)
(7). The pIg2pIgR interaction is a central step in the formation
and export of secretory IgA (SIgA) and secretory IgM (SIgM)
antibodies to mucosal surfaces (8).
This article discusses the putative importance of human NALT
for the immune function of the upper airways. Accumulating evidence suggests a role of Waldeyers ring in humoral immunity of the
regional mucosae and associated glands; some of the NALT-derived
and activated B cells seem to seed preferentially these secretory effector sites (9, 10). Therefore, administration of vaccines via the
nasal route has an interesting potential of enhancing regional immunity and does at the same time stimulate systemic immunity.

NAIVE B CELLS ENTER TONSILS VIA HIGH

ENDOTHELIAL VENULES
Migration of lymphoid cells is strictly regulated by the expression
of multiple adhesion molecules and receptors for chemoattractants (chemokine receptors) that interact with corresponding
ligands on endothelial and stromal cells (11). The extravasation
of mainly naive, or antigen-unexperienced, T and B cells into
inductive lymphoid compartments takes place through specialized postcapillary so-called high endothelial venules (HEVs)
and is regulated by similar molecular principles in the systemic
and the mucosal immune system (Figure 1).
The naive lymphocytes express CD62L (L-selectin) that interacts with the adhesion molecule peripheral lymph node addressin
expressed by HEVs in both lymph nodes and tonsils (12). In
GALT, however, peripheral lymph node addressin is replaced
by a CD62L-binding adhesion molecule named mucosal addressin
cell adhesion molecule-1 (6, 9). The chemokines involved at the
level of HEVs are secondary lymphoid tissue chemokine (CCL21)

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Figure 1. Main adhesion molecule and

chemokine-regulated steps of T- and B-cell migration to, and positioning within, mucosaassociated lymphoid tissue (MALT). Naive T and
B cells enter both MALT and regional lymph
nodes via high endothelial venules (HEVs). The
chemokines involved (right panel) are secondary
lymphoid tissue chemokine (SLC, CCL21) and
Epstein-Barr virusinduced molecule 1 ligand
chemokine (ELC, CCL19), both produced by
stromal cells and redistributed to the luminal
face of HEVs, as indicated, to attract preferentially CCR71 naive T cells and, probably less actively, B cells. SLC may also be involved in the
exit of lymphoid cells from MALT via draining
lymphatics as depicted. Naive B cells are
CXCR51 and, at least in mice, extravasate
mainly via modified HEVs presenting CXCL13
(also called B-cell attracting chemokine [BCA]1 in humans) juxtaposed to, or inside of, lymphoid follicles; they are next attracted to the
mantle zone where BCA-1 is deposited on dendritic elements, such as the follicular-dendritic
cell (FDC) tips. The distribution of this chemokine, together with B and T cells, is shown by three-color immunofluorescence staining in the upper
left panel, and the receptor distribution is similarly shown in the lower left panel. Also, follicular B-helper T (TFH) cells (CXCR51) are attracted to the
follicle by similar interactions (e.g., white arrow, lower left panel ). B cells are primed just outside the lymphoid follicle by interaction with cognate
T cells in clusters with antigen-presenting cells, and the B cells may mutually act on the T cells, as indicated. The activated B cells then reenter the
follicle as germinal center founder cells to give rise to CCR71 B cells after interactions with FDCs and TFH cells (Figure 2). This temporal upregulation of CCR7 allows the further activated B cells to leave the follicle as memory and effector cells by attraction to extrafollicular SLC and ELC.
Immunofluorescence pictures adapted from Carlsen and coworkers (13). HLA-II HLA class II molecules; TCR T-cell receptor.

and Epstein-Barr virus (EBV)induced molecule 1 ligand chemokine (CCL19). We have observed an important species difference
in that both CCL21 and CCL19 are produced by stromal cells in
humans and become redistributed to the luminal face of HEVs,
whereas CCL21 is actually produced by the HEV endothelium itself
only in mice (12). Both these chemokines attract preferentially
CCR71 naive T cells, and apparently less actively naive B cells (9).
Naive B cells are CXCR51 and extravasate, according to
mouse experiments, mainly via modified HEVs presenting
CXCL13 (in humans also called B-cell attracting chemokine-1).
These HEV-like vessels occur juxtaposed to, or inside of, lymphoid follicles (9). The B cells are next attracted to the mantle
zones where CXCL13 is deposited on stromal or dendritic elements, such as the tips of the follicular dendritic cells (FDCs)
(13), an expression pattern that we have found both in human
GALT and tonsils (Figure 1). The origin of CXCL13 in GCs
remains unclear but seems to be mainly macrophage-like
dendritic cells (DCs) (14). Also, follicular B-helper T (TFH) cells
are attracted to the follicles by similar receptor2ligand interactions (Figure 1). The B cells are initially primed just outside the
lymphoid follicle by interaction with cognate T cells and antigenpresenting cells, mainly interdigitating DCs (9); they then reenter
the follicle and end up as CCR71 GC cells after interactions
with FDCs and TFH cells (see below). Primed and selected B cells
are able to leave the follicle as memory and effector cells because
of their CXCR5 down-regulation and temporal CCR7 upregulation, allowing their attraction to the corresponding extrafollicular chemokines (9).

B CELLS ARE ACTIVATED IN GCs TO BECOME MEMORY

AND EFFECTOR CELLS
Primary follicles of secondary lymphoid organs, such as the tonsils, consist mainly of recirculating naive B cells positive for surface IgD and IgM (sIgD1sIgM1), both coexpressed isotypes

exhibiting the same specificity for antigen. These CXCR51 lymphocytes pass into the spaces of the network formed by the
CXCL13-bearing FDCs (Figure 1). It is still unclear why both
sIgD and sIgM must be expressed to render B cells antigenreactive. Likewise, the nature and origin of FDCs are obscure,
but the existence of these cells and their accumulation in the
primary follicles depends on the presence of B cells. Thus, animals depleted of B cells, or mice with severe combined immunodeficiency do not have follicular aggregates of FDCs (15).
In contrast to lymph nodes, tonsils are not encapsulated and
lack afferent lymph, but the reticular crypt epithelium contains
many DCs that can transport exogenous antigens to the extrafollicular T-cell areas and to the B-cell follicles. Interdigitating DCs
function as antigen-presenting cells in the extrafollicular primary
immune responses and occur abundantly around HEVs, often in
clusters with T cells that are mainly of the CD41 phenotype (4).
These lymphocytes consist of both naive (CD45RA1) and memory
(CD45R01) subsets, and some express the high-affinity IL-2 receptor (CD25) as a sign of recent activation (16). Altogether, the tonsils
seem able to mount both primary and secondary T-cell responses.
The GC reaction is initiated by stimulation of naive B cells immediately outside the lymphoid follicles through cognate help from
activated CD41 T cells (Figure 1). These helper T cells have received processed foreign antigen from interdigitating DCs in the
context of class II molecules of the major histocompatibility complex, in humans also called HLA class II, such as the classical
HLA-DR, -DQ and DP molecules. The activated B cells can
pick up antigen and further present it to the cognate T cells in
an interaction that provides mutual support (Figure 1). Some of
these B cells then colonize the follicles and act as founder cells
for GCs (Figure 2).
Secondary follicles with GCs result from further stimulation
of B cells, first via their interactions with FDCs on which native
antigens are retained in the form of immune complexes. By this
activation the founder B cells are induced to proliferate (Figure 2).

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Figure 2. Immune events taking place in the

dark and light zones of germinal center in tonsillar secondary lymphoid follicle. The germinal
center founder cells receive their initial stimulation through cognate interaction with activated CD41 helper T cells just outside the
lymphoid follicle before they enter it to become
centroblasts (Figure 1). Further details are given
in the text. CD40 ligand (CD40L)/CD40 costimulatory molecules; FDC follicular dendritic
cell; GCDC germinal center dendritic cell;
HLA-II HLA class II molecule; IC immune
complex; sIg surface immunoglobulin; TCR
T-cell receptor; TFH follicular B-helper T cell.

A variety of adhesion molecules and other receptor proteins are

involved in these interactions (16).
The GCs can be divided into various more or less well-defined
morphologic compartments (15). B-cell stimulation in the GC
dark zone gives rise to exponential growth recognized by the
monoclonal antibody Ki-67 (Figure 2) directed against a nuclear
proliferation marker (16). The resulting centroblasts hypermutate
their Ig V-region genes and give rise to centrocytes; these cells die
by apoptosis in the light zone unless they are rescued selectively
by their ability to bind with high affinity to antigens present on
FDCs, take up such antigens, and process and present them via
HLA class II molecules to TFH cells, which can be identified by
their high level of CXCR5 expression (Figure 1).
The point in ontogeny when CD41 helper T cells become
TFH cells, and whether these cells represent a separate T-cell
lineage, remains unsolved. Cell-surface markers and transcription
factors are currently under investigation (1719). Tonsillar TFH
cells have been claimed to express CD57 in addition to CXCR5,
but this is apparently not always the case. The best additional
markers seem to be inducible T cell costimulator (CD278),
secretion of the immune activator IL-21, and expression of
the costimulatory CD40 ligand (CD154). Cognate interaction
between TFH cells and activated B cells, which express CD40,
seems to be a very important event in the GC reaction (Figure
2). The same is true for the induction of bcl-2 gene products
after immune activation of centrocytes to prevent their apoptosis, a rescuing event largely driven by IL-21 (19).
Although several costimulatory molecules are involved in the
synapse between TFH cells and HLA class IIexpressing B cells
(18, 19), GCs are not formed if the CD40 ligandCD40 interaction is experimentally blocked (20). Importantly, this costimulatory interaction also promotes switching of the Ig CH genes
of B cells from Cm (IgM) to downstream isotypes, and differentiation to plasmablasts and PCs producing high-affinity antibody. A prerequisite for the crucial consequences of cognate
interaction between B and TFH cells, in which activated GC
B cells present processed antigen to the T cells (Figure 2), is
that the B cells express costimulatory B7 (CD80/CD86) molecules, which can bind to CD28 on the TFH cells. A recent experimental study showed that CD28-dependent signaling is
required for optimal TFH cell maturation and expansion (21).
Classical memory B cells (sIgD2sIgM1) with strong B7 expression are moreover found extrafollicularly in human tonsils

related to the crypt epithelium where they may likewise present

antigen to CD281 T cells (22).

VARYING ISOTYPE AND J-CHAIN EXPRESSION

BY TONSILLAR B CELLS
The tonsillar GC reaction normally generates a variable number
of intrafollicular Ig-producing plasmablasts or PCs dominated by
the IgG (5572%) and IgA (1318%) classes (4, 5). A large proportion of these two phenotypes shows concurrent J-chain expression (2643%), and even more so do those GC cells that
produce IgM or IgD (4982%) in healthy palatine tonsils of children (Figure 3A). Thus, the J chaininducing capacity of these
organs is much higher than that of peripheral lymph nodes but
similar to that of mesenteric lymph nodes. The latter are functionally amplification organs for Peyers patches where the concurrent induction of IgA and J chain is remarkably high (6, 9, 23).
The cytokine profiles and other microenvironmental factors
determining isotype differentiation and coexpression of J chain
in B cells remain obscure (9, 23). According to the decreasing
potential hypothesis (24) it seems that clonal maturation in
the course of several proliferative GC cycles results in downregulation of J-chain expression, thus promoting monomer
production by the resulting IgA1 PCs rather than pIgA for
secretory immunity (9). This idea fits with the fact that the
J-chain expression was significantly suppressed both in intrafollicular and extrafollicular IgA1 cells in tonsils of children aged
212 years with recurrent tonsillitis (Figure 3A). These tonsils
were removed because of at least three annual attacks of acute
tonsillitis during the past 2 years (25). Our studies were performed in 1980 and we were fortunate to obtain an age- and
sex-matched unique biopsy control material from a group of
children undergoing surgery for inguinal hernias. None of them
had had any episode of acute tonsillitis, increased frequency of
common colds, or acute otitis media, and their serum levels of
immunoglobulins were normal. Informed consent to perform
biopsy was obtained from their parents (26).
Thus, whereas downstream CH gene switching in GCs of healthy
tonsils gives rise to a relatively high fraction of extrafollicular
IgA1J-chain1 PCs (50% of total IgA1 PCs), most extrafollicular IgG1 PCs show little or no J-chain expression (Figure 3A).
The J chain can only bind to the heavy chains of IgA and IgM,
so in IgG1 and IgD1 PCs it has no apparent function and is
broken down in the cytoplasm (9). Notably, the phenotypic Ig

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Figure 3. Putative B-cell developmental stages in germinal center of

palatine tonsils and adenoids. The pathways from surface Ig (sIg)
expressing B lymphocytes to terminal plasma-cell differentiation may
include coexpression of cytoplasmic J chain (J) and any of the four Ig
classes depicted, but the J chain can only combine with cytoplasmic IgA
and IgM to form polymers (IgA1J, dimeric IgA; IgM1J, pentameric
IgM). (A) Plasmablasts and plasma cells in germinal center (GC) and
extrafollicular compartment near crypts show cytoplasmic coexpression of J chain in the proportions (%) indicated. Tissue samples were
from children with clinically healthy or diseased (recurrent tonsillitis)
palatine tonsils. An extrafollicular area immunostained for IgA and J
chain is illustrated to the right (see color key) to show double expression
(IgA 1 J chain, yellow fluorescence; arrow). Data adapted from Korsrud
and Brandtzaeg (25, 26). (B) The J chainexpressing B cells terminate to
some extent their differentiation locally in the extrafollicular compartment (small inclined arrows at right) but may preferentially show a potential for homing to distant secretory effector sites, particularly in the
region of the upper respiratory and digestive tracts. Effector B cells with
little or no J-chain expression become extrafollicular plasma cells (or
may join the systemic immune compartment; not indicated).

class distribution was similar in healthy and diseased tonsils

(26), so the only striking tonsillitis-associated change in the local
B-cell system was suppressed J-chain expression in IgA1 PCs
(Figure 3A). There was a similar but nonsignificant trend in
hypertrophic adenoids removed because of obstructive symptoms in a group of age-matched children (26).
The fact that tonsillar IgA1 PCs are mainly of the IgA1 isotype (at least .95%), along with the presence of IgD1 PCs,
supports the notion that tonsillar B-cell differentiation takes
place mainly with classical downstream CH gene switching together with some nonclassical switching to IgD (5, 6, 10, 23).
A regionalized microbial impact on B-cell differentiation is suggested by the unique sIgD1IgMCD381 centroblast subset identified
in the dark zone of tonsillar GCs (27). This subset shows

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deletion of the Sm and Cm gene segments, therefore selectively

giving rise to IgD-producing PCs by the so-called nonclassical
switching. Compartmentalized B-cell dispersion explains the
relatively high frequency of IgD-producing PCs normally occurring in this region (10), and particularly the striking PC replacement with this class that often is seen in IgA deficiency (23, 28).
Most strains of Hemophilus influenzae and Moraxella catarrhalis, which are frequent colonizers of the nasopharynx, express
outer-membrane IgD-binding factors that can activate sIgD1
B cells by crosslinking sIgD of the B-cell receptor (29). In this
manner it seems likely that sIgD1 tonsillar centrocytes are stimulated to proliferate and differentiate polyclonally, thereby
driving V-gene hypermutation and Sm/Cm gene deletion (30).
Such regional microbial influence on B-cell differentiation is
supported by our observation that Sm/Cm deletion is more
frequently detected in diseased than in clinically healthy tonsils and adenoids (10); and extrafollicular IgD-producing PCs
are, as mentioned previously, relatively numerous in recurrent
tonsillitis and adenoid hyperplasia (4, 5). However, there are
large individual variations, and the IgD class percentage reported
in some publications (up to 2025%) is much too high (3032).
We found the mean proportion of IgD-producing PCs to be
well below 5% of all isotypes in the extrafollicular compartment
(4).
It is interesting that sIgD1IgM2 B cells generated by nonclassical switching seem to express predominantly V-gene repertoires that may allow considerable crossreactivity, including
autoimmunity, but understanding the biologic significance of this
observation requires further studies (33). Although numerous
antimicrobial and other IgD antibody activities have been measured both in mouse and human serum, the protective or pathogenic role of circulating IgD has only recently been explored (34).
Because IgD does not activate the classical complement pathway, it is likely that such antibodies can block defense functions
of IgG and IgM within the mucosae and reduce the immuneexclusion efficiency of SIgA and SIgM antibodies in the upper
airways in the face of bacterial infections that drive local IgD
production (28). The ability of IgD to bind to monocytes and
macrophages and basofils (its cross-linking thereby inducing release of proinflammatory cytokines, such as IL-1, IL-6, IL-8, and
tumor necrosis factor-a) may add to the pathogenic potential of
IgD antibodies (32, 35). Of note, selective IgA-deficient patients
showing substantial replacement with only IgM- and IgG-producing
PCs in their nasal mucosa have less clinical problems in their
airways than those with abundant mucosal IgD-producing PCs
(36).

DISSEMINATION OF ACTIVATED TONSILLAR B CELLS

TO SECRETORY TISSUE SITES
Because the J chain is a key peptide in the formation of pIgA and
pentameric IgM that can bind to the pIgR expressed basolaterally on secretory epithelia (8), tonsillar B-cell differentiation
exhibits features compatible with precursor generation for the
secretory immune system. It may be envisioned that only a fraction of the pIgA-expressing plasmablasts that exit from GCs will
terminate their differentiation as extrafollicular PCs (5, 6); instead, many of them may home to regional secretory effector
sites for terminal differentiation to pIgA-producing PCs there
(Figure 3B). The adenoids possess, in addition, a local secretory
immune system because patches of the crypt epithelium express
pIgR/SC (4, 5). This is not the case in the palatine tonsils where
only passive paracellular transfer of IgG and IgA takes place
through the reticular crypt epithelium. Because this epithelium
is of squamous type in the palatine tonsils, it is additionally
protected by its production of the antimicrobial peptide called

Pulmonary Perspective

calprotectin or L1 protein (4, 5). Therefore, topical surface protection, including antibody-mediated immunity in the crypts,
differs between the two lymphoid organs.
Nasal and bronchial mucosae, and salivary and lacrimal glands,
contain an IgA11 and IgD1 PC distribution in their stroma similar
to that of the extrafollicular tonsillar compartment (9). This fact
supports the notion that such regional secretory tissues are seeded
by GC-derived B-cell blasts from Waldeyers ring (5, 9, 10, 23). Its
immune-inductive function would hence be similar to that of rodent NALT (37). Regionalization of MALT also concerns the gut
because the intestinal lamina propria receives most of its activated
B cells from Peyers patches and other GALT structures, such as
the numerous solitary or isolated lymphoid follicles (6, 9, 23). A
possible minor blast contribution to the airways from bronchusassociated lymphoid tissue remains possible. Although isolated
lymphoid follicles do not regularly exist in the normal adult human lung (23, 38), such bronchus-associated lymphoid tissue structures were found in all of nine large bronchial autopsy specimens
of children aged 215 years who died from traumatic causes and
had no airway disease (39). Likewise, in large autopsy specimens
of nasal mucosa occasional scattered isolated follicles were seen in
38% of children below 2 years of age (40). These follicles were
most likely induced postnatally like rodent NALT (3).
Dichotomy of the mucosal immune system between the gut
and the airways has also been suggested by B-cell homing studies
in the rat (41, 42). Furthermore, activated human tonsillar B cells
were found to migrate to the lung, but not to the gut mucosa,
when transferred to mice with severe combined immunodeficiency (43). Notably, direct immunization of the palatine tonsils,
and for nasopharyngeal tonsils particularly vaccination by the
nasal route, gave rise to local B-cell responses but the induced
circulating specific B cells did not enter the small intestinal mucosa (44). Moreover, in infants dying of sudden infant death
syndrome, the palatine GCs were shown to be overactivated as
revealed by an increased number of IgG1 and IgA1 plasmablasts
and PCs (45). These activated B cells apparently seeded regional
secretory effector sites, such as the parotid glands, in excessive
numbers to become local PCs (46). It has also been documented
in several human studies that nasal immunization induces specific
IgA antibodies in nasopharyngeal secretions, in addition to enhancing systemic immunity (6, 9, 47, 48).
Attempts have been made to track directly the dissemination of
B-cell blasts from Waldeyers ring by means of molecular markers.
Because the human herpesvirus EBV preferentially establishes
persistent infection of tonsillar memory and effector B cells, their
migration to other organs has been mapped by DNA analysis for
latent EBV infection (49). On the basis of this approach, B-cell
trafficking was suggested to take place from Waldeyers ring to
peripheral blood and to a lesser extent into systemic lymphoid
organs, such as mesenteric lymph nodes and the spleen (49). Using another DNA marker, namely a deletion of the IgM CH gene,
we confirmed and extended these results by showing that activated tonsillar B cells undergoing so-called nonclassical switching to IgD1J-chain1 plasmablasts by means of this deletion,
preferentially home through cervical lymph nodes to the upper
airways and associated glands (9, 10, 23).
The extravasation of activated memory and effector B and
T cells into effector tissues takes place through the local microvascular endothelium and is controlled in a site-specific manner. This
process is much better defined for the intestinal lamina propria
than for other secretory tissues (9, 11, 23). Thus, the B-cell homing
dichotomy between the gut and the upper aerodigestive tract
alluded to previously clearly has a molecular basis in terms of
adhesion molecules and chemokines (Figure 4). Endothelial mucosal addressin cell adhesion molecule-1 is expressed by the microvasculature throughout the gut lamina propria, where it binds

1599

GALT-derived memory and effector T- and B-cell blasts with

high surface levels of the integrin a4b7 (9, 11, 23).
Homing to the small intestinal lamina propria is, in addition, determined by the chemokine receptor CCR9, which interacts with its
ligand TECK (CCL25) produced preferentially by the epithelium
in that part of the gut. Conversely, homing to the colonic lamina
propria is fine-tuned by CCR10 interacting with MEC (CCL25).
The latter molecular pair is apparently also important for homing
of tonsillar B-cell blasts to the upper respiratory tract (10, 50).
However, these cells show poor gut-homing properties because
of low levels of surface a4b7 but express, instead, CD62L (Figure
4). This adhesion molecule enables such NALT-derived cells to
enter not only regional secretory tissues related to the airways but
also peripheral lymph nodes and to some extent the uterine cervix
mucosa (9, 10). Integration of mucosal immunity induced in Waldeyers ring with systemic immunity is further enhanced by the
expression of CCR7 on tonsillar B-cell blasts (9, 10).

EFFECT OF ADENOTONSILLECTOMY ON
REGIONAL IMMUNITY
In the United States, approximately 400,000 surgeries annually
can be ascribed to removal of palatine tonsils or adenoids. The
indication for such operations has shifted from infection to upper
airways obstruction. The frequency of tonsillectomy has therefore
declined significantly and progressively since the 1970s. This development can probably also be ascribed to the increasing awareness of the immune function of Waldeyers ring. The information
reviewed in the previous sections provides strong support for the
notion that this lymphoid tissue functions as inductive NALT in
humans and supplies secretory effector sites of the aerodigestive
tract with activated pIgA precursor cells (Figure 4). To evaluate
clinically this notion, it is important to study the effect of adenotonsillectomy on the regional SIgA levels. The pioneering report
by Ogra (51) showed that combined tonsillectomy and adenoidectomy in children reduced the level of IgA antibody to poliovirus threefold to fourfold in their nasopharyngeal secretions and
delayed or abrogated their local SIgA response to subsequent
live oral poliovaccine. Notably, however, this result could partially have reflected an abolished local secretory immune function
because of removal of the adenoids (5).
Jeschke and Stroder (52) performed tonsillectomy in children
and found that their serum Ig and salivary IgA levels decreased for
up to 3 years. Furthermore, although DAmelio and coworkers (53)
observed no salivary IgA reduction (but decreased serum IgA) in
previously tonsillectomized adults (1624 yr old), Cantani and coworkers (54) found in children that salivary IgA and serum IgA (and
less so IgG and IgM) were significantly reduced 4 months after
combined adenotonsillectomy. Subsequent studies in tonsillectomized children showed, however, elevated salivary Ig levels after
34 years (55), whereas no effect was found in tonsillectomized
young adults after 6 months except for a slight reduction of total
IgM and salivary IgG antibodies to Streptococcus mutans and EBV
(56).
Altogether, there is a need for more extensive immunologic
studies focusing collectively on the adenoids and palatine tonsils.
Considerable redundancy of inductive lymphoid tissue in Waldeyers ring (such as the lingual tonsils) might mask a potentially
unwanted immunologic effect of adenotonsillectomy. This possibility is supported by studies that have reported reduced SIgA
levels in saliva from children with pharyngitis involving recurrent
tonsillitis (57) or adenoid hyperplasia (58), perhaps reflecting
decreased global pIgA induction in Waldeyers ring caused by
inflammation-induced down-regulation of J-chain expression as
seen in recurrent tonsillitis (Figure 3A). It should also be noted
that the cervical lymph nodes apparently function as reserve

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Figure 4. Putative scheme for compartmentalized mucosal B-cell homing from inductive (top) to effector (bottom)
sites in humans. Depicted are more or less preferred pathways (graded arrows) presumably followed by mucosal B
cells of any isotype activated in nasopharynx-associated
lymphoid tissue (NALT) represented by Waldeyers lymphoid ring (including palatine tonsils and adenoids), and
bronchus-associated lymphoid tissue (BALT), versus gutassociated lymphoid tissue (GALT) represented by Peyers
patches, appendix, and colonic-rectal isolated lymphoid
follicles. The principal homing receptor profiles of the respective B-cell populations, and compartmentalized adhesion and chemokine cues directing their extravasation at
different effector sites, are indicated (pink and blue panels).
The gland-associated distribution of plasma cells (green),
after terminal differentiation of extravasated mucosal B
cells, is schematically depicted at the bottom.

immune-inductive organs draining the nasopharyngeal and oropharyngeal region.

DISEASE- AND AGE-RELATED CHANGES OF TONSILLAR

IMMUNE FUNCTION
Recurrent tonsillitis, and to a lesser extent adenoid hyperplasia, is
associated with decreased J-chain expression by tonsillar plasmablasts and PCs (Figure 3A); their putative contribution to the
regional SIgA system is thereby compromised (Figure 3B). Moreover, recurrent tonsillitis seems, in an irreversible manner, to speed
up the age-related involution of the tonsils as immunologic organs
in terms of reduced B-cell differentiation to PCs (4).
The underlying immunoregulatory alterations might be related
to increased shedding of antigen-transporting membrane (M) cells
in the reticular crypt epithelium, thereby also influencing the regulated balance between expansion of early (J-chain positive) and
mature (J-chain negative) effector B-cell clones (4, 9). The microenvironmental conditions necessary for tonsillar proliferation
of early B-cell clones might depend on the presence of M cells
and particular subsets of helper and regulatory T cells. Increased
shedding of M cells changes the way in which foreign material is
presented to the lymphoid tissue, and relatively more direct passage through the reticular crypt epithelium could conceivably
result in a different level or mode of antigen processing and presentation that would preferentially favor the expansion of mature
(i.e., recycled through GCs), rather than early, B-cell clones
according to the decreased potential hypothesis (24).
Even healthy tonsils are to a large extent involved in the generation of mature memory and effector B-cell clones as evidenced
by the predominance of extrafollicular J chainnegative IgG1 PCs
(Figure 3A). In older children and adults with recurrent tonsillitis,
expansion of both early and mature memory clones is apparently
decreased. This development is indicated by reduced numbers of
Ig-producing PCs in the intrafollicular and extrafollicular tonsillar
compartments (4). In addition to possible changes in helper and
regulatory T cells, an underlying mechanism could be a decreased
regulated translocation of antigens into the tonsils. The number of
active M cells is largely reduced in recurrent tonsillitis; even direct
passage of foreign material may be hampered as the reticular
epithelium becomes covered by a stratified and partly keratinized
lining. Similar changes occur in clinically normal tonsils above 25
years of age (4).
Altogether, inflammatory conditions continuing after the age of
about 10 years seem to accelerate the aging process of tonsils.

However, recurrent tonsillitis may significantly change the tonsillar

immunocompetence even before that age (Figure 3A). The observed alterations may be irreversible because they were revealed
in periods when the patients had been without inflammatory

Figure 5. Presumed immunobiology underlying induction of T (violet)

and B (green) cells after nasal vaccine administration, and local generation of secretory IgA (SIgA) antibodies by export via the polymeric Ig
receptor (pIgR). 1 delivery device for nasal vaccine administration
(nasal spray, drops, or OptiMist); 2 adjuvanted uptake of vaccine
antigen through nasal mucosa; 3 immune-induction in adenoids
and palatine tonsils (human NALT); 4 antigen targeting and migration
of mucosal dendritic cells (DCs); 5 immune induction and amplification in regional (cervical) lymph nodes by antigen-loaded DCs and macrophages (Mfs); 6 compartmentalized homing and extravasation of
NALT-induced T and B cells to secretory effector sites in airways, gut, and
uterine cervix; and 7 local production and pIgR-mediated external
transport of dimeric IgA to generate SIgA. As discussed in the text,
NALT-derived B cells preferentially extravasate at regional effector sites
and in systemic lymphoid organs, while showing only poor homing
capacity for the small intestinal lamina propria (Figure 4).

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TABLE 1. VACCINATED SUBJECTS (% OF N 1519) WITH HEMAGGLUTINATION INHIBITION

SERUM ANTIBODY TITER $ 40 BEFORE AND AFTER TWO DOSES (3 WK) OR FOUR DOSES (6 WK)
OF INACTIVATED WHOLE-VIRUS INFLUENZA VACCINE, AND SHOWING A SIGNIFICANT NASAL IgA
RESPONSE AFTER FOUR DOSES
Vaccine
Delivery

Serum:
Preimmunization

Serum: After
Two Doses

Serum: After
Four Doses

Nasal Fluid
IgA Response

32%
32%
28%
29%

89%
79%
94%
76%

100%
94%
100%
87%

P 0.0003
P 0.0006
P 0.0006
No response*

Nasal spray (OptiMist)

Nasal spray (conventional)
Nasal drops
Oral spray

Based on data from References 79 and 80.

* No increase of IgA antibody titer in nasal fluid, but a slight increase in whole saliva.

symptoms for at least 4 weeks (4, 25). Altered immune function

may contribute to the recurrence of tonsillitis, and a vicious cycle
may then develop. It is noteworthy, however, that considerable
immunologic activity persists even in diseased palatine tonsils and
adenoids of children, so the described functional changes cannot
by themselves justify surgical removal of these lymphoid organs.
Therefore, a conservative attitude toward adenotonsillectomy seems
to be immunologically justifiable, especially at an early age.

VACCINATION AGAINST PATHOGENS BY

THE NASAL ROUTE
The potential advantage of nasal immunization is illustrated by the
protection achieved against influenza. At present, parenteral vaccination is recommended in vulnerable subjects, but this approach
induces little or no cross-protection. Thus, there is a continuing
need for interpandemic manufacturing of the actual vaccines;
when a genomic drift occurs in a virus, the vaccine strain has to
be replaced, and it usually takes at least 6 months before a new
vaccine is available. Conversely, many studies in experimental animals and humans have demonstrated that nasal vaccination gives
rise to cross-protection against drifted strains (48). With the available live attenuated influenza vaccine for intranasal administration (FluMist; MedImmune, Gaithersburg, MD), good protection
was achieved despite the fact that the epidemic strain was not
part of the vaccine (59). Cross-clade immunity against experimentally applied HIV in mice has been reported after nasal
DNA prime followed by nasal peptide boost; the vaccine contained epitopes of clade B, but high and long-lasting serum antibody titers against the neutralizing gp41 ELDKWAS epitopes
from clades A, B, C, and D were observed (60).
In nasal mucosa of unvaccinated adult subjects, antibodyproducing PCs with specificity for influenza virus are present
(61), but it remains unknown whether they are induced by previous (subclinical) infection or reflect cross-reactivity of the mucosal IgA system. Notably, parenteral immunization with an
inactivated trivalent virus vaccine did not result in an increase
of influenza-specific PCs in nasal mucosa (62), although an IgA
response was elicited in tonsils and saliva (63, 64).
A dense population of putative antigen-presenting cells with
a macrophage or DC phenotype exists in and below the normal surface epithelium of human nasal mucosa (65). The previously mentioned results suggest that to stimulate a regional immune response,
a vaccine should be targeted both against these cells, which may
migrate to the cervical lymph nodes, and against the crypts with
M cells characteristic of NALT structures, probably the adenoids
in particular (Figure 5). Such local B-cell induction apparently
imprints the necessary homing properties of the primed cells to
extravasate efficiently in airway mucosa and associated glands
and give rise to secretory immunity at these regional effector
sites (10, 66). The NALT-derived plasmablasts may to some extent reach the uterine cervix mucosa but do not express sufficient

gut-homing properties (Figure 4) to enter consistently the intestinal lamina propria, particularly not in the small bowel (9, 10).
The amount of antigen reaching the lymphoid tissue of Waldeyers ring and cervical lymph nodes after parenteral immunization is clearly insufficient to induce a nasal immune response,
although some SIgA antibodies may occur in nasopharyngeal
secretions. This most likely reflects local production in the
adenoids where, as pointed out previously, epithelial expression
of pIgR/SC exists, in contrast to the palatine tonsils (4, 5). Not
unexpectedly, there is some communication in terms of memory
and effector B-cell distribution between the mucosal and the
systemic immune systems, particularly so in cervical lymph nodes
and Waldeyers lymphoid ring because of shared homing molecules, as discussed in a previous section (10).
The efficacy and effectiveness of the trivalent, cold-adapted live
attenuated nasal spray influenza vaccine (CAIV-T; MedImmune in
cooperation with Wyeth, Madison, NJ) have been documented both
in healthy children and adults (67). Although nasal vaccination also
efficiently induces systemic immunity, a combination of intranasal
and parenteral immunization may be preferable for optimal protection when an inactivated influenza vaccine is used (68). Alternatively, the effect of subunit vaccines applied topically can be
enhanced by incorporation into liposomes or with the addition of
a nontoxic mucosal adjuvant (Eurocine; Eurocine Vaccines AB,
Karolinska Institutet, Solna, Sweden). Adjuvantation of nasal
vaccines with mucoadhesive polymers, such as chitosan derivatives, has been promising in mouse experiments (69), particularly when combined with antigen-loaded nanoparticles (70).
The initial optimism regarding Escherichia coli heat-labile
enterotoxin as an adjuvant in humans (71) vanished with the
occurrence of Bells palsy after nasal application of an adjuvanted inactivated influenza vaccine (Nasalflu; Berna Biotech
AG, Basel, Switzerland) (72). This problem apparently reflects
the possibility for toxins to enter the central nervous system
from the olfactory bulb or cause irritation and swelling of nerves
going through bony canals to the brain. Other adjuvants, such as
the hydrophobic outer-membrane protein preparations (proteosomes) from Neisseria meningitidis, may be an efficient and safe
alternative (7375). In mice, the uptake of proteosomes can be
enhanced by incorporation of the Toll-like receptor 2 ligand
PorB (76), but it remains to be shown whether Toll-like receptors are expressed on M cells or other parts of the follicleassociated epithelium in human NALT. Finally, virus-derived
particles may function without adjuvants as demonstrated for
a trivalent inactivated whole-cell influenza vaccine (77), probably because of enhanced targeting to M cells and DCs. However, a more recent clinical trial reported superior efficacy of
the live attenuated influenza vaccine in children 1259 months
of age, both for antigenically well-matched and drifted viruses
(78).
An inactivated whole-virus monovalent influenza vaccine
was recently tested in Oslo, Norway, with different devices for

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intranasal or intraoral spray application, and exhibited promising

results for induction of antibodies both in serum and nasal secretions (Table 1). Mild side effects were deemed to be acceptable
(79, 80). Most importantly, the same intranasal vaccine also
induced cellular immunity in addition to the desirable twotiered antibody response, mucosal and systemic. A serum hemagglutination inhibition titer of 40 or higher, which is considered
a protective level, was obtained in most volunteers after two
vaccine doses given 1 week apart; and an additional memory
effect was revealed after three or four doses in that 100% of
the individuals had acquired protective antibody titers. This result was best achieved by vaccine application in nasal drops
or with a special breath-activated device (OptiMist; Optinose
AS, Oslo, Norway), whereas oral spray only induced serum
antibodies.

4.

5.

6.

7.
8.

9.

CONCLUSIONS
The enormous innate drive of the mucosal immune system does
not only enhance diversity but also memory (48). However, with
regard to NALT, the situation in humans and mice seems to be
significantly different. First, NALT of rodents is an organized
bell-shaped structure in the floor of the nasal cavity, and its
organogenesis is different from that of GALT and human palatine and nasopharyngeal tonsils (3, 81). Both these types of
tonsils have deep and branched antigen-retaining crypts,
whereas rodent NALT has a smooth surface, like GALT in
all mammalian species (23). This disparity probably explains
that GCs develop shortly after birth in tonsils as in the heavily
microbe-exposed GALT structures, whereas rodent NALT
requires an infection or another danger signal, such as cholera
toxin, to drive GC formation (82, 83).
The relative lack of innate stimulation is therefore probably the
reason that a nonreplicating rotavirus vaccine adjuvanted with
a mutant E. coli toxin did not induce a substantial memory response in murine NALT (84). By contrast, human NALT with its
antigen- and microbe-retaining crypts is liable to polyclonal stimulation for enhanced development of B-cell diversity and memory. In addition, the crypt epithelium is activated by microbial
products to secrete the B-cell activating factor of the tumor necrosis family (BAFF) and the cytokine thymic stromal lymphopoietin (TSLP). Class switch and broad reactivity of local B cells is
promoted by thymic stromal lymphopoietin when it activates
BAFF-producing DCs (85). Together, these features of Waldeyers
ring constitute an intriguing basis for the current interest in exploiting the nasal route (Figure 5) for vaccine administration to combat
a variety of diseases (86). The adjuventation and delivery systems
of inactivated nasal vaccines should be as tissue-compatible as
possible. Many approaches are explored and several have been
tested in phase I clinical trials (87). Side effects have to be carefully
monitored, but nasal vaccine administration seems to be much less
risky than pulmonary delivery by aerosol technology (88).

10.

11.
12.

13.

14.

15.
16.
17.

18.
19.
20.
21.

22.

23.

Disclosure Statement: The author does not have a financial relationship with a
commercial entity that has an interest in the subject of this manuscript.

24.

Acknowledgment: Hege Eliassen and Erik K. Hagen provided excellent assistance

with the manuscript and figures.

25.

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