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Nature Reviews Immunology | AOP, published online 17 October 2014; doi:10.1038/nri3754
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Atypical MHC class II‑expressing
antigen-presenting cells: can anything
replace a dendritic cell?
Taku Kambayashi1 and Terri M. Laufer2
Abstract | Dendritic cells, macrophages and B cells are regarded as the classical
antigen-presenting cells of the immune system. However, in recent years, there has been a rapid
increase in the number of cell types that are suggested to present antigens on MHC class II
molecules to CD4+ T cells. In this Review, we describe the key characteristics that define an
antigen-presenting cell by examining the functions of dendritic cells. We then examine the
functions of the haematopoietic cells and non-haematopoietic cells that can express MHC
class II molecules and that have been suggested to represent ‘atypical’ antigen-presenting cells.
We consider whether any of these cell populations can prime naive CD4+ T cells and, if not,
question the effects that they do have on the development of immune responses.
Department of Pathology
and Laboratory Medicine and
Division of Rheumatology,
Department of Medicine,
Perelman School of Medicine,
University of Pennsylvania,
Philadelphia, Pennsylvania
19104, USA.
2
Philadelphia Veterans Affairs
Medical Center, Philadelphia,
Pennsylvania 19104, USA.
Correspondence to T.M.L.
e-mail:
[email protected]
doi:10.1038/nri3754
Published online
17 October 2014
1
Dendritic cells (DCs), B cells and macrophages constitutively express MHC class II molecules and are regarded
as the ‘professional’ antigen-presenting cells (APCs) of
the immune system (FIG. 1). However, the universe of
APCs, seemingly so well established, has recently been
changing as more and more cell types are proposed
to express MHC class II molecules and have antigenpresenting functions (FIG. 1; TABLE 1). A number of
haemato­poietic cell types have been suggested to present antigens on MHC class II molecules to CD4+ T cells,
including mast cells, basophils, eosinophils, neutrophils,
innate lymphoid cells (ILCs) and CD4+ T cells themselves.
In addition, MHC class II expression has been detected on
non-haematopoietic cell types in the periphery, such as
endothelial cells, epithelial cells and lymph node stromal
cells (LNSCs). MHC class II‑expressing cells also support
thymocyte development and tolerance induction in the
thymus. However, for the purposes of this article, we focus
on how atypical MHC class II+ cells interact with mature
CD4+ T cells that have exited the thymus. In this Review,
we delineate the qualities that define a true APC and then
examine whether any of the atypical MHC class II+ cell
populations have these functional capabilities.
Classical APC characteristics
A consideration of the properties of DCs that facilitate
the activation of naive CD4+ T cells provides a comprehensive list of characteristics that a bona fide APC
should have (BOX 1). Specifically, in addition to their
antigen processing and presentation capabilities, and
their expression of co-stimulatory molecules, DCs
express pattern recognition receptors (PRRs) that mediate activation in response to pathogens. Following PRRmediated maturation, DCs can migrate to the lymph
nodes to promote the activation of naive T cells. Indeed,
DCs have been shown to be both necessary and sufficient for the activation of naive T cells1,2. The APC that
initiates an immune response must make multiple decisions — for example, whether to actively respond to or
tolerate the antigenic challenge, and what type of active
immune response to induce. DCs clearly predominate in
the induction of T cell proliferation. Interestingly, however, DCs are not required for the development of CD4+
T cell responses under certain conditions3,4, suggesting
that other APCs may be able to replace the function of
DCs. Thus, we consider whether any non-classical APC
can prime naive CD4+ T cells, and discuss the roles that
these cells may have in modulating DC‑dependent and
DC‑independent immune responses.
Mast cells as APCs
Mast cells are tissue-resident innate immune cells that are
strategically localized at mucosal and submucosal sites
in close proximity to the external environment. They
have established roles in allergic disease. Following the
activation of high-affinity Fc receptor for IgE (FcεRI),
mast cells degranulate and release pre-stored immuno­
modulatory molecules — such as tumour necrosis factor
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Professional APCs
Atypical APCs
Mast cells
DCs and macrophages
B cells
Key features
• Phagocytic
• Express receptors for apoptotic
cells, DAMPs and PAMPs
• Localize to tissues
• Localize to T cell zone of lymph
nodes following activation (DCs)
• Constitutively express high levels
of MHC class II molecules and
antigen processing machinery
• Express co-stimulatory molecules
following activation
Key features
• Internalize antigens via
BCRs
• Constitutively express
MHC class II molecules
and antigen processing
machinery
• Express co-stimulatory
molecules following
activation
Basophils
Eosinophils
ILC3s
Key features
• Inducible expression of MHC class II molecules
• Antigen-presenting functions limited to
specific immune environments (especially
type 2 immune settings)
• Lack of compelling evidence that they can
activate naive CD4+ T cells in an antigenspecific manner
Figure 1 | Key antigen-presenting functions of professional and atypical antigen-presenting cells. The figure compares
and contrasts the features of ‘professional’ and ‘atypical’ antigen-presenting cells (APCs). Professional
include
dendritic
Nature APCs
Reviews
| Immunology
cells (DCs) and, with a lesser role, macrophages and B cells. These cells constitutively express MHC class II structural proteins
and associated proteins, and antigen-processing machinery. By contrast, atypical APCs — such as mast cells, basophils,
eosinophils and innate lymphoid cells (ILCs) — do not constitutively express MHC class II molecules, but can upregulate
the expression of MHC class II under certain conditions. Basophils, eosinophils and mast cells that express MHC class II may
contribute to T helper 2 cell differentiation, but there is less evidence that they can prime naive CD4+ T cells. B cell receptor;
DAMP, damage-associated molecular pattern; PAMP, pathogen-associated molecular pattern.
(TNF) and histamine — which can promote T cell activation both directly and indirectly, through stimulation of
APCs. Furthermore, mast cell-deficient mice have defective CD4+ T cell responses in experimental auto­immune
encephalomyelitis5 and in Leishmania major infection6,
suggesting that CD4+ T cell responses could be altered
in the absence of mast cells.
Experimental autoimmune
encephalomyelitis
An experimental model for
the human disease multiple
sclerosis. Autoimmune disease
is induced in experimental
animals by immunization with
myelin or peptides derived
from myelin. The animals
develop a paralytic disease
with inflammation and
demyelination in the brain
and spinal cord.
Expression of MHC class II molecules by mast cells. Mast
cells can directly present antigens to T cells. Both rodent7,8
and human9,10 mast cells have been reported to constitutively express MHC class II molecules. The expression of
MHC class II by mast cells correlated with their ability
to present antigen in vitro to naive CD4+ T cells and to
T cell hybridomas. Cytokines such as interleukin‑4 (IL‑4),
interferon‑γ (IFNγ) and granulocyte–macrophage
colony-stimulating factor (GM‑CSF; also known as
CSF2) further enhanced the APC function of mast
cells11,12. However, these reports were contradicted in
follow‑up studies by the same group showing that the
activation of antigen-specific T cells still occurred when
the T cells were cultured with MHC-mismatched mast
cells13. Moreover, co‑culture of mast cells with spleno­
cytes resulted in antigen-independent activation of
T cells14, which was probably mediated by immuno­
logically active exosomes released by mast cells15 (BOX 2).
These results were consistent with subsequent studies
showing that MHC class II molecules mainly reside
in intracellular lysosomal compartments of mast cells,
rather than at the cell surface16.
More recently, several groups have reported that
resting or FcεRI-activated mast cells do not express
MHC class II molecules, either on the cell surface
or intracellularly 17–20. The apparent discrepancies
between the earlier and more recent studies may
reflect the different protocols that have been used
to generate mast cells. The earlier studies generated
mast cells from short-term bone marrow cultures
(~3 weeks) that were supplemented with IL‑3 alone21.
By contrast, later studies used long-term bone
marrow cultures that were supplemented with both IL‑3
and stem cell factor (SCF; also known as KIT ligand).
Many of the cells derived from the short-term cultures
expressed FcεRI but lacked the mast/stem cell growth
factor receptor KIT22, suggesting that these cultures
also contained non-mast cells, including basophils3,23
and FcεRI+ DCs24,25, both of which expressed MHC
class II but not KIT. By contrast, bone marrow cells
grown in cultures supplemented with IL‑3 and SCF
for more than 6 weeks yielded mast cells that express
high, homogeneous levels of both KIT and FcεRI18,20.
Alternatively, the maturation status of mast cells may
have affected their APC function, as only mast cells
from bone marrow cultures less than 3 weeks old were
able to present antigen to T cells26.
Although resting bone marrow-derived mast
cells from long-term cultures do not constitutively
express MHC class II, its expression can be induced on
these cells. After activation of bone marrow-derived
mast cells with Toll-like receptor 4 (TLR4) agonists
and, to a lesser extent, TLR2 agonists in the presence
of IFNγ, a large proportion of cells expressed MHC
class II and other molecules that are associated with
antigen presentation — namely, MHC class II transacti­
vator (CIITA), the invariant chain (Ii) and H2‑DM20.
Similarly, although freshly isolated mast cells from the
peritoneal cavity lacked MHC class II expression, these
mast cells expressed MHC class II after in vitro treatment with IL‑4 and IFNγ27. MHC class II+ mast cells
were also found in lymph node sinuses; treatment of
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Table 1 | Features of ‘atypical’ antigen-presenting cells
Atypical APC
Features of atypical APC
Ability to promote T cell activation
Expression of
co-stimulatory
molecules
Lymph node
migration by
APC
Factors that induce
expression of MHC
class II on APC
+
+/– (CD80 and
CD86)
+
Ii and HLA‑DM
TLR agonists, IFNγ,
GM‑CSF, IL‑4 and Notch
+/–
+/–
+ (mouse)
– (human)
+
FcεRI, IL‑3 and papain
Ii and HLA‑DM
Eosinophils
+
+
CD80 and CD86
+
IL‑3, IFNγ and GM‑CSF
Unknown
Neutrophils
+/–
+/–
CD80 and CD86
+
Co‑culture with T cells,
GM‑CSF and IFNγ
Unknown
ILC2s
+
Unknown
Unknown
Unknown
Unknown
Unknown
ILC3s
–
Tolerance induction
–
+
Unknown
Ii and HLA‑DM
CD4 T cells
–
Tolerance induction
–
+
TCR stimulation
Unknown
LNSCs
Tolerance
induction
Unknown
ICOSL (subset)
NA
TLR agonists and IFNγ
Ii and HLA‑DM
Endothelial cells
–
+
CD137L, OX40L
and ICOSL
–
IFNγ
Unknown
Epithelial cells
Tolerance
induction
+
–
–
IFNγ and microbial
stimuli
Ii and HLA‑DM
Naive T cells
Activated T cells
Mast cells
+/–
Basophils
+
Expression of other
MHC class II‑related
genes
APC, antigen-presenting cell; CD137L, CD137 ligand (also known as TNFSF9); FcεRI, high-affinity Fc receptor for IgE; GM‑CSF, granulocyte–macrophage
colony-stimulatory factor; ICOSL, ICOS ligand; IFN, interferon; Ii, invariant chain; IL, interleukin; ILC, innate lymphoid cell; LNSC, lymph node stromal cell;
NA, not applicable; OX40L, OX40 ligand (also known as TNFSF4); TCR, T cell receptor; TLR, Toll-like receptor.
mice with lipopolysaccharide or infection with L. major
increased the number of mast cells in the draining
lymph nodes and further upregulated MHC class II
expression20. The apparent constitutive expression of
MHC class II by lymph node mast cells may be owing
to the fact that these cells have matured and migrated
following their activation in the tissue. Alternatively,
endogenous ligands that induce the expression of MHC
class II on mast cells may exist at specific anatomical
sites. The latter hypothesis is supported by data identi­
fying Notch–Delta-like protein 1 interactions as an
inducer of mast cell MHC class II expression19. The
requirement for IFNγ and either TLR-mediated or
Notch-mediated signalling for MHC class II expression
by mast cells may be related to the roles of these pathways in inducing the transcription factor PU.1, which
regulates the CIITA promoter 28.
Functions of MHC class II on mast cells. After MHC
class II was expressed by activated bone marrow-derived
mast cells, they could process and present soluble protein
antigens to previously activated CD4+ T cells in vitro, but
they could not prime naive T cells20, perhaps because they
lacked co‑stimulatory molecule expression. However,
given that lymph node-resident and peri­toneal mast
cells that have been activated by IL‑4 and IFNγ express
both CD80 and CD86 (REFS 20,27), it is possible that some
mast cells can present antigens to naive T cells in vivo. In
addition to activated T cells, mast cells appear to preferentially expand antigen-specific regulatory T (TReg) cells
rather than naive T cells20. The activation of TReg cells by
MHC class II+ mast cells may contribute to the protective effects of mast cells in mediating tolerance to skin
allografts and in preventing nephrotoxic serum neph­
ritis, which are processes that were proposed to involve
IL‑9 production by TReg cells to recruit mast cells to the
graft or injury site29,30. It has been shown that endogenous
proteins are effectively presented on MHC class II molecules of mast cells20 and as such, many of the peptides
presented by mast cell MHC class II may be self antigens
that stimulate TReg cells.
Does IgE support mast cell antigen-presenting functions?
Antigen-specific IgE molecules enhance APC function
in a subset of human DCs that express FcεRI by shuttling incorporated antigens to the MHC class II pathway31. FcεRI could also potentially facilitate the uptake
of antigens into mast cells for more efficient antigen
presentation. Indeed, large particulate antigens were
taken up by mast cells through an IgE-dependent
mechanism both in vitro and in vivo32. Moreover, earlier studies using cultured mast cells demonstrated
that the uptake of antigens through antigen-specific
IgE molecules enhances the T cell-stimulating capacity of mast cells18,22,33. However, in the latter scenario,
the activation of T cells was not mediated through
direct antigen presentation by mast cells, as the mast
cells used in these experiments failed to express MHC
class II 17,18,20. Rather, mast cells transferred FcεRIincorporated antigens to DCs that secondarily presented the antigens to T cells18. Antigens incorporated
by mast cells through FcεRI were found to colocalize with secretory compartments, and were released
by the mast cells upon reactivation32. Thus, although
FcεRI may transport antigens to the MHC class II
pathway under specific conditions22,33, mast cells that
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Box 1 | What are the key properties of an antigen-presenting cell?
Burnet’s ‘clonal selection’ theory presupposes that the T cell repertoire contains
a vast number of T cell clones expressing unique antigen receptors. Thus, an effective
adaptive immune response requires a second cell to select and expand those few T cell
clones that express ‘useful’ antigen receptors. Ralph Steinman termed this quality
‘immunogenicity’ (REF. 145), and crucial work by McDevitt, Sela and Humphrey146–148
showed that immunogenicity required ‘immune response’ genes, which mapped to
the MHC locus. Zinkernagel and Doherty149 first demonstrated that the presentation
of viral antigens was MHC restricted and multiple laboratories subsequently showed
that MHC class I and class II proteins present peptide fragments that are derived from
antigens to the T cell receptor (TCR). Thus, immunogenicity requires that proteins are
processed and presented on MHC molecules to be recognized by T cells.
However, T cell activation requires more than the simple expression of MHC molecules
by antigen-presenting cells (APCs). The activation of a naive T cell requires interaction
with an APC that provides multiple signals (see the figure): ‘signal 1’ is delivered through
interaction of the TCR with peptide–MHC complexes; ‘signal 2’ involves co-stimulatory
molecules; and ‘signal 3’ is mediated by instructive cytokines. The ability to deliver these
three signals is the defining characteristic of a professional APC150.
The search to identify APCs focused on cells that could induce B cell and T cell
responses in vitro and in vivo. This antigen-presenting function was initially ascribed to
macrophages, because the crucial ‘accessory cell’ found in these assays was adherent,
as are macrophages151. However, in 1973, Steinman and Cohn152 identified a different
population of adherent cells with large pseudopods in the mouse spleen; they
subsequently demonstrated that these ‘dendritic cells’ (DCs) were the most potent
inducers of T cell proliferation in primary mixed lymphocyte responses153. Additionally,
DCs are particularly well adapted to process proteins into the peptides that are
presented by MHC class II molecules. They express the MHC class II structural α- and
β-chains; however, they also express the associated chaperone invariant chains,
HLA‑DM and HLA-DO, which regulate peptide loading, and the complement of
lysosomal proteases and cathepsins that can operate in the acidic phagolysosomal
pathway (recently reviewed in REF. 154). Although many analyses probe for expression
of the invariant chain (Ii), HLA-DM and HLA-DO, there have been very few stringent
evaluations of the phagolysosomal pathways of atypical APCs.
Basophils modulate T cell responses
Basophils are a rare population of granulocytes that
comprise ~1% of circulating peripheral leukocytes, and
they are structurally and phenotypically related to mast
cells. Like mast cells, basophils contain abundant granules with pre-formed inflammatory mediators that can
be immediately released upon crosslinking of FcεRI34–37.
Important new reagents have recently been used to show
that basophils and mast cells have functionally unique
and non-overlapping roles in IgE-mediated and IgGmediated allergic inflammation in mice38–43. In addition,
it has been suggested that basophils are central to the differentiation of T helper 2 (TH2) cells. Indeed, a number of
studies have suggested that basophil-derived IL‑4 is crucial for the development of TH2 cell responses to cysteine
proteases, allergens and extracellular parasites4,23,42,44–50.
Basophils as APCs. Although basophil-derived IL‑4
can skew T cells to a TH2 cell phenotype, it had been
presumed that antigen-specific T cell activation by
professional APCs, such as DCs, is still necessary for
TH2 cell induction. To examine the requirement for
DCs in antigen presentation during TH2 cell differentiation, a number of groups have used mice expressing
the human diphtheria toxin receptor (DTR) under the
Cd11c promoter (Cd11c–DTR mice); in these mice,
diphtheria toxin treatment results in the ablation of
CD11c‑expressing cells. When bone marrow from
Cd11c–DTR mice was used to reconstitute irradiated
wild-type mice, diphtheria toxin treatment of these chimeric mice did not alter TH2 cell responses to papain
or Trichuris muris 3,4. Furthermore, papain injection or
DAMPs
T. muris infection of mice in which MHC class II
and PAMPs
expression was restricted to DCs3,4,51 did not result in a
TH2 cell response. Together, these results suggested
MHC
PRR
class II
CD4
that MHC class II expression by DCs alone was neither
Peptide
necessary nor sufficient for TH2 cell induction.
This suggested that another MHC class II‑expressing
Signal 1: antigencell type might be required for TH2 cell induction in
specific interactions
TCR
these models. Indeed, basophils were found to constiSignal 2: co-stimulatory
tutively express MHC class II, CD80 and CD86, and the
molecules
expression of these molecules was further upregulated
CD40L
by activation with IL‑3 (REF. 23) or papain3. Moreover,
CD40
Signal 3: instructive
co‑culture of ovalbumin (OVA)-specific naive CD4+
cytokines
T cells with antigen-pulsed basophils alone was suffiIL-12
IL-12R
cient to induce T cell proliferation and TH2 cell differentiation in vitro3,4,23. Basophils could also present antigens
Activated DC
CD4+ T cell
to T cells in vivo, as antigen-specific naive CD4+ T cells
displayed a TH2 cell phenotype in CIITA-deficient mice
CD40L, CD40 ligand; DAMP, damage-associated molecular pattern; IL‑12, interleukin‑12;
Nature
Reviews
| Immunology (which lack MHC class II expression) injected with
IL‑12R, IL‑12 receptor; PAMP, pathogen-associated molecular pattern;
PRR,
pattern recognition
peptide-pulsed basophils23. Basophils were found to
receptor.
effectively incorporate soluble but not particulate antigens by macropinocytosis3, which was further enhanced
have captured antigens may also act as reservoirs by antigen-specific IgE molecules bound to FcεRI
of antigen that can be presented to T cells by other on basophils. IgE-coated basophils efficiently incorAPCs. Therefore, evidence from in vitro studies sup- porated specific antigens in vivo 52, and the addition
ports both direct and indirect roles for mast cells in of antigen-specific IgE molecules to CD4+ T cell and
antigen presentation. However, although some mast basophil co‑cultures augmented the induction of TH2
cells certainly express MHC class II when analysed cells23. However, it is unclear whether the augmented
directly ex vivo, whether they truly present antigen T H2 cell response mediated by IgE was owing to
in vivo remains to be determined.
enhanced antigen presentation or increased IL‑4
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Box 2 | A confounding role for exosomes?
Naive T cells are usually activated by T cell receptor (TCR)-dependent contact with
peptide–MHC complexes on intact antigen-presenting cells (APCs). However, APCs
may also release antigen-presenting vesicles, or exosomes. Exosomes are secreted
membrane vesicles that form within late multivesicular endosomal compartments and
are released into the environment following fusion of the multivesicular bodies with
the plasma membrane.
Exosomes can be secreted by numerous cell types including dendritic cells (DCs),
B cells, mast cells and microglial cells. They are also secreted by several different
tumour cell types. Intestinal epithelial cells (IECs) may also secrete MHC class II‑bearing
exosomes; putative exosomes carrying IEC-specific proteins have been identified by
immunogold analysis of intestinal tissue sections155.
Immunologically active exosomes were first described by Thery and Amigorena156,157,
who noted that exosomes derived from DCs contain immunologically relevant
proteins such as MHC class I, MHC class II and CD86, as well as the heat shock protein
HSC73, which is involved in peptide delivery. Exosomes bearing peptide–MHC
complexes can stimulate naive T cells in vitro; however, Amigorena and his colleagues
have suggested that exosomes must be recaptured by endogenous DCs for antigen
presentation to naive T cells.
We discuss in the main text how mast cells may contribute to the immune response
via the transfer of exosomes to DCs. Similarly, MHC class II‑loaded exosomes may be
secreted by IECs and act as sources of antigen for tissue-resident or migratory DCs158,159.
Thus, the interpretation of many in vivo studies on the immunological functions of
different cell types — including some types of DCs — is confounded by the possibility
that peptide–MHC complexes from non-conventional cells may simply be transferred
to DCs via exosomes for presentation to T cells.
production by FcεRI-activated basophils. Together, these
results suggested that basophils are a source of IL‑4 and
may act as the primary APC in the activation of T cells
in a variety of TH2‑type immune settings.
Mixed lymphocyte
responses
A tissue-culture technique
for testing T cell reactivity.
The proliferation of one
population of T cells — induced
by exposure to inactivated
MHC-mismatched stimulator
cells — is determined by
measuring the incorporation
of 3H-thymidine into the
DNA of dividing cells.
Macropinocytosis
A type of endocytosis (or
phagocytosis) that occurs
during the engulfment of
apoptotic cells. During
macropinocytosis, large
droplets of fluid are trapped
within the membrane
protrusions (ruffles) or
phagocytic arms.
Different findings in different systems. More recently, the
view that basophils might be the primary APC for TH2 cell
responses in vivo has been challenged by studies suggesting that previous results may have been confounded by
the depletion methods that were used. First, the ablation
of DCs in Cd11c–DTR mice — as opposed to in wild-type
chimeric mice reconstituted with Cd11c–DTR bone marrow — markedly reduced the papain-induced TH2 cell
response53. As a proportion of skin-resident migratory
DCs are radioresistant 54–57, diphtheria toxin treatment
of wild-type mice reconstituted with bone marrow from
Cd11c–DTR mice could have spared migratory DCs in
the skin that were sufficient to induce TH2 cell responses.
Similarly, Cd11c–Aβb mice lack MHC class II expression
on migratory skin Langerhans cells and dermal DCs, and
this may confound the findings from studies using these
mice51. Indeed, surgical excision of the injection site a few
hours after antigen challenge also attenuated the TH2 cell
response, suggesting that radioresistant skin DC populations — either Langerhans cells or dermal DCs — might
be important51. As Langerhans cell depletion by expressing DTR under the control of the promoter of the gene
encoding langerin (also known as CD207) had no effect
on TH2 cell induction, it was concluded that dermal DCs
were responsible for antigen presentation in this setting51. Dermal DCs incorporated the largest amount of
injected antigen and were the most potent at inducing
T cell proliferation in ex vivo co‑culture experiments51.
However, DCs alone were unable to skew T cells to a
TH2 cell phenotype ex vivo and required the addition of
basophils to the co‑cultures51. Thus, it was concluded that
although DCs present antigen to T cells, basophils were
required for TH2 cell polarization.
A similar caveat to these models lies in the basophil
ablation methods that were used. In a house dust mite
(HDM) allergen model, which induces a TH2 cell response
in the lungs, different results were obtained when basophils were depleted using different antibodies — namely,
MAR‑1 and Ba103 (REF. 24). Treatment with MAR‑1,
which is specific for FcεRI, had a stronger effect than
Ba103 (which recognizes CD200R3) in attenuating
TH2 cell polarization in HDM-challenged mice, although
both antibodies reduced basophil numbers to an
equivalent extent 24. The authors showed that in addition to basophils, MAR‑1 also ablated a population of
FcεRI+ DCs, whereas Ba103 only depleted basophils.
Furthermore, basophils from HDM-challenged mice were
poor at presenting antigen ex vivo compared to FcεRI+
DCs isolated from the same lymph nodes, suggesting
that DCs mediated antigen presentation. This argument
was supported by experiments showing that diphtheria
toxin treatment of Cd11c–DTR mice abrogated TH2 cell
responses upon HDM challenge24. Two other groups also
questioned the role of basophils in TH2 cell responses by
using novel mouse strains that constitutively lack basophils owing to mast cell protease 8‑driven expression of
Cre recombinase58,59. Papain-induced TH2 cell responses
were intact in these basophil-deficient mice but not in
DC‑deficient mice, suggesting that DCs but not basophils had an important role in papain-induced TH2 cell
polarization58,59. These results are in contrast to the aforementioned studies demonstrating an important role for
basophils in papain-induced TH2 cell polarization3,42,53.
However, a more recent study targeted DTR expression
to basophils under the control of regulatory elements in
the gene encoding IL‑4 and found that basophils were
necessary for TH2 cell differentiation to peptide antigens,
but dispensable for priming to protein antigens60. It is
clear that the immunological effects of acute or constitutive depletion of basophils are quite dependent on the
method of depletion and the biological setting.
Do human basophils show APC functions? Given
the contradictory data generated in mouse systems,
researchers soon began exploring the antigen-presenting
capabilities of human basophils. In humans, basophils
comprise <1% of circulating granulocytes and they
express FcεRI, CD203c (also known as ENPP3) and
CD123 (also known as IL‑3RA). Resting blood basophils are HLA‑DR negative and early studies showed that
short-term activation with allergens, FcεRI engagement
or TLR2 ligands did not induce MHC class II expression61–63. However, subsequent studies suggested that
human basophils could express MHC class II in certain
disease states (for example, in patients with lupus neph­
ritis), in inflamed tissues that had high levels TH2‑type
cytokines64, and in response to culture with IL‑3 (REF. 65).
However, even these cultured human basophils could
not present allergens61 or exogenous peptides to induce
the activation of human T cells, perhaps because they
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lacked expression of relevant co-stimulatory molecules65.
It remains possible that the bone marrow-derived and
tissue basophils that are purified from mice are not well
represented among the blood-derived human basophils
that were used in these studies. Nonetheless, there is not
yet any compelling evidence that human basophils can
present antigens to CD4+ T cells.
Superantigens
Proteins that bind to and
activate all T cells that express
a particular set of Vβ T cell
receptor genes.
Eosinophils as APCs
Eosinophils are a population of circulating granulocytes
that have long been associated with TH2 cell responses.
They are elicited in response to IL‑5 production during allergic inflammation and parasitic infections66–69,
and they are equipped with an arsenal of cationic proteins and inflammatory mediators with anti­helminth
effector functions70. In addition to their effector role,
eosinophils may also be involved in the modulation of TH2 cell immune responses. Similar to basophils, eosinophils have constitutive activity at the IL4
locus38,40,71 and they are the major source of IL‑4 upon
challenge with Schistosoma mansoni eggs69. Moreover,
eosinophils release chemokines that are crucial for the
recruitment of T cells to the lungs during allergic airway
hyperresponsiveness72–74.
Like mast cells and basophils, eosinophils can also
express MHC class II. Expression of MHC class II by
eosinophils was first reported in sputum and broncho­
alveolar lavage samples from patients with asthma in the
early 1990s75,76. Although freshly isolated eosinophils
from blood were devoid of MHC class II expression, its
expression could be induced following in vitro culture
with activated T cell-conditioned media, GM‑CSF, IL‑3,
or a combination of IL‑3 and IFNγ77–80. Similar observations have been made in mice, where airway or lymph
node eosinophils constitutively express MHC class II81–84.
Although mouse eosinophils in the peritoneal cavity
are MHC class II negative, expression of MHC class II
is induced following the culture of these cells with
GM‑CSF85–87. HLA‑DR in human eosinophils localizes to
detergent-resistant lipid rafts that have been suggested
to enhance antigen presentation by professional APCs88
and, in both humans and mice, MHC class II‑expressing
eosinophils are capable of presenting superantigens,
peptides and protein antigens to T cell hybridomas and
T cell lines78,79,85,86,89. These data suggest that eosinophils
have the potential to function as APCs.
Evidence supporting a role of eosinophils in antigen
presentation in vivo came from studies demonstrating
that eosinophils elicited by repeated airway antigen
challenge migrate from endobronchial areas to the
draining mediastinal lymph nodes81. These eosinophils were predominantly found in the T cell zones of
the draining lymph nodes and they formed clusters
with antigen-specific T cells87. Lymph node eosinophils
expressed MHC class II, CD80 and CD86 and could
restimulate memory T cells from antigen-challenged
mice81,90. Moreover, intratracheal transfer of antigenpulsed eosinophils resulted in the enhanced proliferation of antigen-experienced CD4+ T cells in the draining
lymph nodes, suggesting that eosinophils could induce
antigen-specific T cell proliferation. Similar results were
obtained using mice adoptively transferred with antigenspecific naive T cell receptor (TCR)-transgenic T cells,
suggesting that eosinophils could also activate naive
T cells87. The activation of T cells by eosinophils seemed
to be MHC class II dependent, as the intraperitoneal
injection of Strongyloides stercoralis antigen-loaded
wild-type eosinophils, but not MHC class II-deficient
eosinophils, resulted in the increased production of
TH2 cell-associated cytokines91.
However, some studies have argued that eosinophils
might be incapable of efficiently processing protein
antigens. Although human eosinophils expressed MHC
class II after IL‑3 stimulation in vitro and could stimulate
antigen-specific primed T cells when pulsed with peptides, they were unable to do so when pulsed with whole
antigen80. Similarly, whole protein-pulsed mouse eosinophils could not stimulate antigen-specific naive T cells,
although some proliferation of T cells was observed
when the eosinophils were pulsed with peptides84.
A recent study contradicted these findings and showed
that mouse eosinophils were in fact efficient APCs for
naive antigen-specific T cells both in vitro and in vivo91.
They attributed the previously reported lack of antigenprocessing ability of eosinophils to the use of NH4Cl
to lyse red blood cells. In their experiments, eosinophils were unable to activate antigen-specific T cells if
they were exposed to NH4Cl, potentially owing to its
lyso­s omotropic activity. Although this method was
used by one of the previous studies84, red blood cells
were removed by density gradients and centrifugation
in the other 80. Alternatively, differences in the human
and mouse systems may contribute to the discrepancies
that were seen in the latter study.
Although some controversy still remains as to their
antigen-processing ability, many studies have convincingly demonstrated that MHC class II and co‑stimulatory
molecules are expressed by airway-associated and lymph
node eosinophils, and by cytokine-activated circulating
eosinophils77–84. Moreover, eosinophils localize to the
same areas as classical APCs, as they migrate to T cell
zones in the lymph nodes during airway hyper­sensitivity
reactions81,87. Recently, mice that express DTR driven
by eosinophilic peroxidase have been generated, but
T cell-dependent responses in such mice have only been
analysed in an asthma model. At least in an allergic airway disease model, eosinophil depletion had no effect
during the antigen sensitization phase92, suggesting that
T cell responses are intact in the absence of eosinophils.
The generation of new methods to selectively ablate
MHC class II expression on eosinophils will be helpful
to further investigate the in vivo role of eosinophils as
APCs in other disease settings.
APC functions of neutrophils
So far, we have examined the potential antigenpresenting functions of mast cells, basophils and eosinophils. However, neutrophils are the most abundant type
of granulocyte and are worth considering. Neutrophils
are rapidly recruited to sites of tissue damage where they
extrude neutrophil extracellular traps (NETs), produce
antimicrobial peptides, phagocytose microorganisms
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REVIEWS
and recruit other immune cells to clear pathogens.
Thus, neutrophils are generally regarded as professional
phagocytes that are involved early in the response to
tissue injury and infection. However, there is increasing evidence that neutrophils may also modulate the
adaptive immune response through the production
of chemokines and cytokines that recruit DCs to sites of
inflammation93. Thioglycollate-elicited peritoneal neutro­
phils in mice express CD80, but neither CD86 nor MHC
class II94. However, co-culture with CD4+ T cells may lead
to a minimal level of cell-surface MHC class II expression and the ability to process OVA protein and activate
OVA-specific CD4+ T cells94. Similarly, neutrophils purified from the colons of colitic mice were MHC class II+
and, when pulsed with OVA peptide, could also activate
CD4+ T cells. Human neutrophils could express HLA‑DR
and could stimulate super­antigen-dependent T cell
activation; however, they could not re‑activate tetanus
toxoid-specific T cells95.
Despite the suggestion that neutrophils may be
professional APCs, the interpretation of these data is
quite complicated. First, although neutrophil preparations in these studies are 95–98% pure, the presence
of only a few contaminating DCs in either the neutrophil or T cell preparation could be sufficient to mediate DC‑driven T cell activation. Second, neutrophils
induced by Pseudomonas aeruginosa airway infection lack
MHC class II expression, despite expressing CD80 and
CD86 (REF. 96). Finally, it is notable that in many of these
reports, MHC class II expression occurs in GM‑CSF-rich
environments. In this setting, these ‘differentiated’ neutro­
phils could easily be confused with inflammatory TNF
and iNOS producing (TIP)-DCs97 or a recently described
‘neutrophil–DC hybrid’ cell that shows characteristics of
both cell types98. Thus, there is not compelling evidence
that neutrophils can function as true APCs.
Neutrophil extracellular
traps
(NETs). A set of extracellular
fibres produced by activated
neutrophils to ensnare
invading microorganisms.
NETs enhance neutrophil
killing of extracellular
pathogens, while minimizing
damage to host cells.
ILCs and CD4+ T cells as APCs
In recent years, there has been growing interest in ILCs,
which function as rapid sources of cytokines early during immune responses. ILCs lack rearranged antigen
receptors but otherwise resemble CD4+ T cells in their
developmental pathways, transcription factor profiles
and cytokine-producing patterns. Type 1 ILCs (ILC1s),
ILC2s and ILC3s produce TH1-, TH2- and TH17‑type
cytokines, respectively. Recent evidence implicates ILCs
as crucial regulators of innate immunity and inflammation at barrier surfaces, including the skin, airways and
gastrointestinal tract (reviewed in REF. 99).
In addition to their early effects on innate immune
responses, there is increasing evidence that ILCs may
directly interact with CD4+ T cells. One group reported
that ILC3s can express CXC-chemokine receptor 5
(CXCR5) and CC-chemokine receptor 7 (CCR7), and
localize to secondary lymphoid organs where they may
control the maintenance of memory CD4+ T cells100,101.
More directly, genome-wide transcriptional profiling of
ILC3s showed that they express genes that encode MHC
class II structural components, as well as proteins that
are necessary for antigen presentation, including Ii and
H2‑DM102. In this report, a subset of ILC3s had surface
expression of MHC class II and could acquire, process
and present exogenous antigens in vitro but did not
induce proliferation of CD4+ T cells. It was suggested
that ILCs probably do not activate naive CD4+ T cells;
nevertheless, cell-specific deletion of MHC class II
expression in retinoic acid receptor-related orphan
receptor-γt (RORγt)-expressing ILC3s led to the dysregulation of CD4+ T cells specific for commensal bac­teria
and the loss of intestinal epithelial integrity 102. Thus,
these authors proposed that MHC class II expression
on ILCs contributed to intestinal homeostasis by countering professional APCs to prevent the inappropriate
activation of commensal-specific CD4+ T cells. In contrast with these initial observations, a second laboratory
examined the function of splenic ILC3s from the same
strain of mice lacking expression of MHC class II in
ILC3s103. Interestingly, in a different animal facility, this
strain of mice did not develop any intestinal pathology,
suggesting a crucial role for microbial exposure103. They
also reported that, in contrast to ‘tolerizing’ intestinal
ILC3s, splenic ILC3s respond to IL‑1β by upregulating
both MHC class II and co-stimulatory molecules, and
thus contribute to CD4+ T cell proliferation. Perhaps
ILC3s localized to different environments have different
effects on CD4+ T cell activation and differentiation.
In agreement with these observations, we recently
showed that CD4+ T cells regulate the number and function of ILC3s in the mouse intestinal lamina propria in
an MHC class II‑dependent manner104. Although we did
not show that this was directly regulated by MHC class II
expression on the ILCs, we did find that the level of
MHC class II expressed by ILC3s in the intestinal lamina
propria was determined by the presence of functional
CD4+ T cells104. Thus, MHC class II‑expressing ILC3s
may regulate and also be regulated by CD4+ T cells.
There may also be such cross-regulation between
ILC2s and TH2 cells. ILC2s were first characterized as
cells that expressed MHC class II and inducible T cell costimulator (ICOS)105. They are resident in the lungs and
other mucosal tissues, and IL‑2 produced by TH2 cells
may enhance cytokine production by ILC2s106. More
importantly, purified lung ILC2s were shown to present
peptide to TCR-transgenic CD4+ T cells in vitro106; however, ILC2s could not present OVA protein in this study.
A more recent examination found that peptide-loaded
MHC class II+ ILC2s induced TH2 cell differentiation
in vitro; ILC2s could also internalize and process protein antigens, but in vitro CD4+ T cell proliferation could
not be detected107. Nonetheless, MHC class II+ ILCs contributed to the clearance of intestinal helminths107. The
study of MHC class II‑dependent functions of ILC2s
and ILC3s is a rapidly evolving area and the data remain
contradictory. Importantly, the systems studied to date
involve ablation of cell-specific MHC class II expression
rather than directly assaying the APC function of ILC3s
in the absence of antigen presentation by DCs.
Among other non‑B cell lymphocytes, CD4+ T cells
also have the ability to express MHC class II and present antigens to other CD4+ T cells. Although CD4+
T cells from mice do not express MHC class II, human
CD4+ T cells express HLA‑DR molecules upon TCR
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Lymph node
stromal cell
Endothelial cell
Intestinal epithelial cell
Constitutive MHC class II
expression
MHC
class II
Inducible MHC class II
expression
Inducible MHC class II
expression
Function of MHC class II expression unclear
but may have a role in tolerance induction
Figure 2 | Epithelial cells and stromal cells express MHC class II molecules. Vascular endothelial cells, multiple
types of epithelial cells (including intestinal and pulmonary epithelial cells) and lymph nodeNature
stromalReviews
cells can| Immunology
express
+
MHC class II molecules. Although these cells have been shown to activate CD4 T cells in vitro, their expression
of MHC class II molecules in vivo is thought to be more relevant for the induction and maintenance of immune tolerance.
activation108,109. Antigen presentation by CD4+ T cells
seems to have a dominant tolerance-inducing effect on
other CD4+ T cells. Peptide-loaded T cell clones can
activate each other to induce proliferation. However,
these T cells are defective in response to restimulation110, suggesting that antigen-presenting CD4+ T cells
induce anergy. As MHC class II molecules on CD4+
T cells are loaded with self peptides111, it has been postulated that activation by HLA‑DR molecules expressed
by CD4 + T cells may serve to limit self peptidereactive CD4+ T cells. The lack of MHC class II expression by mouse CD4+ T cells has hampered progress in
investigating the antigen-presenting role of CD4+ T cells.
Non-haematopoietic cells as APCs
Up to this point, we have focused on haematopoietic
cells — predominantly myeloid cells — that might act
as APCs. However, there is an extensive literature on the
antigen-presenting abilities of radioresistant endothelial
cells and epithelial cells in particular clinical settings,
especially autoimmunity and transplant tolerance (FIG. 2).
Lymph node stromal cells. The T cell–APC interactions
that mediate T cell activation do not occur in free space
but in tightly regulated anatomic regions of lymph nodes,
which are defined by the presence of non-haematopoietic
stromal cells of mesenchymal and endothelial origin.
LNSCs can be divided into subclasses on the basis of
their surface expression of the glycoproteins CD31 (also
known as PECAM1) and podoplanin (also known as
GP38), and their localization within lymph nodes. These
subclasses include fibroblastic reticular cells (FRCs), folli­
cular DCs (FDCs), lymphatic endothelial cells (LECs),
blood endothelial cells (BECs), pericytes and a small
proportion (<5%) of otherwise undefined stromal cells.
For many years, it was presumed that the sole function on non-FDC LNSCs was to provide the structural
‘backbone’ for secondary lymphoid organs. However,
the stroma clearly contributes to adaptive responses
(as reviewed in REF. 112) by concentrating antigens in the
lymph nodes and directing DC trafficking. Additionally,
multiple groups have demonstrated that LNSCs ectopically express tissue-specific antigens, including transgenedirected neoantigens and endogenous tyrosinase,
and that they can induce the deletion of self-reactive
CD8+ T cells113–117. The mechanisms for the expression of
tissue-specific antigens are not completely clear. Thymic
medullary epithelial cells express autoimmune regulator
(AIRE), which is a transcriptional activator that promotes
the expression of tissue-specific antigens to regulate central deletional tolerance. The Anderson laboratory used
an Aire-driven transgenic reporter to identify a population of lymph node cells that expressed AIRE and tissuespecific antigens117. Interestingly, these AIRE+ cells
expressed low levels of CD45 and CD11c, and were radiosensitive, suggesting that they were a haematopoietic population that was distinct from other LNSCs117. It is not clear
if AIRE is expressed at significant levels in LNSCs, although
a related transcriptional regulator, deformed epidermal
autoregulatory factor 1 (DEAF1), may be114,115.
LNSCs could either be intrinsically tolerogenic cells
or may simply express MHC class I in the absence of
co-stimulatory molecules and, as such, induce the deletion of self-reactive CD8+ T cells113,114. However, the
ImmGen consortium has shown that FRCs, LECs and
BECs can upregulate surface expression of MHC class II
molecules under inflammatory or infectious conditions118. In this setting, it has been demonstrated that
peripheral AIRE+ cells, but not radioresistant LNSCs,
can also mediate deletional tolerance of autoreactive
CD4+ T cells117. Importantly, there are no data suggesting that CD4+ T cell deletion induced by either LNSCs or
AIRE+ cells is mediated independently of DCs. Indeed,
a recent report suggested that LNSC-mediated deletion
of CD4+ T cells is dependent on peptide–MHC class II
complexes that are acquired from DCs119. Thus, it is
possible that these cells are similar to basophils in
modulating antigen presentation by DCs.
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Endothelial cells and epithelial cells. Both human and
mouse vascular endothelial cells and tissue-resident
epithelial cells can express MHC class I and class II
molecules, and there is some evidence that interactions
with CD4+ T cells may be involved in some autoimmune
diseases and in graft rejection. Vascular endothelial cells in
human allografts can express both MHC class I and class II
molecules, and can present processed protein antigens to
T cell clones120,123,124. However, there is no evidence either
in vitro or in vivo that human endothelial cells can express
CD80 or CD86, which are necessary to stimulate naive
alloreactive T cells120. Both rat and mouse allograft models
suggest that DCs in the graft (‘passenger leukocytes’) are
necessary to initiate graft rejection121,122. However, memory
cells have decreased requirements for co-stimulation and
one group has shown in xenograft models that human
memory T cells — specifically the effector memory subset — can directly respond to graft vascular endothelium
to mediate rejection123,124.
Epithelial cells — including intestinal epithelial cells
(IECs), airway epithelial cells and keratinocytes — are
uniquely positioned at the interface between the host
immune system and an environment teeming with antigens, including pathogenic microorganisms and food
antigens, as well as commensal microorganisms. Thus,
decisions about whether to generate pro-inflammatory
or tolerizing responses must continuously be made at
mucosal surfaces. Similar to vascular endothelial cells,
epithelial cells can express MHC class II and may be
uniquely poised to regulate T cell responses to mucosal
antigens. Most work has focused on IECs and lung
alveolar epithelial cells; however, we have previously
suggested that MHC class II expression restricted to
keratinocytes could mediate autoimmune skin disease125.
Multiple reports suggest that both mouse and human
ileal IECs constitutively express MHC class II molecules.
Do these cells have the machinery to present antigen?
Much of the work suggesting that IECs can process and
present antigen has been limited to in vitro studies using
cell lines or primary cells treated with IFNγ. These studies do show that in the presence of IFNγ, the appropriate
machinery — including Ii, H2‑DM and proteases —
can be expressed by IECs in the small intestine and by
oesophageal epithelial cells126. Interestingly, IECs isolated
from patients with inflammatory bowel disease (IBD)
or from animal models of IBD express higher levels of
MHC class II127–129. Work using IEC-like cell lines has
shown that these cells can stimulate T cell hybridomas
or clones in vitro130–135. However, the evidence that naive
T cells can be stimulated by IECs in vivo is less compelling. Indeed, there is no clear evidence that IECs express
sufficient levels of the appropriate co-stimulatory
molecules to activate naive CD4+ T cells127–129.
There are accumulating data suggesting that IECs may
contribute to the differentiation of TReg cells and the maintenance of tolerance. Neoantigen targeted to IECs induced
the expansion of antigen-specific TReg cell populations
in a manner that was independent of DC depletion136.
A more recent in vivo study examining transgenic mice
in which MHC class II expression was restricted to either
IECs or DCs showed that antigen presentation by DCs,
but not by IECs, could drive CD4+ T cell-dependent intestinal inflammation137. The requirement for MHC class II
was examined by selectively deleting expression of CIITA
in non-haematopoietic cells, including IECs138. In these
mice, colitis induced by IL‑10 blockade was much more
severe than in co‑housed wild-type littermates; disease
was attributed to increased numbers of local TH1 cells
and an imbalance in the relative numbers of effector
T cells and TReg cells. Similar to investigations of IECs in
the small intestine (the focus of most published work),
it was also found that MHC class II+ colonic epithelial
cells could not stimulate naive CD4+ T cells in vitro;
rather, they suppressed the activation of CD4+ T cells
by professional APCs138,139. The mechanism for this was
not clear, although it was independent of transforming
growth factor-β.
In both mice and humans, as many as half of the MHC
class II+ cells in the lungs are of non-haematopoietic
origin140,141. Non-haematopoietic cells include vascular
and alveolar epithelial cells. Most data suggest that adaptive immunity to respiratory pathogens is predominantly
mediated by haematopoietic APCs, including migratory DCs and alveolar macrophages; however, there
are data suggesting that epithelial cells might modulate
these responses. For example, type II alveolar epithelial
cells comprise only 4% of the epithelium140 but they are
strategically located to respond to airborne pathogens,
and they produce antimicrobial peptides and proinflammatory mediators such as complement components141,142. Type II alveolar epithelial cells in both mice
and humans express MHC class II that can be upregulated
by IFNγ and microbial stimulation143. Both endogenous
neo-self antigens and exogenous antigens — for example,
from Bacille Calmette–Guérin (BCG) — can be processed
and presented by type II alveolar epithelial cells143. MHC
class II expression on pulmonary non-haematopoietic
cells may contribute to decreased inflammation and
acceptance of orthotopic lung transplants144. In transgenic systems, type II alveolar epithelial cells can prime
antigen-specific CD4+ T cells. Interestingly, the T cell
response is skewed towards differentiation of forkhead
box P3 (FOXP3)-expressing CD4+ T cells143. In this regard,
type II alveolar epithelial cells may be similar to vascular
endothelial cells. Thus, endothelial cells and epithelial cells
may be additional cell types that express MHC class II and
modulate the outcome of DC–CD4+ T cell interactions.
Concluding remarks
There are a large number of haematopoietic and
non-haematopoietic cell types that can express MHC
class II molecules and present antigens to CD4+ T cells.
However, MHC class II expression alone is not sufficient
for full APC function, as APCs need to be able to process antigens, migrate to secondary lymphoid organs and
express co‑stimulatory molecules. In this regard, professional APCs, such as macrophages and B cells, could
potentially replace the function of DCs in certain situations. However, the non-professional APCs that have
been discussed in this Review are unlikely to replace
DCs, because there is little compelling data (if any) that
these cell types are able to activate naive CD4+ T cells.
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Rather, we propose that these non-conventional APCs
modulate immune responses that have been initiated
by DCs. This is especially true for non-haematopoietic
cells that may mediate the deletion of autoreactive T cells
or may stimulate TReg cells. The requirement for additional non‑DC APCs is quite context dependent — for
example, basophils, mast cells and eosinophils have
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Acknowledgements
Work in the laboratory of T.M.L. is supported by a VA Merit
Award. Research in the laboratory of T.K. is supported by fund‑
ing from the US National Institutes of Health and the American
Asthma Foundation.
Competing interests statement
The authors declare no competing interests.
DATABASES
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