By
From the * Institut National de la Santé et de la Recherche Médicale, Contrat Jeune Formation
95-01, Institut Curie, Section Recherche, 75005, Paris, France; and Biologie moléculaire de gène,
Institut National de la Santé et de la Recherche Médicale U277, Institut Pasteur, 75015, Paris,
France; and § Laboratoire d'immunologie des pathologies infectieuses et tumorales, Institut Cochin
Génétique Moléculaire, Institut National de la Santé et de la Recherche Médicale U445, 75014, Paris, France
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Abstract |
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T cell receptors on CD4+ lymphocytes recognize antigen-derived peptides presented by major
histocompatibility complex (MHC) class II molecules. A very limited set of peptides among
those that may potentially bind MHC class II is actually presented to T lymphocytes. We here
examine the role of two receptors mediating antigen internalization by antigen presenting cells,
type IIb2 and type III receptors for IgG (FcRIIb2 and Fc
RIII, respectively), in the selection
of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant
Fc
RIIb2 or Fc
RIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through Fc
RIIb2 and Fc
RIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through Fc
RIII, but not through Fc
RIIb2.
The capacity to present these latter epitopes was transferred to a tail-less Fc
RIIb2 by addition
of the Fc
RIII-associated
chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the
chain cytoplasmic tail resulted in the selective loss of presentation of these
epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens.
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Introduction |
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Antigen receptors on CD4+ helper T lymphocytes recognize short peptides presented by class II molecules of MHC (1, 2). Antigenic peptides are generated by proteolytic degradation in the endocytic pathway, where they associate with MHC class II molecules. Only a very limited set of peptides among all the potential peptides is actually loaded onto MHC class II molecules in APCs (3). The mechanisms underlying the selection of peptides for MHC class II-restricted antigen presentation are yet unclear. However, we know that two independent complex processes of intracellular transport towards endosomes are crucial for MHC class II-restricted antigen presentation: the traffic of MHC class II molecules and the delivery of antigens (4, 5).
MHC class II intracellular transport has been analyzed in
detail. Newly synthesized MHC class II molecules reach
endosomes, either directly from the trans-golgi network or
after a short appearance at the plasma membrane, in association to the invariant (Ii)1 chain (5). Ii is then degraded and
the class II-associated Ii chain peptide (CLIP) is replaced by
an antigenic peptide under the control of HLA-DM (6). It
has recently become clear that an alternative, Ii chain-independent pathway for MHC class II transport to endosomes
also exists. Indeed, MHC class II molecules may reach the
endocytic pathway from the cell surface by endocytosis (7),
due to internalization signals present in the cytosolic domain of the MHC class II chain (8). Newly synthesized
and recycling MHC class II molecules may present different peptides (9). Accordingly, we have previously shown
that different antigen receptors may also selectively target
antigens for presentation by either of these MHC class II
presentation pathways (10).
In contrast, very little is known about the endocytic
transport of antigen receptors. Physiologically, antigens are
delivered to the endocytic pathway by different families of
receptors, which strongly increase the efficiency of MHC
class II-restricted antigen presentation (11). In B lymphocytes, surface Ig mediates both cell activation and the uptake of specific antigens (12), while the expression of a particular endocytosis-deficient receptor for the Fc portion of
IgG (FcRIIb1) prevents efficient presentation of irrelevant
IgG-complexed antigens (13). Interestingly, the epitope
specificity of surface Ig positively and negatively influences the presentation of various T cell epitopes (14, 15).
In monocytes and dendritic cells, receptors for IgG
(FcRs), in addition to mannose receptors, mediate antigen
internalization and strongly increase the efficiency of presentation to specific T cells (16). Two different Fc
Rs,
type IIb2 and type III (Fc
RIIb2 and Fc
RIII) are expressed in dendritic cells. Fc
RIIb2 is a monomeric receptor that, like Fc
RIIb1, mediates the inhibition of cell activation when cocrosslinked to surface Ig (13). Fc
III is an
heterotrimer consisting of an
chain and a dimer of
chains, which couple the receptor to cytoplasmic effectors
of signal transduction (17).
To individually analyze the function of these two receptors, we expressed them by cDNA transfection into an FcR-negative B cell lymphoma cell line (13, 18, 19). We have
previously shown that the amino acid sequence, called immunoreceptor tyrosine kinase activation motif (ITAM),
in the Fc
III-associated
chain responsible for cell activation is also involved in receptor internalization (18, 19). In
addition, mutation of either of the two tyrosine residues
in the ITAM of the
chain inhibits both cell activation and
ligand internalization (19). Thus, in contrast to Fc
RIIb2, which contains no ITAM, is not tyrosine phosphorylated,
and does not induce cell activation, Fc
RIII associates with
and activates cytosolic tyrosine kinases after engagement by
its ligand (18). In addition to this functional diversity of the
two receptors, their expression is also selectively regulated,
since TNF-
and IFN-
increase the expression of Fc
RIII
and inhibit that of Fc
RIIb2 in monocytes (20).
The diversity in the signals required of FcRIIb2 and
Fc
RIII internalization, as well as the differential regulation of their expression in APCs, suggest that the two receptors may have different antigen-presenting functions. We
here examine the ability of Fc
RIIb2 and Fc
RIII to induce the presentation of various T cell epitopes from two
different antigens, CI
repressor and hen egg lysozyme (HEL).
Internalization of antigen-antibody complexes through
FcRIII induced the efficient presentation of all the T cell
epitopes tested, whereas Fc
RIIb2 only induced the presentation of a few. Point mutation of leucine 35 to alanine
(L35A) in the cytoplasmic tail of Fc
RIII
chain blocked
signal transduction without affecting the internalization of
immune complexes. This mutation also blocked the presentation of the epitopes that were only presented after internalization by Fc
RIII. In contrast, the presentation of all the epitopes that were efficiently generated after internalization by Fc
RIIb2 was not affected. Thus, the nature of
the receptor that mediates antigen internalization influences
the selection of the epitopes presented to T lymphocytes.
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Materials and Methods |
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B Lymphoma Cell Lines.
The B lymphoma IIA1.6 is a type II FcT Cell Hybridomas.
Culturing of the T cell hybridomas and antigen presentation assays were performed in RPMI 1640 containing 10% FCS, 10 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 × 10
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Assays for Antigen Presentation.
Antigen presentation was assessed by culturing transfected IIA1.6 cells together with specific T cell hybridomas for 18-20 h in the presence of various concentrations ofAssays for Cell Activation.
Cell activation through Fc receptors or endogenous membrane immunoglobulins was determined as previously described (19). In brief, triplicates of each B lymphoma cell (105 cells/well in 100 µl) were stimulated either through Fc receptors using preformed ovalbumin immune complexes, made as described above, or through endogenous membrane immunoglobulins using F(abImmune Complex Internalization.
The internalization of immune complexes was assayed as previously described (19). In brief, the cells were washed once in internalization buffer (RPMI, 5% FCS, 10 mM glutamine, 5 mM sodium pyruvate, 50 mM 2-ME, and 20 mM Hepes, pH 7.4) and incubated with horseradish peroxidase (HRP) anti-HRP immune complexes for 2 h at 0°C (107 cells/ml). Immune complexes were prepared as a 10× solution in internalization buffer (HRP 50 µg/ml and a polyclonal rabbit anti-HRP antibody at 400 µg/ml) for 30 min at 37°C. After fixation of HRP immune complexes, the cells were washed three times in internalization buffer and incubated at 37°C for various times (2 × 106 cells/ml). Internalization was stopped by adding cold internalization buffer and the cells were washed once in PBS. Duplicates of each time point were either left in PBS at 4°C to measure cell surface HRP-ICs or incubated in Triton X-100 (0.1%) for 5 ![]() |
Results |
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We have shown previously that internalization of antigen-IgG complexes by FcRIIb2 and III
in IIA1.6 cells (an Fc
R-negative B lymphoma cell line
derived from A20 cells) induces presentation of the dominant epitope of the CI
repressor (12-26 peptide on IAd)
at antigen concentrations 102-104-fold lower than fluid
phase uptake (13, 19). This increase in the efficiency of antigen presentation is, at least in part, due to increased antigen uptake by the APCs since endocytosis-incompetent receptors for IgG (13, 19) were deficient for both internalization of immune complexes and efficient presentation at low antigen concentrations. Although these results underline the
importance of internalization in antigen presentation, they
do not address the possibility that postinternalization intracellular targeting of antigen-receptor complexes is also important for the generation of certain epitopes. If this was
the case, we would then expect that internalization through
particular antigen receptors results in the presentation of
different epitopes to specific T lymphocytes.
To test this possibility, we assessed the ability of cells expressing either FcRIIb2 or Fc
RIII (cells coexpressing
the
and
chains, which we have previously shown to
present the 12-26/IAd epitope at very low antigen-IgG
concentrations) to present another epitope, defined by the
same 12-26 in association with I-Ed. This epitope is not
presented after fluid phase antigen uptake, or in vivo after
immunization with intact antigen (it is therefore a cryptic
epitope), but it elicited an immune response when the mice were injected directly with the peptide (22).
The cell lines expressing either FcRIIb2 or Fc
RIII
were incubated with increasing concentrations of CI
repressor, free or complexed to two different anti-
repressor
mAbs, as previously described (13). Complexing antigen to
IgG allowed receptor-mediated antigen uptake through
Fc
Rs. T cell hybridomas specific for either IAd- or the
IEd-restricted epitopes were also included in the cultures.
Antigen presentation was assessed by measuring IL-2 secretion by the T cell hybridomas.
As shown in Fig. 1 A, the IEd restricted epitope was not
presented after fluid phase uptake (right), whereas the IAdrestricted epitope was presented at high antigen concentrations (left). Confirming our previous results, FcRIIb2 increased the efficiency of presentation of the IAd restricted
epitope by 3-4 logs when the C1
repressor was complexed to IgGs (T cell activation was still observed at 30 ng/ml). Similarly, Fc
RIII-mediated internalization induced presentation of this epitope at concentrations 30-100-fold lower
than fluid phase uptake. Therefore, both type II and III
IgG receptors increased the efficiency of presentation of
this dominant IAd-restricted epitope.
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However, FcRIII was less efficient than Fc
RIIb2
(Fig. 1 A, compare lower and upper left). This difference
was due to the different levels of expression of the two receptors, since Fc
RII and Fc
RIII were expressed at 1.6 × 105 molecules/cell and 2.8 × 104 molecules/cell, respectively (reference 19 and data not shown). When cells expressing 2-3 × 104 Fc
IIb2 per cell were used, the efficiency of presentation was similar to that obtained with
Fc
RIII expressing cells (data not shown).
In contrast to this IAd-restricted epitope, the IEd epitope
was not presented after FcRIIb2-mediated internalization
of immune complexes (Fig. 1 A, lower right). In contrast, efficient presentation of 12-26 on IEd was observed with Fc
RIII-expressing cells after internalization of immune
complexes (upper right). This selectivity of presentation of
the IEd-restricted epitope was not due to a difference between the Fc
RIIb2 and Fc
RIII cell lines in the levels of
surface expression of either MHC class II or other costimulatory molecules, because the 12-26 peptide was presented
with similar efficiencies to the two T cell hybridomas by
the two transfected cell lines (Fig. 1 B). Since engagement
of Fc
III by immune complexes may, under certain circumstances (see below), induce IL-2 secretion by A20 and
IIA1.6 cells (18, 27), we verified that the CI
repressor immune complexes did not induce any IL-2 secretion in
the absence of T cell hybridomas (data not shown).
Therefore, only FcRIII-mediated internalization induced presentation of the 12-26 peptide on IEd. Fc
RIIb2,
which induced very efficient presentation of the same peptide on IAd, was totally inefficient for the presentation of
the IEd-restricted epitope. Therefore, receptor-mediated
antigen internalization is not sufficient for presentation of
all T cell epitopes; the nature of the receptor mediating antigen uptake influences the selection of epitopes presented
to T lymphocytes.
We have shown previously that both
the internalization and the increased efficiency of presentation of the IAd/12-26 dominant epitope in the CI repressor were dependent on the Fc
RIII-associated
chains
(19). Thus, Fc
RIII with a cytoplasmic domain-deleted
chain did not internalize ligand or promote antigen presentation at low concentrations. In addition, when the cytoplasmic domain of the
chain was fused to the extracellular
and transmembrane domains of Fc
RIIb2, cell activation,
immune complex internalization, and efficient antigen presentation were observed (19). Therefore, we next sought to
assess if the presentation of 12-26 on IEd was also dependent on the Fc
RIII-associated
chain.
As expected, the tail-less FcRIIb2, which was not internalized, did not allow the efficient presentation of the
two epitopes (Fig. 2 A, lower panels). In contrast, when the
cytoplasmic tail of the
chain was fused to this tail-less receptor to form an Fc
RII/ic
chimera, efficient presentation of both IAd/12-26 and IEd/12-26 was observed after
immune complex internalization (Fig. 2 A, upper panels).
The cell lines expressing the tail-less Fc
RII and the Fc
RII/ic
chimeras presented the 12-26 peptide with similar efficiencies (Fig. 1 B). Therefore, the cytoplasmic domain of the
chain bears all the information required for
the presentation of the IEd epitope.
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Two different pathways of antigen presentation may result in the
loading of antigenic peptides on MHC class II molecules: the conventional pathway requires newly synthesized and
the so-called alternative pathway requires recycling MHC
class II. Presentation through the conventional and alternative pathways differ by their kinetics and their sensitivity to
protein synthesis inhibitors (as well as by their dependence
on H2-M, the Ii chain, and the cytoplasmic tail of MHC
class II molecules; references 8, 28). It has recently been
shown, that these two pathways may control the presentation of different epitopes from a single antigen (9). We
have also previously shown that internalization through
different receptors may trigger the presentation the same
epitope by either antigen presentation pathways (10).
Therefore, it was possible that FcRIIb2 and Fc
RIII were
also targeting antigens for presentation by different pathways and that the IEd epitope could only be generated in
one of these pathways.
We tested this possibility by assessing the effect of the
protein synthesis inhibitor cycloheximide on the kinetics of
presentation of the IAd and IEd epitopes. FcRIIb2- or Fc
RIII-expressing cells were preincubated in cycloheximide
for 3 h before addition of CI
repressor immune complexes. After incubation of the cells at 37°C for various periods of time, the cells were fixed and T cell hybrids specific for the IAd/12-26 or IEd/12-26 epitopes were added.
After an additional 18 h incubation, the amounts of IL-2
produced by the T cell hybrids were measured in the supernatants.
As shown in Fig. 3, presentation of the IAd epitope in
FcRII- and Fc
RIII-expressing cells, as well as presentation of the IEd epitope by Fc
RIII-expressing cells, all exhibited slow kinetics and were blocked by cycloheximide.
These results indicate that both receptors induced antigen
presentation through the conventional pathway, which requires newly synthesized MHC class II molecules. The difference in the presentation of the IEd epitope is therefore
not due to the targeting of antigen to different antigen presentation pathways by the two receptors.
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Engagement of FcRIII, but not of Fc
RIIb2,
induces the activation of multiple tyrosine kinases, a rise of
cytosolic Ca2+ concentrations and, subsequently, the activation of the transcription of various genes (18). Therefore,
the selective presentation of the cryptic epitope by cells expressing Fc
RIII could be either a direct consequence of a
different intracellular targeting of antigen or an indirect effect of overall cell activation. We next attempted to test the
latter possibility in two different ways.
First, we tested whether cell activation by FcRII/ic
chimera induced a change in surface expression of a putative costimulatory molecule required only for the efficient
activation of some of the epitope-specific T cell hybrids.
The Fc
RIIb2- and Fc
RII/ic
-expressing cells were incubated overnight with or without irrelevant immune complexes (ovalbumin (OVA)-anti-OVA), in order to induce cell activation (we verified that these immune complexes induced efficient cell activation using a phosphotyrosine blot assay and IL-2 secretion, data not shown). The
cells were then fixed and reincubated with increasing doses
of 12-26 peptide and either the IAd or the IEd 12-26
epitope-specific T cell hybrids.
As shown in Fig. 4 A, the peptide was presented to both
T cell hybridomas with similar efficiencies by the unstimulated and the stimulated cells. Therefore, cell activation
through the chain did not induce any modification in the
efficiency of presentation of the 12-26 peptide to the IAd
and the IEd specific T cell hybrids. The effect of antigen internalization through Fc
RIII is therefore related to the intracellular processing of antigen.
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Second, we tested the effect of cell activation on intracellular antigen processing. FcRII/ic
expressing cells
were incubated with soluble CI
repressor in the presence
of irrelevant immune complexes (as in Fig. 4 A) in order to
engage the corresponding receptors, and induce cell activation. The cells were then fixed and reincubated with either
the IAd or the IEd 12-26 specific T hybridomas. As shown
in Fig. 4 B, activation through Fc
RII/ic
did not induce
the presentation of the cryptic epitope after fluid phase antigen uptake.
However, it was still possible that both cell activation
and efficient receptor-mediated antigen uptake were required together for the presentation of the IEd restricted
epitope. To test this possibility, the FcRIIb2 expressing cells were simultaneously incubated with
repressor-containing immune complexes (to allow efficient antigen uptake) and specific anti-mouse IgG2a goat antibodies (to induce cell activation through surface immunoglobulin). The
cells were then fixed and reincubated with the two T cell
hybrids. As shown in Fig. 4 C, cell activation through sIgG
did not induce Fc
RIIb2 expressing cells to present the
cryptic epitope. Therefore, overall cell activation is not sufficient to allow presentation of the IEd-restricted epitope
after Fc
RIIb2-mediated antigen internalization.
We concluded from
the previous series of experiments that the presentation of
the IEd-restricted epitope was not an exclusive consequence of overall cell activation, but rather resulted from
the selective intracellular targeting of antigen by FcRIII.
However, the intracellular traffic of membrane receptors
may also be related to their signal transduction capabilities.
In the case of tyrosine kinase receptors, like epidermal growth factor receptor, the kinase activity is involved in internalization (29) and endosomal trafficking (30). By analogy to PTK receptors, we reasoned that the ability of Fc
RIII (or Fc
RII/ic
) to activate PTKs after cross-linking
(17) might influence its intracellular traffic and thereby allow the generation of the IEd-restricted epitope.
We attempted to address this possibility by searching for
mutations that differentially affected internalization and cell activation by the chain. An alanine scan of the region of
the ITAM (Amigorena, S., and C. Bonnerot, unpublished
results) was performed by sequentially introducing single
point mutations in the cytoplasmic tail of Fc
RII/ic
. The
corresponding cDNAs were expressed stably in the IIA1.6
B lymphoma cells and the resulting transfectants were
tested for their ability to induce cell activation and ligand
internalization after receptor cross-linking by anti-Fc
R mAbs (Amigorena, S., and C. Bonnerot, unpublished results). Thus, we found that mutation of leucine in position
35 to alanine (Fc
RII/ic
L35A) completely abolished cell
activation without affecting receptor internalization.
As shown in Fig. 5 A, FcRII-ic
L35A chimera-expressing cells have lost the ability to secrete IL-2 after engagement of the receptor by immune complexes, whereas secretion of IL-2 was normal after engagement of surface IgG
in the same cells. In contrast, internalization of immune
complexes was not affected by the mutation (Fig. 5 B).
Therefore, leucine 35 in the
chain cytoplasmic domain is
required for cell activation but dispensable for receptor internalization.
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Since FcRII-ic
L35A
was incapable of stimulating IL-2 production or inducing
activation of PTKs (Amigorena, S., and C. Bonnerot, unpublished results) but was normally internalized, we next
tested its ability to induce the presentation of the IAd- and
IEd-restricted 12-26 peptide. As shown in Fig. 6 A, Fc
RII-ic
L35A-expressing cells presented soluble CI
repressor IAd 12-26 epitope with the same efficiency as Fc
RIII-expressing cells. When the cells were incubated with
antigen-IgG complexes, both Fc
RIII-ic
- and Fc
RII-
ic
L35A-expressing cells presented the IAd epitope with
high efficiency. In contrast, only Fc
RII-ic
-expressing cells
presented the IEd 12-26 epitope, whereas absolutely no presentation of this epitope was observed after immune complexes internalization by Fc
RII-ic
L35A-expressing cells
(Fig. 6 A, lower panels). The efficiencies of presentation of
the 12-26 peptide to the IAd and IEd-specific T cell hybridomas were similar for the Fc
RII-ic
- and the Fc
RII-
ic
L35A-expressing cells (Fig. 6 B). The mutation of leucine 35 therefore affected the presentation of the IEd 12-26
epitope much more drastically than the presentation of the
same peptide with IAd.
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The two epitopes described this far concern the same or
overlapping peptides (included in the 12-26 region) associated to either IAd or IEd. We next tested whether the same
effect was found using two other IAd restricted epitopes,
IAd 46-64 and IAd 80-102. As shown in Fig. 7 (upper right),
the 46-64 epitope was efficiently presented after internalization by both FcRIIb2 and Fc
RII-ic
. In contrast, the
80-102 peptide was only presented after internalization by
the Fc
RII-ic
. As in the case of the IEd 12-26 epitope,
the IAd 80-102 epitope was not at all presented after internalization by Fc
RIIb2. In addition, as in the case of the
IEd 12-26 and IAd 12-26, the presentation of the IAd 80-
102, but not of IAd 46-64 epitope, was blocked by the
L35A mutation.
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Is the difference in the repertoire of epitopes presented
after FcRIII or Fc
RII-ic
antigen internalization limited to CI
repressor? To answer this question, we tested a
panel of six T cell hybridomas specific for various epitopes
of HEL. The efficiency of presentation of IgG-complexed
HEL to the different T cell hybrids was compared for the
Fc
RIIb2- and the Fc
RII-ic
-expressing cells. As shown
in Fig. 7 (lower panels), the IAd 11-25 and IEd 108-116
epitopes were efficently presented only after internalization by Fc
RII-ic
-expressing cells. As in the case of the CI
repressor IAd 12-26 and IAd 46-64 epitopes, the L35A mutation inhibited the efficient presentation of these two
epitopes.
As summarized in Table 1, internalization by FcRII-ic
chimeras allowed the efficient presentation of all epitopes
tested, whereas Fc
RIIb2 only presented a subset of these
epitopes. For all the other epitopes tested, Fc
RIIb2 either
allowed no presentation or induced presentation at the
same high antigen concentrations as those which also induced presentation after fluid phase uptake. As a general
rule, the epitopes which were efficiently presented after internalization by either Fc
RIII or Fc
RIIb2 were also efficiently presented by the L35 mutant receptors. In contrast, in the case of the epitopes that were presented after Fc
RIII and Fc
RII-ic
, but not presented after internalization by
Fc
RIIb2, the L35 mutation inhibited presentation. In all
cases, the L35 mutant receptors behaved like the Fc
RIIb2
in terms of epitope presentation.
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Discussion |
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The mechanisms of selection of T cell epitopes within
complex protein antigens are poorly understood. Yet this
choice is critical, on one the hand for shaping the T cell
repertoire during thymic positive and negative selection,
and, on the other hand, for the development of efficient
immune responses. Our results show that the T cell
epitopes that we have analyzed may be classified into two
discrete categories. The first category consists of the
epitopes that were efficiently presented after internalization
by either FcRII or Fc
RIII (which in most cases were
also presented, although less efficiently after fluide phase
uptake), and the second, of the epitopes that were only
presented after internalization by Fc
RIII, or the Fc
RII-
ic
chimeras (at least two of these epitopes were cryptic, i.e.,
not presented after immunization of mice with intact antigen).
Why, then, did FcRIII and Fc
RIIb2/
-chain chimeras induce the presentation of epitopes that were not presented after internalization via Fc
RIIb2 or fluid phase uptake? The selectivity in the presentation of different epitopes
between receptors is certainly not due to quantitative differences in the uptake of antigen by the presenting cells,
because (a) both Fc
RIII and the Fc
RII-chain chimeras were expressed at lower levels than Fc
RIIb2; (b) all receptors mediated the internalization of immune complexes
with similar kinetics and efficiencies (Fig. 5 B); and (c)
Fc
RIIb2 increased the efficiency of presentation of several
epitopes to the same extent or even to greater extents than
Fc
RIII and the Fc
RII-ic
chimera (Figs. 1 and 2).
Therefore, the selectivity in epitope presentation must reside in a qualitative difference between the two receptors.
One of the qualitative differences between type II and III
FcRs is in the induction of cell activation. The
chains
have been shown to mediate activation through Fc
RIII, due
to a conserved ITAM, present in its cytoplasmic tail. In
contrast, Fc
RIIb2 bears no ITAM and does not induce
cell activation. Upon activation through Fc
RIII, IIA1.6
cells produced a cytokine that supports the growth of the
CTLL.2 IL-2-dependent cell line (18). We checked very carefully that the IL2 production measured by the CTLL.2
cells in our antigen presentation experiments was due to
the T cell hybrids and not to the presenting cells. If the
presenting cells were incubated with CI
repressor or
HEL immune complexes in the absence of T lymphocytes,
background proliferation of the CTLL.2 cells was observed
(data not shown). In addition, all the experiments were repeated with paraformaldehyde-fixed APCs, a treatment
that completely prevents cytokine secretion (Fig. 3 and data
not shown). Under these conditions, only the T cell hybrids can produce IL-2.
The coactivation experiments (Fig. 4) show that presentation of the FcRIII-restricted epitopes was not a consequence of overall cell activation. The most convincing result from this point of view may be the one shown in Fig. 4
C. When immune complexes were internalized by Fc
RIIb2-expressing cells in the continuous presence of F(ab)
2 fragments of anti-IgG antibodies, which efficiently induced cell
activation through mIgG (18), no presentation of the Fc
RIII-restricted epitopes was observed. It could be argued here
that cell activation through sIgG might be qualitatively different from cell activation through Fc
RIII. Although we
can not completely exclude this possibility, we found that
direct antigen internalization though sIg (using A20 B cells
expressing anti-DNP sIg and DNP-derivatized CI
repressor) allowed presentation of the same epitopes as Fc
RIII (data not shown). This shows that although presentation of Fc
RIII-restricted epitopes also occurred after
antigen internalization by sIg, overall cell activation through
sIg did not induce the presentation of those epitopes when
the antigen was internalized through Fc
RIIb2. Therefore, antigen must be physically associated to the receptor for the efficient presentation of the Fc
RIII-restricted epitopes.
The simplest and most direct explanation for these results
is that FcRIII targeted antigens to a particular intracellular compartment that Fc
RIIb2 can not access. Several of our
experiments support this possibility and, taken together,
shed light on various aspects of this putative transport path.
First, the differential generation of T cell epitopes after internalization by Fc
RIIb2 and III was not due to selective
antigen targeting to the conventional and recycling presentation pathways, since presentation of both dominant and
cryptic epitopes had slow kinetics and were blocked by cycloheximide. This was an important possibility to test because it has recently been shown that the epitopes presented
by both pathways may be different (9).
Second, a single point mutation (of leucine 35 to alanine)
blocked the presentation of FcRIII-restricted epitopes, without affecting the presentation of the epitopes efficiently
presented after Fc
RIIb2 internalization. In other words,
the chimeric Fc
RII-ic
L35A behaved exactly like Fc
RIIb2
in terms of epitope presentation. Interestingly, L35A mutation
was first identified as blocking cell activation, but not receptor
internalization. Therefore, like Fc
RIIb2, L35A chimeric
receptors are not capable of inducing activation of cytosolic
PTKs. These results suggest that association to tyrosine kinases or tyrosine phosphorylation of the receptor itself is required for the efficient generation of the Fc
RIII-restricted epitopes.
In the case of PTK receptors, like the epidermal growth
factor receptor, the kinase activity has been shown to directly affect intracellular transport, since in addition to internalization (29) transport from endosomes to lysosomes
requires the kinase activity (30). Although the FcRIII-associated
chain contains no intrinsic PTK activity, it has
been shown to associate to Syk after phosphorylation of its
ITAM by PTK of the src family (17). In addition, for
ITAM-containing receptors like Fc
RIII, association to
Syk is required for transport from endosomes to lysosomes
(Amigorena, S., and C. Bonnerot, unpublished results).
However, transport to lysosomes is unlikely to be determinant for presentation of the Fc
RIII-restricted epitopes,
since Fc
RIIb2 efficiently mediated transport of immune
complexes to Percoll heavy, degradative compartments (31).
Several recent studies have analyzed the intracellular locations where peptide loading occurs and showed that multiple endocytic compartments may be involved (32). However, little is known about the actual influence of the intracellular compartments where peptide loading occurs on the generation of particular T cell epitopes (34). Indeed, besides the affinity of peptides for MHC class II molecules, other factors are critical for the formation of T cell epitopes. The proteolytic generation of peptides, or their degradation depends on specific proteolytic enzymes. The loading of different peptides is differentially affected by HLA-DM (35). Therefore, the endosomal environment most likely determines the nature of the peptides loaded onto MHC class II molecules (34).
We have previously shown that a particular population
of vesicles in the cells used here, class II vesicles or CIIV,
are an important site of peptide loading (36, 37). Therefore, it is possible that FcRIIb2 and Fc
RIII (or Fc
RII-
ic
) have differential accessibilities to CIIV, thus accounting for the generation of different peptides after internalization by these two receptors. Directly testing this possibility
will require an extensive analysis of Fc
RIIb2 and Fc
RIII
intracellular transport, which will next be undertaken.
Physiologically, FcRII and Fc
RIII are expressed on
professional APCs such as macrophages and dendritic cells
(20, 38). Furthermore, the relative rates of expression of
these two receptors is regulated by cytokines such as IFN-
and TNF-
(20). Our results therefore suggest that the selection of peptides presented to T cells depends on the antigen receptors expressed by the APCs which are themselves dependent on their microenvironment.
The consequences of antigen receptor expression on the
presentation of different epitopes may also be relevant to
autoimmunity. Indeed, it has recently been proposed that
only dominant epitopes from autoantigens participate in
thymic selection, since cryptic epitopes would not be presented under normal conditions (3, 39). T cells specific for
cryptic epitopes from autoantigens therefore are not tolerized and may become pathogenic if cryptic epitopes are
presented in the periphery for any particular reason. In the
recent past, several groups have shown various mechanisms for unveiling cryptic epitopes in vitro (for review see reference 39). Interestingly, after downregulation of a membrane receptor antigen by its ligand, cryptic epitopes may
be revealed (40). Changes in the hierarchy of epitope presentation may also occur in different APCs or when antigen is complexed to other proteins (such as antibodies)
(15). Our results indicate a novel mechanism of cryptic
epitope unveiling, i.e., receptor-mediated antigen uptake.
The participation of FcRIII in revealing cryptic epitopes in vivo, as well as the possible involvement of FcRs in
epitope spreading and in autoimmunity, have now to be
evaluated.
![]() |
Footnotes |
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Received for publication Received for publication 15 September 1997 and in revised form 2 December 1997..
We are grateful to Drs. Ming-Zong Lai and Eli E. Sercarz for providing us with several anti-CI ![]() |
References |
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