By
From * INSERM CJF 95-01, Institut Curie, Section Recherche, 75005 Paris, France; Unité
d'Immuno-allergie, Institut Pasteur, 75015 Paris, France; and § Laboratoire d'Immunologie des
Pathologies Infectieuses et Tumorales, Institut Cochin de Genetique Moleculaire, INSERM U445,
75013 Paris, France
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Abstract |
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Stimulation of CD4+ helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally
distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-/Ig-
heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its
Ig-
-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary
experimental approaches: mutational analysis of the Ig-
cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide-
MHC class II complexes through antigen targeting by BCR subunits.
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Introduction |
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Antigen-presenting cells (APCs), to stimulate CD4+ helper T lymphocytes, must capture and process exogenous antigens into antigenic peptides which associate with MHC class II molecules in the endocytic pathway (1, 2). The formation of peptide-class II complexes classically occurs in endolysosomal compartments (called MIIC or CIIV), which accumulate newly synthesized class II molecules (3), or in recycling endosomes, which contain mature class II molecules internalized from the cell surface and then recycled back to the plasma membrane (6, 7). Therefore, peptide-class II complexes may be generated at any point throughout the endocytic pathway (8). This study delineates how antigen receptors might determine the delivery of antigens to different compartments and thus could modulate MHC class II-restricted antigen presentation.
B lymphocytes mediate efficient class II-restricted antigen presentation first by facilitating the uptake of soluble
antigens through clonally distributed receptors (BCRs)1
(9). These receptors are multichain complexes composed of a ligand-binding module, the membrane immunoglobulin
(mIg), and a transducing module, the Ig-/Ig-
heterodimer (10, 11), both chains containing a conserved peptidic
motif located in their cytoplasmic tails. This immunoreceptor tyrosine-based activation motif (ITAM [12, 13]) consists of conserved amino acid residues (D2xY2xL7xY2xL)
which couple receptors to intracellular effectors, leading to
cell activation (11) and antigen internalization (14). Antigen recognition by mIg triggers the activation of Src family phosphotyrosine kinases (PTKs), resulting in tyrosine phosphorylation of Ig-
/Ig-
ITAM (17) and the recruitment of PTK Syk by phosphorylated ITAM (18, 19),
which turns on different signaling pathways (11). Concomitantly, the BCR facilitates the endocytosis of soluble antigens and their access to endosomal/lysosomal compartments, where the antigens are degraded into peptides
which then associate to MHC class II molecules to be presented to T lymphocytes (20). Therefore, another important function of the BCR is to address soluble antigens to
the intracellular sites of peptide loading on class II molecules (5, 15). To understand the role of the BCR in antigen presentation, a critical question should therefore be addressed: Do signal transduction effectors, involved in
BCR-mediated B cell activation, determine the presentation of peptide-MHC class II complexes through BCR-mediated antigen internalization?
The functions of BCR subunits in the intracellular targeting of the BCR partially answer this question. Indeed,
the BCR is involved in the delivery of mIg-bound antigens
to newly synthesized MHC class II molecules accumulating
in endosomal compartments (5). The association of the Ig-/Ig-
sheath to mIg may account for the ability of the
BCR to induce both specific endosomal targeting and efficient antigen presentation (14, 15). In the complex structure of the BCR, both Ig-
and Ig-
cytoplasmic domains
contain internalization signals (16), but only the Ig-
cytoplasmic tail was able to target antigens to newly synthesized
MHC class II, whereas Ig-
cytoplasmic tail contains targeting signals that direct antigens towards recycling class II
(16). In addition, the cytoplasmic tails of Ig-
and Ig-
were reported to interact with distinct cytoplasmic effectors
(21) and to trigger different signaling pathways (22). Thus,
the cytoplasmic domain of Ig-
interacts with specific cytoplasmic proteins that might be necessary for the intracellular targeting of the antigen-bound BCR to MHC class II
compartments. Two related questions were addressed specifically: Does BCR-mediated antigen targeting to class II
compartments have any immunological consequences for
the selection of specific T cell epitopes, and what are the
cytoplasmic effectors of this particular endosomal sorting of
antigen-bound BCR to class II compartments? We show
that the delivery of mIg-bound antigens, through Ig-
, to
newly synthesized MHC class II molecules induced the
presentation of a large spectrum of T cell epitopes derived from
repressor (C1), hen egg lysozyme (HEL), or ovalbumin (OVA). In contrast, antigen targeting to recycling
class II through the Ig-
cytoplasmic tail led to the presentation of only some of these epitopes. We then identified a
signal in the Ig-
cytoplasmic tail that was responsible for
the presentation of these T cell epitopes and the activation
of syk tyrosine kinase. Endogenous syk activation and presentation of T cell epitopes, induced through the Ig-
cytoplasmic tail, were abolished by the overexpression of a syk
mutant devoid of kinase domain, which had no effect on antigen presentation induced by the Ig-
cytoplasmic tail.
Therefore, the recruitment of syk tyrosine kinase by the Ig-
BCR subunit in the endosomal pathway may be an important step in potentiating secondary B cell immune response
by addressing antigens to MHC class II compartments.
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Materials and Methods |
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B Lymphoma Cell Lines.
The B lymphoma IIA1.6 is an FcStable Expression of Syk Dominant Negative Mutant.
The syk dominant negative mutant was constructed by joining the EcoRI-KpnI fragment of rat syk cDNA (27) to a KpnI-XbaI PCR fragment amplified from a hemagglutinin (HA)-tag-containing plasmid. The sequence coding for the HA-tag is recognized by the mAb 12CA5. The resulting truncated syk has been sequenced, and corresponded to the first 260 amino acids of the rat syk cDNA fused to the sequence GSGYSYDVPDYA. This construct was then inserted in an Sr-driven expression vector bearing the puromycin gene resistance. After linearization with ScaI restriction enzyme, 50 µg of DNA was used to electroporate B cells expressing FcT Cell Hybridomas.
T cell hybridomas and cells used in antigen presentation assays were cultured in RPMI 1640 containing 10% FCS, 10 mM glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2
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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 of antigens complexed or not with specific antibodies. TheDetection of Tyrosine Kinase Activation.
Cells were preincubated at 4°C with or without 20 µg/ml of 2.4G2 for 30 min, and then washed twice with RPMI 1640. 5 × 106 cells were then stimulated with prewarmed F(ab')2 fragments of mouse anti-rat antiserum (50 µg/ml) at 37°C for 2 min. As a positive control, untreated cells were stimulated with prewarmed F(ab')2 fragments of anti-IgG antibodies (30 µg/ml) for 2 min at 37°C. The stimulated cells were washed and resuspended in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100) containing a cocktail of protease inhibitors and tyrosine phosphatase inhibitors (5 mM NaF, 5 mM EDTA, 1 mM Orthovanadate). Phosphoproteins were immunoprecipitated with agarose-coupled antiphosphotyrosine antibodies PT-66 (Sigma Chemical Co.) or a rabbit antiserum raised against the 11 COOH-terminal amino acids from human syk coupled with KLH. The phosphoproteins were then run on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membrane (Millipore Corp.) to be detected with the antiphosphotyrosine mAb Py20 coupled with HRP (ICN Biomedicals, Orsay, France). Chemiluminescence was detected with a commercial kit (Boehringer Mannheim Corp.) by exposure of the filters to X-omat film (Eastman Kodak Co., Rochester, NY). The filters were then stripped by a 30-min incubation at 50°C in 50 mM Tris, pH 6.8, 2% SDS, 100 mMIC Internalization.
The cells were washed once in internalization buffer (RPMI 1640, 5% FCS, 10 mM glutamine, 5 mM sodium pyruvate, 50 mM 2-ME, and 20 mM Hepes, pH 7.4) and incubated with HRP-anti-HRP IC for 2 h at 4°C (107 cells/ml). IC 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 IC, 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 for each time point were either left in PBS at 4°C to measure cell surface HRP IC, or incubated in Triton X-100 (0.1%) for 5 min at room temperature to measure the total amount of HRP IC. The HRP was revealed by adding substrate buffer (0.5 mg/ml OPD [Sigma Chemical Co.] and 0.12% H2O2 in 0.05 M phospho-citrate buffer, pH 5.0) at 4°C. The reaction was stopped with 6 N HCl, and the change in color was determined spectrophotometrically at 492 nm. ![]() |
Results |
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The binding of exogenous antigens to the BCR potentiates antigen uptake and degradation in B lymphocytes. To determine if BCR-mediated
antigen uptake has any consequences for the presentation of various T cell epitopes, we compared antigen presentation after fluid phase or BCR-mediated antigen uptake using A20 B cells expressing or not expressing TNP-specific
IgM. The phage repressor (C1) was used as antigen.
TNP-coupled (3 µg/ml) or -uncoupled C1 (30 µg/ml)
was incubated for various times with A20 cells expressing anti-TNP IgM (A20 anti-TNP). The cells were then fixed,
and C1 presentation was detected with two T hybridomas
specific for the same peptide in association with IA or IE of
the H2d haplotype: the hybridoma 24.4, which recognized
a dominant T cell epitope (IAd 12-26), and the hybridoma
26.1, which recognized a cryptic T cell epitope (IEd 12-26)
(28). These two T cell epitopes were detected on anti-TNP A20 cells after 2 h of incubation with TNP-coupled
antigens (Fig. 1 a). The incubation of presenting cells with
the protein synthesis inhibitor cycloheximide for 3 h before
and during incubation with TNP-coupled C1 completely
abolished BCR-mediated antigen presentation (Fig. 1 a). In
contrast, fluid phase uptake of uncoupled C1 (Fig. 1 a) only
led to the presentation of the IAd 12-26 epitope after a lag
time of 4 h. Under these conditions, antigen presentation
was partially resistant to the cycloheximide treatment.
Therefore, BCR-mediated antigen internalization has a
dual function in antigen presentation: by addressing antigens to newly synthesized MHC class II molecules, BCR
induces the presentation of T cell epitopes which are not
presented after fluid phase antigen uptake.
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The cytoplasmic tail of the BCR Ig- subunit is able to
target antigen to newly synthesized MHC class II (16).
Therefore, we investigated whether the direct targeting of
C1 through the cytoplasmic tail of Ig-
is able to induce
the presentation of the dominant, IAd 12-26, and the cryptic, IEd 12-26, T cell epitopes. IIA1.6 B cells (Fc
R-defective variant of A20 B cells) expressing Fc
R chimeras containing the cytoplasmic tail of Ig-
or Ig-
(Fc
R/Ig-
and Fc
R/Ig-
, respectively [22]) were incubated with C1
(3 µg/ml) complexed with two anti-C1 mAbs (15 µg/ml
each) to target C1 on Fc
R chimeras. After various periods of culturing at 37°C, the presenting cells were fixed and incubated with the two T cell hybridomas 24.4 and 26.1. The presentation of IAd 12-26 and IEd 12-26 T cell epitopes
occurred with similar kinetics on Fc
R/Ig-
-expressing cells (Fig. 1 b) and A20 anti-TNP (Fig. 1 a). In addition,
the incubation of presenting cells with cycloheximide inhibited antigen presentation induced by direct targeting of
C1 through the Ig-
cytoplasmic tail (Fig. 1 b). Surprisingly, similar experiments with Fc
R/Ig-
-expressing cells
revealed that endosomal targeting of C1 to recycling class
II by the Ig-
cytoplasmic tail (16) was not able to induce
presentation of the cryptic IEd 12-26 T cell epitope,
whereas the dominant IAd 12-26 T cell epitope was efficiently and quickly presented by recycling class II molecules (no effect of cycloheximide treatment; Fig. 1 b).
Using HRP-anti-HRP rabbit IgG IC as a multivalent
ligand of FcR chimeras, we observed that both Ig-
and
Ig-
chimeras internalized IC with similar efficiency and
kinetics (Fig. 1 c). Therefore, the incapacity of the Ig-
chimera to induce presentation of the IEd 12-26 epitope
was not due to inefficient ligand internalization. These results suggested that the BCR induced presentation of a dominant and a cryptic T cell epitope by newly synthesized
class II molecules through a targeting signal contained in
the cytoplasmic tail of Ig-
.
Therefore, a function of the BCR in antigen presentation might be to induce the presentation of a large spectrum of T cell epitopes by targeting antigens to newly synthesized class II. To address this question, we used a panel
of T cell hybridomas specific for various peptides deriving
from C1, HEL, or OVA, restricted by the IA or IE H2d
class II molecules. The efficiency of presentation of IgG-complexed antigens was compared in FcR/Ig-
- and
Fc
R/Ig-
-expressing cells (Table 1). The targeting of
these different proteins through the cytoplasmic tail of Ig-
induced the presentation of all epitopes tested, i.e., C1 IAd
48-64 and IAd 80-102, HEL IEd 108-116 and IAd 44-62, and the IAd-restricted OVA epitopes recognized by the
3DO54.8 and the 3DO18.3 T cell hybridomas. In contrast,
the processing of the same IC by Fc
R/Ig-
-expressing
cells led only to the presentation of some of these T cell
epitopes (Table 1). Antigen targeting by the Ig-
or Ig-
cytoplasmic tails through newly synthesized or recycling
class II molecules, respectively, did not lead to the presentation of the same peptides. These results raised the question, What is the intracellular mechanism of specific presentation
of these T cell epitopes by Ig-
and BCR-mediated antigen
internalization?
The Ig- and Ig-
cytoplasmic tails induce the activation of different signaling pathways (22) and
interact with distinct cytoplasmic effectors (21). Therefore,
the selective presentation of epitopes by B cells expressing
anti-TNP-specific IgM or Fc
R/Ig-
chimera might be
either a direct consequence of a different intracellular targeting of antigen, or an indirect effect of B cell activation which might induce surface expression of putative costimulatory molecules required for the efficient activation of T
cell hybridomas.
To distinguish between these possibilities, the FcR/
Ig-
- and Fc
R/Ig-
-expressing cells were incubated with
or without irrelevant IC (OVA, 15 µg/ml; anti-OVA rabbit IgG, 50 µg/ml), to induce B cell activation (not
shown). After 24 h, the cells were then fixed and incubated
with increasing concentrations of 12-26 peptide and either
the 24.4 or the 26.1 T cell hybridoma. The peptide was
presented to both T cells with similar efficiency by the unstimulated and the stimulated cells (Fig. 2 a). Similar results
were obtained when the cells were fixed after 6 or 12 h incubation with IC (not shown). Therefore, the signaling
pathways induced through Ig-
and Ig-
did not modify
the efficiency of presentation of the 12-26 peptide by IAd
and IEd molecules.
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Nevertheless, the BCR or Ig- cytoplasmic tail could
induce signaling events that modified the composition of
processing compartments, leading to the presentation of the
IEd 12-26 epitope. The effect of Ig-
-induced cell activation on intracellular processing of C1 was assessed by coincubating Fc
R/Ig-
-expressing cells with irrelevant IC (as
shown in Fig. 2 a) in order to trigger cell activation
through the Ig-
cytoplasmic tail, and with soluble C1 (30 µg/ml), which typically only induced the presentation of the IAd 12-26 epitope (see Fig. 1 a and Fig. 3 b). After 24 h,
the cells were fixed and incubated either with 24.4 or 26.1 T cells (Fig. 2 b). B cell signaling through Ig-
did not lead
to the presentation of the IEd 12-26 epitope and did not increase the presentation of the IAd 12-26 epitope after the
fluid phase uptake of the C1 (Fig. 2 b).
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However, fluid phase antigen uptake is not very efficient
in B cells, and could be a limiting step that leads to failure
to generate enough peptides to induce the presentation of
the IEd 12-26 epitope. To test this possibility, the FcR/
Ig-
-expressing cells were simultaneously incubated with
IgG1-complexed C1 (to allow efficient generation of 12-26 peptide) and with specific anti-IgG2a antibodies (to induce
B cell activation through endogenous mIg). The cells were
then fixed and incubated with the two T cell hybridomas.
BCR stimulation did not induce Fc
R/Ig-
-expressing cells to present IEd 12-26 epitope and did not increase presentation of the IAd 12-26 epitope (Fig. 2 c). Therefore, the
stimulation of the whole BCR was not sufficient to allow
presentation of the cryptic IEd 12-26 epitope after C1 internalization through the Ig-
cytoplasmic tail; however,
the 12-26 peptide was efficiently generated since it was
presented by IAd molecules, although the direct targeting
of C1 to the TNP-specific BCR was able to induce presentation of the same T cell epitope (Fig. 1 a). In addition,
the failure of the Ig-
chimera to induce presentation of
cryptic IEd 12-26 epitope was not due to a lower sensitivity
of the T cell hybridoma, as suggested by the results obtained for fixed presenting cells (shown in Fig. 2 a), since
similar results were obtained with unfixed cells (Table 1).
Indeed, on unfixed cells, the two T cell hybridomas detected the IAd 12-26 and IEd 12-26 T cell epitopes with
similar sensitivity (see Fig. 3 c, and data not shown). These
data suggested that the BCR Ig-
subunit contains a signal
that can recruit proteins involved in the antigen targeting
toward endosomal compartments, where the 12-26 peptide may be generated and associated to IAd and IEd MHC class
II molecules.
The two major functions assigned to the Ig-/Ig-
heterodimer are intracellular signaling and receptor internalization, both dependent on
ITAMs contained in their cytoplasmic tails. To discriminate between these two functions of the BCR subunits, we
analyzed antigen presentation through Fc
R/Ig-
chimeras containing mutated tyrosine residues, previously shown
to prevent the activation of tyrosine kinases (25).
First, we analyzed IC internalization by the FcR/Ig-
chimera in which tyrosine residues of Ig-
ITAM were replaced by alanines (Fc
R/Ig-
Y23-A Y34-A). HRP-
anti-HRP rabbit IgG IC were bound to wild-type and mutated Fc
R/Ig-
-expressing cells at 4°C, then the cells
were cultured at 37°C for various times to allow receptor internalization. Internal and cell surface fractions of IC
were determined by an enzymological assay. As shown in
Fig. 3 a, both wild-type and mutated Ig-
chimeras internalized IC with similar kinetics. Tyrosine residues of Ig-
ITAM, as for Ig-
(37), did not appear to be required for
the internalization of multivalent ligands.
Therefore, we evaluated whether mutation of these tyrosine residues, which inhibit B cell activation, altered the
presentation of the dominant, IAd 12-26, and the cryptic,
IEd 12-26, T cell epitopes. Cells were incubated with various concentrations of C1 complexed or not with two anti-C1 mAbs (15 µg/ml each) and the two T cell hybridomas.
Although wild-type and double-mutated FcR/Ig-
chimeras were able to induce presentation of the IAd 12-26 epitope at low concentrations of IgG-complexed C1, no
presentation of the cryptic IEd 12-26 epitope was detected
when IC were internalized through the Fc
R/Ig-
Y23-A
Y34-A chimera (Fig. 3 b). We verified that stimulation of
the 24.4 and 26.1 T cell hybridomas was obtained at similar
concentrations of 12-26 peptides with Fc
R/Ig-
- or Fc
R/Ig-
Y23-A Y34-A-expressing cells (Fig. 3 c). In
addition, we obtained similar results with B cells expressing
Ig-
chimera where the first (Fc
R/Ig-
Y23-A) or the
second (Fc
R/Ig-
Y34-A) tyrosine residue of Ig-
ITAM was mutated to alanine (Table 1). The mutation of
tyrosine residues of the Ig-
ITAM also affected the presentation of IAd 48-62 HEL epitopes, but had no effect on
the IEd 108-116 T cell epitope derived from HEL when
the antigen was complexed with specific antibodies (Table
1). Therefore, we concluded that mutations that abolished
the ability of the Ig-
cytoplasmic tail to induce B cell activation also blocked the intracellular targeting of antigens,
determining the presentation of some, but not all, T cell
epitopes.
After BCR stimulation, tyrosine kinases phosphorylate ITAM tyrosine residues, enabling
them to associate with the two SH2 domains of syk tyrosine
kinase (18, 19). Since mutation of the tyrosine of Ig-
ITAM inhibits the generation of some T cell epitopes, we
further evaluated the role of syk tyrosine kinase in antigen
presentation through the BCR Ig-
subunit.
First, we tested whether the double mutation of both tyrosine residues contained in Ig- ITAM affected syk activation through the cytoplasmic tail of Ig-
. Wild-type and
mutated Fc
R/Ig-
-expressing cells were stimulated for
2 min at 37°C through Fc
Rs (using the anti-Fc
R mAb,
2.4G2), or through endogenous mIgG2a (using F(ab')2 fragments of rabbit anti-mouse IgG), or, in a control experiment, with irrelevant F(ab')2 fragments of mouse anti-
rat IgG. Cell lysates were prepared, and phosphotyrosine
proteins were immunoprecipitated with agarose-coupled
PT66 and analyzed by immunoblotting with HRP-coupled antiphosphotyrosine mAb Py20 or rabbit anti-syk
antisera. As shown in Fig. 4 a, B cell stimulation through
endogenous BCR or the Ig-
chimera induced the phosphorylation of similar proteins, among them the syk tyrosine kinase (Fig. 4 b). Mutated Ig-
chimera lacked the
ability to induce phosphorylation of syk as well as of other
kinases substrates, although endogenous mIgs were functional in these cells (Fig. 4). In addition, the kinase activity
of syk was increased after Fc
R/Ig-
stimulation but not
after cross-linking of the tyrosine-mutated Ig-
chimera as
measured by phosphorylation of a specific substrate of syk
kinase, a GST fusion protein containing HS1 polypeptide
(reference 36; Fig. 4 b). Therefore, the Ig-
cytoplasmic
tail is able to activate syk tyrosine kinase and to mediate the
presentation of numerous T cell epitopes via a targeting signal located in the Ig-
ITAM.
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To further evaluate the role of syk tyrosine kinase in antigen presentation by B cells, we devised a strategy to specifically block the recruitment and activation of syk by the
Ig- subunit of the BCR. For this purpose, an inactive
form of syk was stably expressed in Fc
R/Ig-
-expressing
B cells. A cDNA encoding the two SH2 domains of syk
tagged with an HA peptide but lacking its kinase domain
was inserted in an expression vector bearing the puromycin
resistance gene. After transfection, expressing cells were selected with puromycin and cloned. The clones were
screened for the expression of HA-tag by intracellular
FACScan® analysis using FITC-coupled anti-HA mAb
(not shown). Positive clones were further characterized by
Western blotting using a rabbit antiserum specific for a
peptide contained in the NH2 portion of the syk SH2 domains present both in dominant negative mutant (32 kD)
and in endogenous syk (70 kD; Fig. 5 a). The activation of
endogenous syk kinase through the Ig-
cytoplasmic tail
was then analyzed in the A2 and G2 clones.
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For this purpose, the two clones were left unstimulated
or stimulated through the Ig- chimera or endogenous
mIgG2a, then phosphoproteins were immunoprecipitated
and analyzed by Western blotting using Py20 and anti-syk
antibodies. Overexpression of the syk dominant negative
mutant prevented the phosphorylation of various intracellular proteins, as well as endogenous syk kinase, induced
through the Ig-
cytoplasmic tail. Although global tyrosine phosphorylation was affected, syk phosphorylation was
only marginally decreased after B cell stimulation through
endogenous mIg (Fig. 5 b). Similarly, we found that overexpression of syk SH2 domains led to a substantial inhibition of kinase activity in syk immunoprecipitates obtained
after cell stimulation through the Ig-
cytoplasmic tail, whereas only a marginal effect was found after endogenous
mIg stimulation, although the same quantity of syk protein
was immunoprecipitated (Fig. 5 c). These results suggested
that syk could be activated through an Ig-
-dependent and
-independent pathway.
To verify this hypothesis, we examined the effect of
overexpression of syk dominant negative mutant on syk
activation through the cytoplasmic tail of Ig-. Clones
overexpressing the syk dominant negative mutant were
obtained in B cells expressing the Fc
R/Ig-
chimera. The
E8 and E10 clones, shown in Fig. 6 a, were analyzed. Syk
kinase activity and phosphorylation could not be inhibited by overexpression of syk SH2 domains after stimulation of
the Ig-
chimera (Fig. 6 b). In conclusion, both subunits
of the Ig-
/Ig-
heterodimer were able to activate syk
tyrosine kinase, but only syk activation through Ig-
was
inhibited by the syk dominant negative mutant. This model
allowed us to evaluate the role of syk kinase in MHC class
II-restricted antigen presentation through the subunits of
the BCR.
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Using HRP-anti-HRP rabbit IgG IC, we first established that the overexpression of the syk dominant negative
mutant did not affect the internalization of cross-linked Ig-
chimera (Fig. 7 a) or Ig-
chimera (not shown). The presentation of C1 was next analyzed in cells overexpressing
the syk dominant negative mutant. No presentation of the
cryptic IEd 12-26 epitope was detected when IC were internalized through the Fc
R/Ig-
chimera in cells overexpressing the two syk SH2 domains, whereas presentation of
the dominant IAd 12-26 epitope was only slightly reduced
(Fig. 7 b). No effect of the syk dominant negative mutant
was observed with Fc
R/Ig-
-expressing cells (Fig. 7 b),
although the cells stimulated the 24.4 and 26.1 T cell hybridomas at similar concentrations of 12-26 peptide (Fig. 7
c). In addition, overexpression of the syk dominant negative mutant slowed down presentation of the IAd 12-26 epitope after the internalization of IgG-complexed C1
through the Ig-
chimera (Fig. 7 d ). Therefore, the overexpression of the syk dominant negative mutant seemed
able to modulate the presentation of T cell epitopes which
are specifically generated by antigen targeting to newly
synthesized, such as the cryptic IEd 12-26, T epitope issuing from the C1. The syk tyrosine kinase, a major effector
of the BCR-induced signaling pathway, therefore, is also
involved in BCR-mediated antigen presentation by newly synthesized MHC class II molecules.
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Discussion |
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Capture of antigens by BCRs is the first step in antigen
entry into the endosomal pathway, where antigenic peptides are generated and associated with newly synthesized
MHC class II molecules. We report here that antigen targeting to newly synthesized class II molecules through the
BCR or Ig--associated subunit induces the presentation
of a larger spectrum of T cell epitopes than antigen targeting to recycling class II molecules. Using mutagenesis analysis of the Ig-
cytoplasmic tail and stable overexpression in B cells of a syk dominant negative mutant, we describe
evidence that signal transduction effectors of B lymphocyte
activation are involved in the generation of peptide-MHC
class II complexes.
How do Ig- and Ig-
cytoplasmic tails induce presentation of different T cell epitopes? Quantitative differences
in the rate of antigen internalization can be excluded, since
the Ig-
chimera was expressed at a lower level than the
Ig-
chimera (22), and since both chimeric receptors mediated internalization of IC with similar kinetics and efficiency (Fig. 1 c [16]). In contrast, the kinetics of antigen
presentation induced by the Ig-
and Ig-
cytoplasmic tails
demonstrated that they target antigens to newly synthesized
or recycling pools of MHC class II molecules, respectively. Analysis of various T cell epitopes provides new evidence
to further distinguish these two antigen-presentation pathways. Thus, antigen internalization via the Ig-
, but not
the Ig-
, chimera stimulated the DO18.3 hybridoma (Table 1), which recognizes an IAd-restricted OVA T cell
epitope specifically generated in invariant chain-positive
presenting cells (32, 38). In contrast, the invariant chain-
independent epitope recognized by DO54.8 hybridoma (32) is presented after antigen internalization through the Ig-
or Ig-
chimera. These data suggest that the Ig-
cytoplasmic tail targets antigens to endosomal compartments,
where class II molecules transiently accumulate through an
invariant chain-dependent mechanism which characterizes
newly synthesized class II molecules. In addition, the data
also supported the hypothesis that some T cell epitopes
cannot be generated in an alternative antigen-presentation pathway defined by compartments where class II molecules
cycle with the cell surface (6, 7). Indeed, antigen targeting to recycling class II molecules through Ig-
did not induce
the stimulation of DO18.3 hybridoma or other T cell hybridomas specific for cryptic or subdominant epitopes derived from the
repressor and HEL. Therefore, the two
MHC class II-restricted antigen pathways can be functionally distinguished on the basis of T cell epitopes they are
able to efficiently generate. However, the complete BCR as well as the Ig-
chimera address antigen to the newly
synthesized pool of MHC class II molecules, presenting a
large spectrum of peptides. Therefore, the Ig-
subunit is
dominant over Ig-
in this process and accounts for the
ability of the BCR to induce efficient antigen presentation
during secondary in vivo immune responses. This conclusion raised a new question: What is the intracellular mechanism of BCR- or Ig-
-mediated antigen presentation?
Two distinct functions have been assigned to Ig- and
Ig-
: first, to address antigen to different pools of MHC
class II molecules (16), and second, to induce different signaling pathways (22, 25). Therefore, the overall cell activation induced by Ig-
or the complete BCR might be responsible for the ability of the Ig-
chimera to induce
stimulation of numerous T cells. However, this hypothesis
is unlikely because, first, the stimulation of 12-26 IAd- and
IEd-restricted T cell hybridomas by Ig-
chimera-expressing cells was not modified by chimera cross-linking, and
second, the overall cell activation induced by BCR cross-linking was not able to increase presentation of the IAd 12-26 epitope or to induce presentation of the IEd 12-26 epitope
by Ig-
chimera-expressing cells. These data indicated that
potential alterations in antigen processing during cell activation do not account for induction of presentation of
these T cell epitopes. In addition, we excluded the possibility that BCR or Ig-
chimera cross-linking increased cell
surface expression of MHC class II or costimulatory molecules such as B.7 or CD40 ligand (not shown). In contrast,
mutagenesis analysis identifies, in the Ig-
cytoplasmic tail,
a targeting signal able to induce efficient antigen presentation of numerous peptides. Indeed, mutation of tyrosine
residues contained in the Ig-
ITAM, which completely
abolish signal transduction (25, 39, 40), affects Ig-
-mediated antigen presentation, whereas similar mutation in Ig-
ITAM did not affect BCR internalization and BCR-mediated antigen presentation (37). These results indicate that
tyrosine residues of Ig-
and Ig-
ITAM are differently involved in the recruitment of cytoplasmic effectors mediating antigen presentation.
Tyrosine mutants of the Ig- ITAM also provide a new
tool to dissociate the two major functions of the BCR,
ligand internalization and signal transduction, since these
mutations block activation of most of the tyrosine kinases
but had no effect on receptor internalization. In addition,
these mutations specifically alter the presentation of some,
but not all, T cell epitopes and inhibited the phosphorylation and activation of syk tyrosine kinase. The role of the
kinase activity of syk in BCR-mediated antigen presentation was investigated using B cells overexpressing the two
SH2 domains of syk. This mutant protein may be recruited
by tyrosine-phosphorylated ITAM and should act as a
dominant negative mutant of ITAM function. Indeed, in
cells overexpressing the syk mutant, the activation of endogenous syk kinase was blocked after Ig-
chimera stimulation and was inhibited after BCR stimulation, whereas
syk activation through Ig-
was not affected by overexpression of the syk mutant. It is probable that the ITAMs of
both BCR subunits activated syk by different mechanisms
as suggested by previous data indicating that the Ig-
and
Ig-
cytoplasmic tails interact with distinct cytoplasmic effectors (21) and activate different signaling pathways (22).
An attractive hypothesis is that only the phosphorylated ITAM of Ig-
directly interacts with the SH2 domains of
syk and induce syk activation, whereas the activation of syk
through the Ig-
cytoplasmic tail is determined by the interaction of another molecule. Whatever the intracellular
mechanism of syk activation through the two BCR subunits, our data clearly show that the overexpression of the
syk mutant differentially affects antigen presentation through
Ig-
and Ig-
. This is in agreement with previous data showing that the mutation of Ig-
ITAM tyrosine residues
did not affect Ig-
-mediated antigen presentation (37),
whereas similar mutations in Ig-
ITAM only affected the
presentation of some, but not all, T cell epitopes (Table 1).
Therefore, there is compelling evidence that functionally
distinguishes classic and alternative pathways of antigen
presentation. Different peptides associate with recycling
and newly synthesized pools of class II molecules which are
specifically targeted by signals contained in the cytoplasmic tails of antigen receptors. Although these signals are located in a similar ITAM, they can be functionally distinguished.
Mutation of tyrosine residues in ITAM and overexpression
of the syk dominant negative mutant did not affect antigen
presentation using recycling class II molecules through the
Ig-
cytoplasmic tail, whereas both alter antigen presentation using newly synthesized class II through the Ig-
cytoplasmic tail. These data indicate that although both Ig-
-
and Ig-
-phosphorylated ITAMs activate syk, the kinase
domain of syk seems to be involved only in the targeting of
antigen receptors to newly synthesized class II molecules
which accumulated, before cell surface arrival, in endocytic
compartments called CIIV (for Class II vesicles [3]).
What is the function of the kinase domain of syk in
BCR-mediated antigen presentation? The phosphorylated
ITAMs of the Ig-/Ig-
sheath interact and activate syk as
well as other cytoplasmic proteins (21). Recently, syk was
shown to associate with or to activate the regulatory subunit of the phosphatidylinositol 3-kinase (PI3 kinase), p85
(41), which activates the catalytic subunit, p110 (42). The
target of this enzyme is the inositol ring bond to the fatty
acids constituting most cell membranes. The role of PI3 kinases in intracellular transport has been demonstrated by
analysis of yeast mutants (vps 34, yeast equivalent of the PI3
kinase) deficient for transport to the vacuole (yeast equivalent of lysosome), and by using wortmannin, inhibitor of
endosomal transport (42). The sequential recruitment of
syk and PI3 kinase, after receptor aggregation and ITAM
phosphorylation, could be a crucial step in endosomal sorting of the BCR, as demonstrated previously for PTK receptors. The kinase domain of the epidermal growth factor
receptor (EGFR), is required in receptor sorting toward
multivesicular endosomal compartments (43). In the case of
the platelet growth factor receptor (PGFR), the sorting of the receptor toward lysosomes is determined by the interaction of its kinase domain with PI3 kinase (44). Although
the precise mechanism by which syk kinase determines antigen presentation remains unclear, our results allow us to
propose two nonexclusive hypotheses for syk kinase activity in endosomal sorting of antigen-bound BCR. Since the
syk dominant negative mutant specifically affected antigen
presentation using newly synthesized class II molecules, syk
might be required for the endosomal sorting of antigen-bound BCR to class II compartments by the direct activation of the kinase domain of syk or by the recruitment of
cytoplasmic effectors, such as PI3 kinases. In a second
mechanism, the recruitment and activation of syk kinase
might alter the structure and composition of endosomal
compartments involved in antigen processing. In either case, it appears likely that syk has a novel and important
function in intracellular trafficking and antigen presentation.
![]() |
Footnotes |
---|
Address correspondence to C. Bonnerot, INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France. Phone: 33-1-42-34-63-88; Fax: 33-1-42-34-63-82; E-mail: bonnerot{at}curie.fr
Received for publication 16 January 1998 and in revised form 28 April 1998.
D. Lankar and V. Briken contributed equally to this work.We are grateful to R. Grynsteen for critical reading of the manuscript, and to all of the members of INSERM CJF 95-01 for useful discussions.
This work was supported by grants from INSERM, Institut Curie, the Association de Recherche contre le Cancer (ARC), and the Ligue Nationale Contre le Cancer. V. Briken was supported by a European Community fellowship.
Abbreviations used in this paper BCR, B cell antigen receptor; GST, glutathione S-transferase; HA, hemagglutinin; HEL, hen egg lysozyme; HRP, horseradish peroxidase; IC, immune complex(es); ITAM, immunoreceptor tyrosine-based activation motif; mIg, membrane immunoglobulin; PI3 kinase, phosphatidylinositol 3-kinase; PTK, phosphotyrosine kinase; SH2, src homology 2 domain.
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