By
Section of Immunobiology, Yale University School of Medicine, and Howard Hughes Medical Institute, New Haven, Connecticut 06510
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Abstract |
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Major histocompatibility complex (MHC) class II molecules can present peptides derived from
two different sources. The predominant source of peptide in uninfected antigen presenting
cells (APCs) is from self-proteins that are synthesized within the cell and traffic through the
MHC class II compartment. The other source of antigen is endocytosed proteins, which includes both self- and foreign proteins. Foreign protein antigens generate adaptive immune responses, whereas self-peptides stabilize the MHC class II heterodimer on the cell surface, allowing positive and negative selection of thymocytes. Therefore, self-antigens play an
important normal role in shaping the T cell receptor repertoire as well as a pathological role in
autoimmunity. To determine whether processing and presentation of self-antigens by MHC
class II molecules differs depending on whether the antigen is supplied through synthesis within
the cell or by endocytosis, we used a T cell clone against an E peptide presented by I-Ab to
show that processing through these two routes can differ. We also show that mice can be tolerant to the epitope formed through the endogenous route, but responsive to the epitope that
can be formed through endocytosis. This suggests that negative selection occurs primarily
against antigens that are synthesized within the APC, and that endocytosed self-antigens could
serve as autoantigens. Finally, we also demonstrate that lipopolysaccharide-activated B cells are
defective for uptake, processing, and presentation of this self-antigen, and that this correlates
with the increased expression of the costimulatory molecules B7.1 and B7.2. This may provide
a model for studying the onset of an autoimmune response.
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Introduction |
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Peptides that are presented on MHC class II molecules are derived from two different sources, and they serve two different functions. Under normal conditions, most MHC class II molecules are occupied by peptides derived from antigens that are synthesized within the APC itself. Peptides that are derived from endogenously synthesized proteins traffic through the endocytic compartment and are primarily derived from other MHC class I and class II molecules (1). These self-peptides allow stable expression of the MHC class II molecules on the cell surface (2), so that these complexes can be used for positive and negative selection of thymocytes (3). The other protein source for MHC class II associated peptides are proteins that must be internalized via endocytosis. Pathogens provide a source of antigen which must be endocytosed, and peptides of this type are also presented by MHC class II molecules to naive T cells, so that the organism can mount an appropriate adaptive immune response against the pathogen.
This implies that two types of antigen can be loaded through the endocytic pathway. The first type is pathogen derived, whereas the second is endocytosed self-antigens. This raises the question of whether processing of proteins that gain access to the MHC class II compartment after endogenous synthesis differs from processing of the same proteins which gain access to the MHC class II compartment through endocytosis. Although some data exists that suggests that processing of endogenously synthesized and endocytosed exogenous antigen may differ (4), essentially no data exist that specifically address this question. Such differences could have important implications both for positive and negative selection of the TCR repertoire and for autoimmunity.
Studies in recent years have focused on identifying the compartment(s) in which antigen processing and peptide loading onto MHC class II molecules occurs, referred to as the MHC class II compartment (MIIC)1 or the class II loading vesicle (CIIV) (5, 6). From these studies and others examining mechanisms of MHC trafficking, proteolysis of the MHC class II invariant chain (Ii), and peptide loading has emerged a more complete model for MHC class II trafficking. MHC class II molecules are transported as a complex with Ii to an endosomal compartment. Ii directs this localization and retains the complex in this compartment until Ii is cleaved to a form called the class II Ii peptide (CLIP) that is still bound in the groove of the MHC molecule but is no longer anchored to the membrane (7). At this point, Ii can still prevent binding of other peptides to MHC class II, but can no longer direct its trafficking. MHC class II itself, however, contains signals that direct it to the endocytic compartment (10). These signals may then direct the localization of MHC class II to MIIC/CIIV, perhaps even directing its formation (11). In the CIIV, DM can catalyze the removal of CLIP and the binding of peptide (12). Rapid transport of the MHC-peptide complex to the cell surface then occurs. This rapid transport is suggested by the kinetics of transport of SDS stable MHC- peptide complexes from CIIV to the cell surface (5), and the fact that large intracellular pools of MHC class II are not detected in early endosomal compartments under normal conditions (13). Rapid transport to the cell surface may preclude further processing of the MHC-peptide complex under the variable conditions potentially available along the endosomal pathway.
In any case, the finding that endogenously synthesized
antigen is presented by MHC class II has raised the question of whether endogenous and endocytosed exogenous
antigens are processed and loaded onto MHC class II molecules in the same form (5, 14). A study using the antibody
Y-Ae, which can detect a specific MHC-peptide complex
(I-Ab + E 52-68), shows that this complex forms from
endogenously synthesized molecules in the MIIC (15), as
can the same epitope from exogenously derived peptides.
Thus, what appears to be the same MHC-peptide complex
can form in the same specialized compartment. However,
it is not clear from this analysis if the same product is produced, as T cell recognition of the Y-Ae epitope was not determined. It is not yet clear, furthermore, that endogenously synthesized protein antigens have access to all of the
antigen-processing compartments. To examine this question, we prepared T cell clones by immunization of
C57BL/6J mice that express I-Ab but lack E
. We also immunized B10.A(5R) and B10.A(3R) mice that are similarly
I-Ab positive but possess the E
chain and express the Y-Ae
epitope (16, 17). These studies illustrate three important
points. First, that endocytosed antigen is processed distinctly from endogenously synthesized antigen. Second,
that tolerance exists to endogenously synthesized antigen
but not to antigen internalized by endocytosis. Third, that
the ability of APCs to take up and present antigen is reciprocally regulated with the induction of costimulatory molecules, which may serve to prevent autoimmunization of
existing potentially autoreactive T cells.
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Materials and Methods |
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Peptides.
The peptides were synthesized as previously described (16), purified by HPLC, and checked for accuracy by mass spectroscopy and amino acid analysis by the W.M. Keck Biotechnology Resource Center (Yale University, New Haven, CT). The sequence of EMice.
C57BL/6J(B6), B10.A(3R) (3R), B10.A(5R) (5R), or 107.1 (B6-I-E transgenic) mice were used in the experiments shown here. B6 and 5R mice were obtained from The Jackson Laboratory (Bar Harbor, ME). 3R mice were bred and maintained within our own animal facility. 107.1 mice were obtained from Richard Flavell (Yale University).T Cells.
ET Cell Clone and Hybrid Assays.
All proliferation assays were set up using 2 × 104 cloned T cells/well in 96-well plates. 2-3 × 105 irradiated or mitomycin C-treated splenocytes/well were used as APCs. All proliferation assays were cultured for 40-48 h at 37°C, and then pulsed with 1 µCi per well [3H]thymidine (6.7 Ci/mmol; Amersham Corp., Arlington Heights, IL) for 6-12 h. The plates were then harvested and counted. Hybrid assays, measuring IL-2 production by CTLL assay, were performed as described (16). All T cell and hybrid assays were performed in duplicate or triplicate. Each experiment shown is representative of multiple assays. For LPS assays, 4 × 106 splenocytes/ml were cultured with 10 µg/ml LPS (Difco Labs., Detroit, MI) for 24 h and were then harvested, irradiated (2,000 rads), washed, counted, and cultured with peptides and T cells.Antibodies.
The antibodies used in these experiments were Y-Ae (17), the anti-I-Ab antibody Y3JP (18), and the anti-I-E antibody Y17 (19). FACS® (Becton Dickinson, Mountain View, CA) staining and analysis was performed as previously described (16). Y-Ae was detected using FITC-labeled Fc-specific goat anti-mouse IgG (1:500; Sigma Chemical Co., St. Louis, MO). The anti-CD28 antibody 37N51was used to provide costimulation in some experiments. Culture supernatant was added to T cells and APCs at the optimal dilution of 1:30 (final concentration) before the addition of the cross-linking antibody, goat anti- hamster IgG (Caltag Labs., San Francisco, CA) at 1 µg/ml.Antibody Stimulation.
In mAb stimulation assays, mAbs were added to the APCs as purified mAb or tissue culture supernatants 30-60 min before the addition of the T cells.Inhibitors of Antigen Processing.
100 µM chloroquine, 100 µM primaquine, or 1.5 mM ammonium chloride was used to block antigen processing. The inhibitors were added to irradiated splenic APCs at twice the concentration listed above for 30-45 min at 37°C before dilution to the concentration listed above by peptide antigen. The APCs were then cultured overnight in the presence of the inhibitors and the peptide before we washed the APCs and added T cells. Anti-CD28 and the cross-linking antibody goat anti-hamster were added to all wells to provide costimulation. The APCs, having been irradiated on the previous day, probably would not be able to provide the necessary costimulation for the T cells. The APCs were treated overnight because long pulses with EFixation.
APCs were fixed at <3 × 107 cells/ml in 0.2% paraformaldehyde for 5 min at room temperature. The cells were then diluted threefold with 0.2 M glycine in Click's medium, pelleted, and washed two times with complete media. Anti-CD28 was added in combination with the cross-linking antibody goat anti-hamster to provide costimulation, as described below.Purification of I-E Protein.
Whole I-E protein was isolated as described for I-Ab, from 100 (B6 × C3H) F2 mice (provided by Richard Flavell). In brief, for this procedure a Y17 sepharose column was added between a nonspecific control column and a Y-Ae column. I-E was eluted from the Y17 column with high pH buffer, and neutralized immediately with 1 M Tris, pH7. This material was dialyzed at 4°C against PBS, and stored at 4°C until use in the assays described above.Immunization of B6 Mice against I-E.
To generate an antigen-specific, receptor-mediated uptake mechanism to enhance presentation of limited quantities of I-E protein, B6 mice were repeatedly immunized with B6/I-E transgenic (strain 107.1, provided by Richard Flavell) splenocytes. Before experiment 1, which is shown in Fig. 8, two B6 mice were immunized intraperitoneally with 2 × 107 107.1 splenocytes. After 5 d the mice were reimmunized intravascularly via retroorbital sinus with 107 107.1 splenocytes. After another 4 d, a B6 spleen was removed from one of the immunized animals, irradiated with 1,000 rads to maintain the APC function of B cells, and used for experiment 1 (Fig. 8, A and B). After ~1 wk, the other mouse was reimmunized intravascularly with 2 × 107 107.1 splenocytes, except this time the 107.1 cells were treated with LPS for 20 h before injection. After another 4 d the B6 spleen was removed, irradiated (1,000 rads), and used for experiment 2 (Fig. 8 C). Spleen from unimmunized B6 was also used.
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Results |
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To compare processing of endogenously
synthesized antigen with the processing of exogenously
supplied antigen, T cell clones were raised in C57BL/6J
(B6) mice by immunization with a synthetic peptide derived from the sequence of MHC class II E chain residues 52-68. B6 mice express the MHC class II molecule I-Ab,
but do not express the peptide donor molecule I-E. This
peptide was chosen because it is known to be presented by
the MHC class II molecule I-Ab when I-E is also synthesized within the cell (1). Additionally, a monoclonal antibody, Y-Ae, specifically recognizes this MHC-peptide complex, allowing its easy detection (1, 17).
All of the clones responded to synthetic peptides and
were restricted by I-Ab as shown by antibody blocking experiments; their response was also blocked by the antibody
Y-Ae (Fig. 1 A). Surprisingly, none of the clones responded
to the I-Ab-E peptide complex presented on 3R splenocytes that express I-E molecules and the Y-Ae epitope (Fig.
1 B). Although the experiment shown here has an unusually strong response of E
6 to 3R APCs in the absence of
synthetic peptide, this response was weak relative to the response of E
6 to APCs plus synthetic peptide. Typically, this response was no greater than the background response
of E
6 to B6 APCs (see Fig. 5). This suggested that processing of endogenously synthesized antigen differed from
processing of exogenously supplied antigen. It may be that
the synthetic peptide which was added to the culture had
been processed to a shorter form that is required for stimulating the E
6 clone. A control hybrid was also raised by
immunization of B6 mice with the peptide E
52-68; this
hybrid is also restricted by I-Ab, and blocked by Y-Ae, but
this hybrid also responded to the endogenously synthesized
and processed ligand on 3R spleen cells (Fig. 1 B).
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The possibility
that either too much or too little ligand was responsible for
the lack of response of the cloned T cell line E6 to the
endogenously generated ligand was ruled out in several initial experiments. Since the Y-Ae antibody recognizes the
synthetic E
peptide bound to I-Ab as well as the endogenously generated complex, FACS® staining with Y-Ae was
used to compare the level of ligand created by addition of
the synthetic peptide to the level of endogenous ligand.
Fig. 1 C shows that even at doses of synthetic peptide sufficient to give plateau stimulation of E
6 (10 µg/ml), the staining with Y-Ae was much lower than that on 3R splenocytes. Therefore, the lack of response of E
6 was not
due simply to overly low levels of the ligand on the APCs.
However, too much ligand can suppress the response of
T cell clones, as is seen in Fig. 2 A. As shown in Fig. 1 A,
E6 T cells responded similarly to synthetic peptide added
to B6, which does not express the endogenous ligand, and
3R, which does express the endogenous ligand. Dose-
response curves of the response of E
6 to peptide on B6 or
3R completely overlap (data not shown). Since addition of
antigen allowed the clones to respond, their lack of response to the endogenously synthesized protein could not
be explained by the phenomenon of high antigen dose suppression.
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The possibility that the clones were
responding to a contaminant in the peptide preparation was
examined. The peptide, E52-68, was synthesized on several separate occasions, purified by HPLC, and checked for
accuracy by amino acid analysis and mass spectroscopy. The main species was determined to have the correct sequence, but, minor contaminants were still present. However, blocking of the response of the clones by the antibody Y-Ae suggests that they are specific for the E
peptide, since this antibody specifically recognizes this peptide when bound to I-Ab (Fig. 1, A-C). Additional HPLC
purifications enhanced the response of E
6, indicating that
E
6 does respond to the correct peptide and not to a contaminant (not shown).
We next asked if E6
responded to a different length of the E
peptide than did
1H3.1. As shown in Fig. 2 A, 10-fold less E
52-66 than
E
52-68 was required to give the same stimulation of
E
6, whereas 1H3.1 responded equally to both peptides
(Fig. 2 B). Further truncation on either end of the peptide
decreased stimulatory capacity for E
6, whereas 1H3.1
could respond to a nonapeptide epitope E
56-64 (Fig. 2 A
and data not shown). This suggested that E
6 required
processing of E
52-68 to E
52-66 in order to respond.
To investigate this further, two experiments were performed to determine whether E6 requires that E
52-68
be processed to a shorter form. The first is shown in Fig. 3.
Splenocytes were either irradiated (designated live), or
fixed in paraformaldehyde before addition of the synthetic
peptides. E
6 could only respond to E
52-68 when presented by live APCs that could still endocytose and process
the peptide. The peptide E
52-66 did not need processing to stimulate E
6, since it could be presented by either live
or formaldehyde-fixed APCs (Fig. 3 A). The control hybrid, 1H3.1, could respond to both peptides on either live
or fixed cells, showing that neither peptide required processing to stimulate 1H3.1, and that E
52-68 could bind
to I-Ab on fixed cells (data not shown). Flow cytometric
staining with Y-Ae, shown in Fig. 3 B, confirmed that
loading of E
52-68 onto I-Ab on live or fixed cells was not
less than loading of the other two peptides. Thus, the inability of E
6 to respond to the longer peptide on fixed
cells could not be explained as an inability of this peptide to
bind I-Ab on fixed cells. This supported the hypothesis that
E
52-68 was the endogenous ligand, to which only 1H3.1
could respond, and that E
52-66 was not produced from
the endogenously synthesized and processed protein.
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We next asked whether drugs that block the acidification
of the endocytic pathway and are known to inhibit processing of exogenous peptides and proteins could also inhibit the response of E6 to E
52-68. APCs were treated
for 30 min with the lysosomotropic agents chloroquine or
ammonium chloride. Peptides were then pulsed onto the
APCs overnight in the presence of the inhibitors. The APCs were finally washed and added to T cells in the presence of cross-linked anti-CD28, added to all wells to provide costimulation needed for T cell responses. Fig. 4
shows that both chloroquine and ammonium chloride inhibited presentation of E
52-68 to E
6, but could not
completely block presentation of E
52-66 to E
6 or presentation of either peptide to 1H3.1. These inhibitors decreased the overall proliferation of the clones to all of the
peptides. However, the background response was also
lower, so the fold response was actually higher in the presence of inhibitors. The flow cytometric analysis with Y-Ae
(data not shown) confirmed that loading of the peptides
onto I-Ab was equivalent in the presence and absence of
the inhibitors. Thus, the lack of response of E
6 to the
longer peptide was not due to a decrease in the ability to
load the longer peptide in the presence of the inhibitors.
Therefore, E
6 appeared to respond selectively to E
52-
66, and could not recognize the naturally processed peptide
E
52-68 in the absence of further processing.
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The ability of live 3R
splenocytes to create the ligand for E6 from the synthetic
peptide E
52-68 (see Fig. 1 A) suggested that these APCs
may also be capable of creating the correct ligand from the
endogenously synthesized proteins if these proteins were processed in the correct compartment(s). To test this hypothesis, anti-I-Ab antibodies were added to 3R or control
B6 splenocytes before addition of E
6 in an attempt to
force the recycling and reprocessing of I-Ab-peptide complexes. The presence of anti-I-Ab antibodies allowed E
6
to respond to the endogenous ligand in the absence of
added exogenous synthetic peptide. This response was specific for the E
peptide-I-Ab complex, since the clones
only responded when both I-Ab and I-E were expressed in
the APCs, either as 3R or as a transgenic E
chain in the
B6/I-E transgenic line called 107.1 (Fig. 5, A-C). Several
anti-I-Ab antibodies including both culture supernatants
and purified mAb could mediate this response (data not
shown), indicating that the response was not due to contamination by other potentially stimulatory antibodies. Additionally, cultured bone marrow dendritic cells from 3R
mice only stimulated E
6 after treatment with anti-I-Ab
antibody culture supernatants (data not shown).
The possibility that the antibodies lead to proliferation of
E6 by stimulating the APCs to express costimulatory signals was tested and ruled out. Providing costimulation in
conjunction with the endogenous ligand, by using 3R LPS
blasts, or cross-linked anti-CD28 with 3R APCs, was not
sufficient to cause proliferation of E
6 (data not shown).
Thus, the most likely explanation for the ability of the
anti-I-Ab antibodies to cause proliferation of E
6 to endogenous ligand was that the antibodies force internalization of I-Ab and thus allow reprocessing of the ligand.
APCs were capable of generating the
epitope required for the E6 response from both exogenously added synthetic peptide and endogenously generated peptide-MHC complexes that were forced to recycle.
Thus, it seemed likely that the lack of response of E
6 to
the unmanipulated endogenous ligand was due to an inability of the complex or individual components to traffic
through the compartment required to generate the ligand
for E
6. Alternatively, APCs may have been able to generate the ligand for E
6 from the peptide E
52-68, but they
may not have been able to generate the ligand for E
6
from whole protein. This possibility was actually already
partially excluded. As discussed in the preceding section, the APCs could generate the ligand for E
6 from the endogenous ligand when it was forced to recycle. In this case,
the APC was able to perform all of the required processing
steps. However, the antibody-forced recycling is an artificial system and did not necessarily indicate that whole antigen added exogenously would gain access to the compartment(s) required to generate the epitope for E
6. Therefore,
it was of interest to determine whether APCs could process
the whole I-E protein to the epitope required for E
6
stimulation when the protein was added exogenously.
I-E was purified from (C57BL/6J × C3H) F2 mice by
immunoaffinity chromatography using the anti-I-E antibody Y17. Fig. 6 A shows that splenic APCs processed the
whole I-E molecule into an epitope that E6 could recognize. The response was very weak compared with the response to synthetic peptide (Fig. 6 B); however, 1H3.1
makes an even weaker response to the exogenous I-E protein. The weak response of 1H3.1 to the whole protein indicated that the weak response of E
6 was due to low concentrations of the antigen and not to differences in the
ability of APCs to create the longer versus the shorter peptide. The response of both E
6 and 1H3.1 to the whole
protein was weaker than the response to the peptide if the
response was plotted against the micromolar concentration
of antigen rather than its mass. This was not unexpected as
far fewer steps are required to generate a T cell epitope
from a peptide than to generate a T cell epitope from
whole protein.
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To enhance the efficiency of presentation of the limited
quantities of whole I-E protein available, an attempt was
made to obtain APCs with the ability to take up the antigen specifically. To accomplish this, B6 mice were repeatedly immunized with splenocytes from the mouse strain
107.1, which, as discussed earlier, is a B6 mouse with an I-E
transgene. Before experiment 1, shown in Fig. 6, B6 mice
were immunized intraperitoneally with 2 × 107 107.1 splenocytes. After 5 d the mice were reimmunized intravascularly via the retroorbital sinus with 107 107.1 splenocytes.
After another 4 d, a B6 spleen was removed from one of
the immunized animals, irradiated with 1,000 rads to maintain the APC function of B cells, and used for experiment 1 in Fig. 6. APCs from unimmunized mice were also used
(data not shown), but no significant difference in the ability
to present I-E protein or peptide was noted in experiment
1. After ~1 week, another mouse was reimmunized with 2 × 107 107.1 splenocytes, except that this time the 107.1 cells
were treated with LPS for 20 h before injection. After another 4 d, the B6 spleen was removed and used for the experiment shown in Fig. 6 C (the response to APCs from
unimmunized mice is shown for experiment 2 in Fig. 6, A
and B). Interestingly, E6 responded to these APCs even
without the addition of peptide or protein antigen (Fig. 6 C).
The APCs must still have been presenting antigen that was
taken up and processed from the 107.1 splenocytes. The caveat to the experiments shown in Fig. 6, A and B, is that
the preparation of purified I-E may contain undetectable
peptide species. However, the ability of APCs from B6
mice immunized with I-E expressing splenocytes to stimulate E
6 suggested that APCs could process the whole I-E
molecule to the epitope required to activate E
6.
These data suggest that bona fide mechanisms of antigen
uptake, such as the surface Ig receptors of B cells, can lead
to the generation of the epitope required for E6 activation. Importantly, this indicates that the MHC class II
epitopes generated from endogenously synthesized antigen
can be different than epitopes that can be created from exogenously provided, endocytosed antigen.
To determine whether B10.A(3R)
mice fail to induce tolerance to E52-66, 3R mice that express the endogenous Y-Ae ligand (I-Ab + E
52-68) or
control B6 mice (I-Ab only) were immunized with either
E
52-66 or E
52-68. Fig. 7 A shows that despite the fact
that 3R mice express large quantities of the Y-Ae epitope
(I-Ab + E
52-68), they are still able to respond to the
peptide E
52-66. These mice, as expected, do not make a
response to the E
52-68 peptide (Fig. 7 B). This peptide,
although processed efficiently in vitro to E
52-66, may
not be processed efficiently enough in vivo to prime a response to E
52-66 in 3R mice. Fig. 7, C and D, shows
that, as expected, B6 mice can respond to either E
52-68 or E
52-66. Thus, 3R (and 5R; data not shown) mice are
tolerant to E
52-68 but are not tolerant to E
52-66. The
response of 3R mice to E
52-66 was inhibitable by Y-Ae,
proving that it involved the same epitope that was recognized by E
6 (Fig. 8); it was also shown to be mediated by
CD4 T cells (data not shown).
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One implication of the above results is that autoantigen processing is likely to occur only when an autoantigen is acquired from the exogenous milieu and not from altered processing of endogenous proteins in APCs, as central tolerance exists to such proteins. Autoimmune disease could only result from the uptake and processing of extracellular antigen, as happens in various disease models. To test this hypothesis, we carried out the following experiments.
Cells Rendered Costimulator Positive Cannot Take Up and/ or Process Antigen.In the experiment shown in Fig. 9, single cell suspensions of spleen were cultured for 24 h with
10 µg/ml LPS in order to induce costimulatory molecules.
In this experiment, macrophages would be depleted by
overnight culture on tissue culture-treated plastic, but dendritic cells would dissociate from the plastic after overnight
culture (20). The cultures were subsequently harvested, irradiated, and washed before addition to peptide and T cells.
The harvested APCs should have been primarily B cells with some dendritic cells. The peptide E52-68, which requires processing to stimulate E
6, cannot be presented by
splenic LPS blasts. The control peptide, E
52-66, is presented to E
6 by LPS blasts, thus indicating that the lack of
response to the longer peptide is not due to a non-specific
toxic effect of LPS. Additionally, the lack of response to
E
52-68 presented by LPS blasts is not due to a decrease in
the ability of this peptide to bind to surface MHC class II,
since this peptide is presented to the control processing-independent hybrid 1H3.1 (Fig. 9 B). Y-Ae staining of
both of these peptides loaded onto LPS blasts is also comparable (Fig. 9 C).
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This general rule of reciprocal regulation of antigen processing and the ability to costimulate T cell growth was
further tested by a time course experiment in which the
ability of APCs to present processing-independent (E52-
66) versus processing-dependent (E
52-68) antigen is
compared at various times after treatment with LPS. APCs
were treated with LPS for 0 or 4 h. After 4 h, the LPS was
washed out and the APCs were incubated for the indicated time period before addition of antigen. Antigen was then
added for 2 h before the APCs were washed, irradiated,
and added to culture wells with T cells or hybrids. The incubations on tissue culture-treated plastic would be expected to deplete the splenocytes of adherent cells (macrophages and dendritic cells), so the APCs studied in this
experiment were primarily B cells (20).
Fig. 10 A shows that presentation of E52-66 to E
6
peaks at 8 h. Presentation of the processing-dependent
peptide E
52-68 to E
6 is greatly decreased at this time
(Fig. 10 B). In Fig. 10 C we show flow cytometric analysis
of the expression of the costimulatory molecule B7-2 at
various time points after LPS stimulation. Expression of this
molecule also peaks at 8 h with this protocol of LPS treatment. Thus, there is a reciprocal correlation between the
ability of splenic B cells to present processing-dependent
antigen and the expression of the costimulatory molecule
B7-2. Although the significance of the difference in responses between time points is questionable, the correlation is repeatable. This experiment does show that the ability of B cells to present processing-dependent antigen can
be negatively modulated by LPS treatment.
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Discussion |
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E6, a T cell clone raised against a peptide derived from
MHC class II E
chain bound to I-Ab, served as a useful
reagent for studying processing of endogenously synthesized versus exogenously provided antigen for presentation by MHC class II molecules. The fine specificity of the T
cell epitope was determined. The T cell required a 15-amino acid peptide, E
52-66, and was unresponsive to the
longer peptide E
52-68 unless E
52-68 was processed.
The E
52-68 epitope that was created from endogenously
synthesized antigen had already been identified (1). The
evidence that E
6 could not recognize E
52-68, despite
the fact that the clone was produced by immunization with
this peptide, came from several sources. First, E
6 prefers
the shorter peptide by about a factor of 10 when they are
presented by viable APCs (Fig. 2 A). Second, E
6 responds
to the longer peptide only when it is presented by viable,
processing-competent APCs (Fig. 3 A), and known inhibitors of antigen processing can block the response to the
longer peptide (Fig 4 A). Third, forced recycling with monoclonal anti-I-Ab antibody produced the epitope for
E
6 in cells that expressed the E
chain and I-Ab (3R, 5R,
and B6-107), but not in B6 cells that do not express the E
chain. Moreover, only antibodies directed at I-Ab could
produce this effect; the I-E-specific antibodies Y-17 and 14.4.4S (data not shown) were unable to produce the E
6
epitope, presumably because they forced internalization
only of I-E and not that of I-Ab (Fig. 5). Finally, when B6
mice were used to present purified I-E molecules, they
could produce the epitope recognized by E
6 (Fig. 6).
Thus, the evidence is overwhelming that the shorter peptide was involved in the epitope recognized by E
6.
To confirm this, we then immunized both B6 and 5R
mice with the peptides E52-68 and E
52-66. The results, shown in Fig. 7, demonstrate that 5R mice, as previously shown (Rudensky, A.Y., and C.A. Janeway, Jr., unpublished data), were tolerant of the E
52-68 peptide,
even when it was injected in strong adjuvant. Likewise, 5R
mice immunized with E
52-68 did not mount a significant response to E
52-66. However, 5R mice primed
with the shorter peptide E
52-66 mounted a vigorous response to E
52-66 as well as a weaker, but still significant,
response to E
52-68. This is presumably due to the processing of the longer E
52-68 to the shorter E
52-66 in
the culture. Thus, 5R mice, which express the endogenously processed E
52-68, are tolerant to it, whereas they are nontolerant to the shorter peptide E
52-66. When B6
mice were immunized with either peptide, they showed
vigorous responses to both peptides, although the response
to priming with the shorter peptide gave a stronger response, and priming with the longer peptide was recalled
more powerfully with the shorter of the two peptides. This
could reflect some difference in the TCR repertoire in B6
mice, as suggested by the earlier finding that most T cells raised by immunization of B6 mice with E
52-68 could
not respond to 5R spleen APCs (Rudensky, A.Y., P. Preston-Hurlburt, and C.A. Janeway, Jr., unpublished observations).
This response was specifically inhibitable with the Y-Ae
monoclonal antibody, proving its specificity (Fig. 8).
Finally, we used the requirement for processing of
E52-68 to examine the relationship between the acquisition of costimulatory capacity and the ability to take up and
process antigen. This revealed that LPS, used to induce B7
expression on spleen-derived APCs, could inhibit the antigen uptake and processing by such APCs (Figs. 9 and 10).
This reciprocal regulation of costimulatory molecules that
are required for naive T cell activation, and processing of
antigen required for antigen presentation to the TCR, may
be a crucially important defense against autoimmune disease. APCs take up self-antigens continuously and are especially likely to do so when stimulated by infection, which
can induce APCs to become costimulatory. Nevertheless,
the ability of an APC to take up and process antigen declines as it becomes B7 positive, perhaps to the point that
the level of autoantigen presented to naive T cells is insufficient for breaking self-tolerance. Although the evidence in
Figs. 9 and 10 is suggestive, more work clearly needs to be
done to explore this result.
An interesting result that emerged from this work was
the role of the two COOH-terminal amino acids in presentation of E52-66 to the T cell clone E
6. This clone is
representative of many cloned T cell lines we have prepared over the years that reacted to the immunizing peptide but not to the naturally processed Y-Ae epitope expressed on 3R and 5R spleen cells. Once we had worked out that E
6 is specific for the E
52-66 peptide, and in
fact ignores the longer, naturally processed peptide E
52-
68, we realized that most of our cloned lines and hybridomas reacted exclusively to the shorter peptide E
52-66.
The problem posed by this result is twofold: first, how are
the two COOH-terminal amino acid residues perceived by
the TCR on the majority of our clones and hybridomas, when they lie at the extreme end of the peptide and are
thus unlikely to contact the TCR at all? Second, why do
most T cells from B6 mice prefer this shorter peptide, even
though immunized with the longer peptide? We will address these two questions separately.
The processing of peptides for presentation by MHC
class II molecules has been studied by many authors. It is
generally concluded that peptides can range in size from a
minimum of 13 amino acids to a maximum of ~25 amino
acids or longer (1, 25, 26). The modal value for peptide
length is 17, exactly the length of E52-68. In truncation
studies carried out by many investigators over the years, the
minimal peptide length that can stimulate most T cell
clones and hybrids is 8-9 amino acids, and indeed, the T
cell hybrid 1H3.1 can respond to the minimal peptide
E
56-64 (AQGALANIA) (Barlow, A.K., R. Medzhitov,
and C.A. Janeway, Jr., data not shown). Nonetheless, the
TCR on E
6, and most clones we have derived from immunization of B6 mice with E
52-68, ignores this longer
peptide in favor of the shorter E
52-66, and can not recognize the minimal peptide epitope AQGALANIA. Recent studies have demonstrated that the COOH terminus
of a peptide makes contact with the TCR V
region (21-
23), and thus its structure should contain clues as to the
recognition of the two residues that have to be removed
from the endogenously processed form. However, the TCR-
chains of E
6 and 1H3.1 are both encoded in
V
6, so it is unlikely that the terminal residues at the very
extreme end of the peptide can make contact with the only
variable residues of the TCR-
chain, those encoded in
the CDR3 region. We are planning to make
chain-only
transgenic mice from E
6 and 1H3.1 in order to attempt
to resolve this mystery (3).
The second mystery is why most of the T cell lines and
clones we have analyzed appear to prefer the shorter peptide, as they are unresponsive to 5R spleen cells that
present the naturally processed epitope known to consist
primarily of the 17-amino acid peptide E52-68. There
are two possibilities to explain this. The first is that the
shorter peptide has a higher affinity for the I-Ab molecule
than the longer peptide, as suggested by its greater immunogenicity in B6 mice (Fig. 7 B) and its greater potency in eliciting proliferative responses from T cell clones (Fig. 2). However, the T cell hybrid, 1H3.1, does not distinguish
these peptides on a molar basis, so the avidity for I-Ab is
probably not the explanation for this difference. The second possibility is based on our recent finding that the
MHC class II-specific repertoire is selected on the basis of
recognition of self-peptide-self-MHC class II complexes
during its development in the thymus (3, 24). The self-peptide repertoire affects both positive and negative intrathymic selection, with the main effect being on positive
selection. We hypothesize that there is a ligand in the B6
thymus that more closely resembles the E
52-66-I-Ab
complex than the E
52-68-I-Ab complex, and that this
ligand selects a majority of T cells that favor recognition of
the former over the latter complex. Evidence for this being
true comes from the ability of Y-Ae treatment of newborn
B6 mice, which are conventionally thought to lack the Y-Ae
epitope altogether, to inhibit development of T cells specific for the E
52-68 and, by inference, E
52-66 specific
cells as well (Rudensky, A.Y., and C.A. Janeway, Jr., unpublished observations). Now that we have analyzed the
nature of the difference between these two cloned T cell
lines, one of which shows an absolute preference for the
shorter peptide, whereas the other can not distinguish peptide length, we are in the process of preparing TCR transgenic mice to analyze selection both in B6 and in I-E+
mice. We predict that the E
6 TCR will show positive selection in B6 and 5R mice, and we have already observed
profound deletion of the 1H3.1 TCR when it is bred to
5R mice.2
In summary, we have deduced the specificity of two
cloned T cell lines, E6 and 1H3.1, and shown them to
differ by the two COOH-terminal amino acids (
KA) of
the peptide E
52-68. The origin of the differences in recognition by these two clones remains to be determined by
further studies, perhaps using immunization of single TCR
chain transgenic mice. As the phenotype of E
6-like clones appears to be dominant in mice primed with the
peptide E
52-68, it remains to be determined whether this
is due to events occurring in the thymus before the immunization event. However, this seems likely to be the case
(3, 24). Finally, we have used this system to infer that costimulation is regulated reciprocally with the ability to take
up and process antigen for potentially autoreactive T cells.
This may be yet another mechanism to avoid the induction
of autoimmune responses.
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Footnotes |
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Address correspondence to Charles A. Janeway, Jr., Section of Immunology, Yale University School of Medicine, 310 Cedar St., LH416, Box 208011, New Haven, CT 06520. Phone: 203-785-2793; Fax: 203-737-1765; E-mail: charles.janeway{at}yale.edu
Received for publication 9 June 1997 and in revised form 4 February 1998.
A.K. Barlow's present address is Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.The authors wish to thank their many colleagues for helpful discussions, especially Alexander Rudensky, Alexander Chervonsky, Ruslan Medzhitov, Christophe Viret, Peter Cresswell, Ira Mellman, Kim Bottomly, Nick Crispe, and Richard Flavell. We thank Kara McCarthy for secretarial assistance.
This study was supported in part by the Howard Hughes Medical Institute and by National Institutes of Health (NIH) grant R37 AI-14579 to C.A. Janeway, Jr. A.K. Barlow was supported in part by NIH training grant T32 AI-07019 and by a fellowship from Miles Laboratories (West Haven, CT).
Abbreviations used in this paper CIIV, class II loading vesicle; Ii, invariant chain; MIIC, MHC class II compartment.
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