Peptide dependency of alloreactive CD4+ T cell responses

Sanjeev K. Mendiratta1, Jean-Paul Kovalik, Seokmann Hong, Nagendra Singh, W. David Martin2 and Luc Van Kaer

Howard Hughes Medical Institute, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, 811 Rudolph Light Hall, Nashville, TN 37232, USA

Correspondence to: L. Van Kaer


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II–peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion.

Keywords: alloreactivity, gene-targeted mouse, H2-M, MHC class II molecules, peptides


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T lymphocytes recognize foreign peptide antigens bound with self proteins encoded by the MHC, a property referred to as self MHC restriction (1). T lymphocytes acquire the capacity to recognize antigens in the context of self MHC molecules during their development in the thymus by the process of positive selection (24). While the mechanism of positive selection clearly provides an explanation for the specificity of T cell-mediated immune responses against foreign antigens, it has been more difficult to explain how the same process can lead to the generation of T lymphocytes reactive with foreign MHC molecules, a phenomenon called alloreactivity (5). In contrast to the low frequency of T cells that recognize foreign antigens in the context of self MHC, as many as 1–10% of all T cells respond to alloantigens. The property of a large number of T lymphocytes to react with foreign MHC molecules forms the cellular basis for the rejection of tissue grafts.

To understand the molecular basis for allorecognition it is important to obtain detailed information about the ligand that is recognized by the TCR expressed on alloreactive T lymphocytes. Until recently it was generally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells (5). However, it is now clear that MHC-bound peptides can influence alloreactive T cell responses (5,6). Direct evidence for this came from studies on MHC class I. Human alloreactive T lymphocytes directed against MHC class I molecules did not always recognize these MHC molecules when they were expressed in murine cell lines (79). Conversely, murine MHC class I molecules expressed in human cells were not always recognized by murine alloreactive cytotoxic T lymphocytes (CTL) (10). In the latter experiments, it was possible to sensitize the human cell line for CTL lysis by addition of murine cyanogen bromide-cleaved cytoplasmic proteins to the cultures. Studies with the antigen-processing-deficient cell lines RMA-S and T2 further showed that very few alloreactive T cell clones can recognize empty MHC class I molecules (1114). Endogenous cellular peptides obtained from cyanogen bromide-cleaved proteins, or through acid extraction of cell lysates or purified MHC class I molecules, often sensitized target cells for recognition by the CTL clones. Combining this approach with HPLC fractionation of peptides indicated that the majority of alloreactive CTL are peptide dependent (12,15). However, since some alloreactive clones recognized more than one peptide peak, it was suggested that alloreactivity may not be very peptide specific. This conclusion was further supported by experiments showing that some alloreactive CTL can be sensitized by addition of non-cellular synthetic peptides to target cells (1619). Although some alloreactive T cell clones with exquisite peptide specificity have been identified (17,1921), this is probably not a general property of alloreactive T cell responses.

Few studies have addressed the role of peptides in the recognition of MHC class II molecules by alloreactive CD4+ T cells. Early studies showed that the recognition of MHC class II by alloreactive CD4+ T lymphocytes could be partially inhibited by addition of exogenous antigens to the antigen-presenting cells (APC) (22,23). It was further demonstrated that some alloreactive CD4+ T cells were able to distinguish between allogeneic MHC class II products expressed in different tissues (24,25). The best evidence for a role of peptide in alloreactive CD4+ T cells responses comes from a study with the antigen processing defective cell line T2. Using class II transfectants of this cell line it was shown that the majority of CD4+ T cells that participate in alloresponses recognized class II conformers that depend on the presence of peptide (26). Although informative, these studies were limited by uncertainties about the nature and complexity of the peptides that were bound by the class II molecules expressed by the transfected cells.

Here we have studied the requirement for peptide in alloreactive CD4+ T cell responses with mice expressing a single MHC class II–peptide combination, H2-Ab plus a peptide (termed CLIP) derived from the class II-associated invariant chain (Ii). The results indicate that recognition of MHC class II by the majority of alloreactive CD4+ T cells is peptide-dependent.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
H2-M mutant mice on a mixed C57BL/6 (B6)x129/Sv background have been described (27). Ii mutant mice on a mixed B6x129/Sv background (28) were obtained from Dr Elizabeth K. Bikoff (Harvard University School of Medicine, Boston, MA), MHC class II mutant mice on a B6 background (29) were obtained from Taconic (Germantown, NY) and ß2-microglobulin-deficient mice on a B6 background (30) were obtained from the Jackson Laboratory (Bar Harbor, ME). All of these mutant mouse strains were maintained and bred under specific pathogen-free conditions in the animal facility at Vanderbilt University School of Medicine. BALB/c (H2d), SJL (H2s), RIII/J (H2r), B6 (H2b) and CBA/J (H2k) mice were purchased from the Jackson Laboratory or Harlan Sprague Dawley (Indianapolis, IN) and maintained in our animal facility.

Peptides
The following peptides were used: ovalbumin(323–339), ISQAVHAAHAEINEAGR; murine CLIP(85–99), KPVSQMRMATPLLMR; cytochrome c(39–65), KTGQAEGFSYTDANKNKGITWGEDTLM; RNase(90–105), SKYPNCAYKTTQANKH; and E{alpha}(52–68), ASFEAQGALANIAVDKA. Peptides were obtained from the biopolymers facility of the Howard Hughes Medical Institute at the University of Texas Southwestern Medical Center (Dallas, TX).

Generation of short-term alloreactive T cell lines
Mice from a panel of allogeneic strains were immunized i.p. with 2x107 irradiated (2000 rad) spleen cells from B6 mice. Ten days later, spleens were harvested and cells were enriched for CD4+ T lymphocytes by staining with anti-CD8 antibodies (2.43) (obtained from Dr Barney Graham, Vanderbilt University) followed by panning on plates coated with rabbit anti-rat IgG (Cappel, Organon Teknika, West Chester, PA). Purified responder cells were then cultured with irradiated splenic APC (4–5x107) from B6 mice for 6 days. Before use in mixed lymphocyte reactions residual CD8+ T cells from these cultures were removed by panning with plate-bound anti-CD8 antibodies.

Mixed lymphocyte reactions
Mixed lymphocyte reactions with responder cells from short-term alloreactive T cell lines were performed by co-culture (2x104) with titrated numbers of irradiated stimulator cells for 24 h at 37°C. Then 1 µCi [3H]thymidine (NEN Life Science Products, Boston, MA) per well was added and cells were further cultured for 16 h. [3H]Thymidine incorporation was measured by harvesting cells with a cell harvester (Tomtec, Orange, CT) and counting the amount of radioactivity with a betaplate reader (EG & G Wallac, Gaithersburg, MD).

For primary or secondary mixed lymphocyte reactions, spleen cells depleted of CD8+ T lymphocytes were used from naive mice or from mice that had been immunized i.p. 10 days earlier with 2x107 irradiated (2000 rad) spleen cells from B6 or H2-M mutant mice. Responder cells (1x105) were mixed with varying numbers of irradiated spleen stimulator cells for 3 days at 37°C. Proliferation of responder cells was measured as above.

Generation of T cell hybridomas
Mice were immunized i.p. with 2x107 irradiated (2000 rad) spleen cells from C57BL/6 or H2-M mutant mice. Ten days later 5x107 spleen cells from these immunized animals were re-stimulated in vitro with 4–5x107 irradiated B6 or H2-M–/– spleen cells for 3 days. Cells were counted and fused with Bw5147 {alpha}ß cells (obtained from Dr Willi Born, National Jewish Center, Denver, CO) using polyethylene glycol 1500 (Boehringer Mannheim, Indianapolis, IN). Hybridomas were obtained by plating fused cells in HAT selection medium (Boehringer). Some of these hybridomas were subcloned by limiting dilution.

The E{alpha}(52–68)-specific T cell hybrid M5 and the RNase(90–105)-specific T cell hybrid MR8 were obtained after immunization of mice with these peptides (N. Singh and L. Van Kaer, submitted).

T cell hybridoma recognition assay
Reactivities of T cell hybridomas were measured by co-culture of a fixed number (1x105) of hybridoma cells with varying numbers of irradiated stimulator cells. In the experiments presented in Figs 3 and 6GoGo varying amounts of synthetic peptides were also added to the cultures. After 24 h of culture, supernatants were collected and assayed for IL-2 content using the IL-2-dependent cell line HT-2. The 50 µl supernatant was mixed with 104 HT-2 cells and incubated at 37°C for 20 h. IL-2-dependent proliferation of HT-2 cells was tested by pulsing the cells with 1 µCi [3H]thymidine and further culture for 16 h. [3H]Thymidine incorporation was measured as described above. Reactivities of hybrids were graded as strong, weak or none when IL-2 production was >50, 10–50 or <10% respectively of the IL-2 produced when the hybrids were stimulated with H2-M+/+ cells.



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Fig. 3. Peptide-selectivity of T cell hybrids. The indicated T cell hybrids (1x105 cells) were cultured with a fixed number of irradiated APC (3x105) from wild-type mice, H2-M mutant mice or Ii mutant mice or APC from H2-M mutant mice pulsed with 10 µg/ml of the indicated synthetic peptides, or with a mixture of these peptides (2.5 µg/ml each). Stimulation of the hybridoma cells was measured by IL-2 production, as assayed by proliferation of HT-2 indicator cells. (A) Reactivities of a set of representative alloreactive T cell hybrids that recognize H2-M+/+ but not H2-M–/– cells. (B) Reactivities of the E{alpha} peptide-specific T cell hybrid M5 and the RNase peptide-specific T cell hybrid MR8.

 


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Fig. 6. Peptide specificity of T cell hybrids. The indicated T cell hybrids (1x105 cells) were cultured with a fixed number of irradiated APC (2.5x105) from wild-type mice, H2-M mutant mice or Ii mutant mice, or APC from Ii mutant mice pulsed with varying amounts of the indicated synthetic peptides. Stimulation of the hybridoma cells was measured by IL-2 production, as assayed by proliferation of HT-2 indicator cells.

 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
H2-M mutant mice express normal levels of the MHC class II molecule H2-Ab, yet most of these complexes are occupied by a single peptide, termed CLIP, derived from the class II-associated Ii chain (27,3134).). This conclusion was based on in vitro biochemical assays, flow cytometry analyses, peptide elution studies and antigen presentation assays (27,3134). From these studies it was estimated that as many as 99.8% of all class II Ab molecules in these mice are occupied by CLIP (34). As expected, cells from these mice are defective in the presentation of exogenous protein antigens to T cells. Surprisingly, however, these cells also showed reduced capacity to present exogenous peptides to T cells (27,31,32). This finding indicates that CLIP cannot be easily displaced with other peptides, such as peptides derived from the culture medium. For these reasons we concluded that APC from H2-M mutant mice are suitable for studying the role of peptides in alloreactive CD4+ T cell responses. We therefore used cells from these mice as targets and stimulators for alloreactive CD4+ T cells.

H2-Ab-specific alloreactive T cell lines respond poorly to cells from H2-M mutant mice
We first wanted to examine if H2-Ab-specific bulk alloreactive CD4+ T cells can react with the Ab–CLIP complexes expressed by H2-M mutant cells. Mice from the BALB/c (H2d), RIII/J (H2r), SJL (H2s) and C57BL/6 (B6) (H2b) strains were immunized with irradiated splenic APC from B6 (H2b) mice. Ten days later spleen lymphocytes from these mice were depleted of CD8+ T cells and re-stimulated in vitro with irradiated splenic APC from B6 mice for 6 days. These bulk alloreactive T cell cultures were then tested for reactivity with APC from B6, H2-M–/–, Ii–/– and class II–/– mice (all from the H2b haplotype). All cultures, except those from syngeneic B6 mice, reacted strongly with stimulator cells from B6 and Ii–/– mice but not with similar cells from MHC class II–/– mice, indicating that the majority of T cells in these cultures were directed against class II Ab complexes (Fig. 1Go). The proliferative response of these short-term alloreactive T cell lines to spleen cells from H2-M mutant cells was remarkably weak, indicating that the reactivity of most T cells in these cultures with class II Ab molecules is strongly influenced by the type of peptide that is bound in the antigen-binding groove of Ab.



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Fig. 1. H2-M mutant cells are poorly recognized by short-term H2-Ab-specific polyclonal alloreactive T cell lines. The indicated allogeneic mice were immunized with 2x107 irradiated spleen cells from B6 mice. Ten days later, spleens from the immunized mice were harvested, CD8+ T lymphocytes were removed and responder cells were re-stimulated in vitro for 6 days with 4–5x107 irradiated B6 spleen cells. These short-term T cell lines were then tested for reactivity with titrated numbers of irradiated splenic stimulator cells isolated from the indicated mouse strains. Reactivity of the T cell lines was measured 24 h later by uptake of [3H]thymidine.

 
Only a small subset of Ab-specific alloreactive T cell hybridomas recognize APC from H2-M mutant mice
To obtain information on the overall frequency of peptide-dependent alloreactive T cells and to address the issue of peptide specificity, we generated large panels of Ab-specific alloreactive T cell hybridomas. Mice from a set of allogeneic strains were immunized with irradiated H2b (H2-M+/+ or B6) spleen cells, responder cells were re-stimulated with H2b spleen cells in vitro and fused with Bw5147 {alpha}ß cells to generate T cell hybridomas. The reactivities of these hybrids with cells from H2-M+/+, H2-M–/–, Ii–/–, ß2-microglobulin–/– (CI–/–) and MHC class II–/– (CII–/–) mice are summarized in Table 1Go, and reactivities of representative hybrids are shown in Fig. 2Go. Hybrids that did not react with H2-M+/+ cells, or that reacted with class II-deficient cells, were excluded from the analysis. The reactivities of these hybrids with the various targets were graded as strong, weak or none, when their response was >50, 10–50 or <10% respectively of their response with cells from H2-M+/+ mice.


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Table 1. Reactivity of randomly selected H2-Ab-specific alloreactive T cell hybridomas
 


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Fig. 2. Reactivity of representative H2-Ab-specific T cell hybrids with APC from a variety of gene-targeted mice. The indicated hybrids (1x105 cells) were incubated with titrated numbers of irradiated splenic stimulator cells from H2-M+/+, H2-M–/–, Ii–/– and class II–/– mice. Stimulation of the hybridoma cells was measured by IL-2 production, as assayed by proliferation of HT-2 indicator cells.

 
The frequency of Ab-specific alloreactive T cell hybrids that were able to recognize H2-M–/– cells varied among the mouse strains from which they were isolated: 20% of the hybrids isolated from CBA/J mice, 14% of the BALB/c hybrids, 0% of the RIII hybrids and 11% of the SJL hybrids (Table 1Go). Since it was shown that H2-M-deficient cells may express residual levels of non-CLIP peptides (33,34), the frequencies measured here may represent an overestimation of the actual number of hybrids that can react with the Ab–CLIP complex. Nonetheless, most hybrids tested in this system appear to depend on MHC-bound peptides for recognition of class II.

Are the hybrids that react with H2-M–/– cells specific for the Ab–CLIP complex? This question can be addressed by comparing the extent of reactivity of these hybrids with H2-M–/–, H2-M+/+ and Ii–/– cells. It has been shown that wild-type H2b cells also express Ab–CLIP complexes, albeit at much lower levels than H2-M mutants (27,3136). Since most hybrids react with H2-M–/– cells weaker than with H2-M+/+ cells (Table 1Go), it is likely that they can recognize Ab molecules occupied by non-CLIP peptides and therefore lack exquisite peptide specificity for Ab–CLIP. This conclusion is further supported by the finding that all of the hybridomas that are reactive with H2-M mutant cells also respond to cells from Ii mutant mice. Ii mutant cells express reduced levels of surface MHC class II molecules that are mostly SDS-unstable and either empty or occupied by low-affinity peptides (28,37,38). Thus, we conclude that these hybrids lack exquisite specificity for the Ab–CLIP complex.

The hybrids that reacted with cells from H2-M+/+ mice but not with cells from H2-M–/– mice are peptide selective. To analyze the specificity of this class of hybrids further, we tested a representative set of these hybrids for reactivity with H2-M–/– cells in the presence of synthetic peptides. Figure 3Go(A) shows that these hybrids did not react with any of the peptides tested and did not react to a mixture of four different peptides (E{alpha}, ovalbumin, cytochrome c and RNase). In contrast, control hybrids strongly reacted with their cognate peptide antigen when added to H2-M–/– cells (Fig. 3BGo). Although the overall reconstitution of class II molecules on H2-M–/– cells in these experiments is probably very low, these results suggest that these hybrids are strongly peptide selective.

Interestingly, some hybrids did not react with either Ii–/– or H2-M–/– cells, while reacting strongly with wild-type H2b cells (Table 1Go). These clones probably represent alloreactive T cells that are strongly peptide selective or even peptide specific, requiring the presence of endogenous Ii-dependent peptides for their reactivity.

Taken together, these hybridoma data indicate that the recognition of MHC class II molecules by most alloreactive T cells is strongly influenced by the type of peptide bound to the MHC groove.

Cells from H2-M mutant mice are inefficient stimulators of alloreactive CD4+ T cell responses
To further study the peptide dependence of alloreactive CD4+ T cell responses, we tested whether cells from H2-M mutant mice can induce alloreactive T cell responses in primary and secondary bulk mixed lymphocyte reactions. APC from H2-M mutant mice were unable to induce a detectable alloreactive T cell response in primary cultures with allogeneic responder cells from all mouse strains tested (Fig. 4AGo). Even when allogeneic mice were first immunized with H2-M mutant cells and spleen responder cells depleted of CD8+ T lymphocytes were tested for proliferation with H2-M mutant stimulator cells, no significant reactivity was observed in most experiments performed (Fig. 4BGo). This indicates that the allogeneic mice tested here contain very few CD4+ T cell precursors that are able to react with Ab–CLIP complexes.



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Fig. 4. APC from H2-M mutant mice are inefficient stimulators of alloreactive CD4+ T cell responses. (A) Cells from H2-M mutant mice as stimulators in primary mixed lymphocyte reactions. Spleen cells from the indicated mouse strains were enriched for CD4+ T cells and 1x105 cells then cultured with titrated numbers of irradiated splenic APC from H2-M+/+ or H2-M–/– mice. Proliferation of responder cells was measured by uptake of [3H]thymidine. (B) Cells from H2-M mutant mice as stimulators in secondary mixed lymphocyte reactions. The indicated mouse strains were immunized with irradiated APC (2x107) from H2-M+/+ or H2-M–/– mice. Ten days later, spleens from immunized mice were harvested and CD8+ T cells were removed. Responder cells (1x105) were then tested for reactivity with titrated numbers of irradiated spleen stimulator cells from H2-M+/+ or H2-M–/– mice respectively. Proliferation of responder cells was measured by uptake of [3H]thymidine.

 
Specificity of alloreactive T cell hybridomas generated from mice previously immunized with H2-M mutant cells
We tested if it was possible to generate alloreactive T cell hybrids with exquisite specificity in their reactivity with Ab–CLIP complexes after immunization of mice from a panel of allogeneic strains with H2-M mutant cells. Spleen cells from immunized mice were re-stimulated in vitro with irradiated H2-M mutant cells and hybrids were generated. Since little proliferation occurred in the in vitro re-stimulation, in most fusions very few hybridomas were obtained. Several fusions were performed for each allogeneic responder strain. Individual hybrids were tested for reactivity with cells from H2-M+/+, H2-M–/–, Ii–/–, ß2-microglobulin–/– (CI–/–) and MHC class II–/– (CII–/–) mice. Hybrids reactive with class II–/– cells and hybrids that did not react with any of the stimulator cells tested were excluded from the analysis. Results are summarized in Table 2Go and data for representative hybrids are shown in Fig. 5Go.


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Table 2. Reactivity of T cell hybridomas obtained after immunization of allogeneic mice with cells from H2-M mutant mice
 


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Fig. 5. Reactivity of representative T cell hybrids isolated from allogeneic mice after immunization with APC from H2-M mutant mice. The indicated hybrids (1x105 cells) were incubated with titrated numbers of irradiated splenic stimulator cells from H2-M+/+, H2-M–/–, Ii–/– and class II–/– mice. Stimulation of the hybridoma cells was measured by IL-2 production, as assayed by proliferation of HT-2 indicator cells.

 
Although these hybrids were generated after immunization and in vitro re-stimulation with cells from H2-M mutant mice, a significant fraction (15–40%) was unable to recognize H2-M–/– cells but did react with H2-M+/+ cells. These hybrids probably represent alloreactive CD4+ T cells that were activated only weakly or perhaps non-specifically during the in vitro culture.

Most of the hybrids that responded to H2-M–/– cells reacted even stronger with H2-M+/+ cells and also recognized cells from Ii–/– mice, indicating that these hybrids are not specific for the Ab–CLIP complex and therefore lack exquisite peptide specificity. Only some of the hybrids from SJL mice did not recognize Ii–/– cells, while reacting with H2-M–/– cells (Table 2Go and Fig. 5Go). These hybrids therefore represented candidates with exquisite CLIP-specificity. We further tested the specificity of two of these hybrids (SJL-M107 and SJL-M110) in peptide-loading experiments, using APC from Ii–/– mice. While these hybrids did not react with Ii–/– cells or Ii–/– cells pulsed with three different non-CLIP peptides (cytochrome c, E{alpha} and ovalbumin), they responded vigorously to Ii–/– cells pulsed with CLIP (Fig. 6Go).

In conclusion, it appears that very few alloreactive CD4+ T cells generated after direct immunization with cells from H2-M mutant mice show exquisite specificity for the Ab–CLIP complex.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have studied the role of MHC-bound peptides in CD4+ alloreactive T cell responses using APC from H2-M mutant mice that express a homogeneous MHC class II–peptide complex, H2-Ab bound by CLIP. We found that cells from H2-M mutant mice were recognized by few Ab-specific alloreactive T cells and that H2-M–/– APC were inefficient stimulators of alloreactive T cell responses. These findings demonstrate that recognition of H2-Ab molecules by most alloreactive CD4+ T cells is strongly influenced by the type of peptide that is bound in the MHC groove.

Peptides could contribute to alloreactivity in a number of ways. First, alloreactive TCR could make direct contacts with peptides embedded in the MHC groove. In favor of this hypothesis, alloreactive T cells with exquisite specificity in their recognition of MHC–peptide complexes have been identified (17,1921). Similarly, some of the hybrids identified in this study appeared to be specific for the Ab–CLIP complex. However, such hybrids were only obtained in one mouse strain (SJL) and only after direct immunization with H2-M-deficient APC. It appears therefore that the Ab–CLIP complex is not very immunogenic in vivo. This may not be true for all MHC–peptide complexes and may be caused by a property of CLIP, not shared with most other peptides. CLIP is a universal class II ligand that binds with many MHC class II molecules and alleles (39). It is possible that our difficulties to induce alloreactive T cell responses against CLIP are caused by deletion of cells with this reactivity in most mouse strains. Nevertheless, our results, together with the studies on peptide-specific alloreactive T cells described previously (17,1921), suggest that at least some alloreactive T cells can make direct contacts with peptides embedded in the MHC groove. As with all forms of T cell recognition (40), there may be a great amount of flexibility in this interaction.

Second, peptides could affect allorecognition by inducing subtle changes in the conformation of the {alpha}-helices of the MHC molecule that are recognized by the alloreactive TCR. Although X-ray crystallography studies have argued that peptides have little effect on the conformation of MHC molecules (41,42), studies with conformation-dependent antibodies have clearly shown that peptides can affect MHC class I (4346) and class II (47,48) conformation. Consistent with these experiments, several studies have suggested that peptide binding to MHC class I molecules can affect TCR recognition by inducing conformational changes in the MHC residues that line the antigen-binding groove (19,49,50). Can the present results be explained by CLIP-induced changes in the conformation of class II Ab molecules? The Ab–CLIP complex is not efficiently recognized by some conformation-dependent antibodies (e.g. BP107) (unpublished; 31,32,51). It seems likely, therefore, that CLIP binding induces subtle changes in the residues that line the Ab peptide-binding groove. In turn, these conformational changes may be recognized by some peptide-dependent alloreactive TCR. Such alloreactive T cells would be able to cross-react with all Ab–peptide complexes that adopt the same conformation as Ab–CLIP. Our observation that most alloreactive hybrids that reacted with Ab–CLIP complexes expressed by H2-M mutant cells also recognized Ii-deficient cells is difficult to explain exclusively on the basis of direct contacts between the CLIP peptide and the alloreactive TCR. We therefore favor the interpretation that both direct contacts with peptide and conformational changes induced by this peptide on the MHC residues that line the antigen-binding groove contribute to the peptide dependence of alloreactive T cell responses.

The frequency of randomly selected anti-H2-Ab-specific alloreactive hybrids that were unable to react with H2-M-deficient cells ranged from 80 to 100%, depending on the strain from which these hybrids were isolated. Since we cannot exclude that these hybrids reacted only with a subset of the peptide–MHC complexes expressed by wild-type cells, these numbers probably represent an overestimation of the overall frequency of peptide-independent hybrids. We can therefore conclude that the vast majority of alloreactive CD4+ T cells are peptide dependent and that only a small fraction of alloreactive T cells are truly peptide independent.

The high frequency of alloreactive T cells, as compared with the low frequency of antigen-specific T cells, has puzzled immunologists for many years. It is particularly intriguing that T cells selected in the thymus to recognize self MHC plus foreign peptide can react so vigorously with foreign MHC plus self peptide. Bevan (52) proposed that this is caused by the high density of the foreign MHC–peptide determinants expressed by allogeneic APC. However, this hypothesis did not take into account that alloreactive T cell responses are highly peptide dependent. How then can the high frequency of alloreactive T cells be explained? Our findings indicate that a single MHC class II–peptide complex, although expressed at normal levels (i.e. at high density), is a very poor target and inducer of alloreactive T cell responses. This suggests that the diversity of the repertoire of peptides normally bound with MHC molecules is critical to explaining the high frequency of alloreactivity. This conclusion is consistent with the large number of studies on class I-restricted CTL (521,53) and with the studies on class II-restricted Th cells (22–26 and this study), which indicate that most alloreactive T cells not only require peptides for reactivity, but are quite peptide selective and occasionally peptide specific. Thus, most alloreactive T cells can distinguish between distinct MHC–peptide complexes and only a small number of alloreactive T cells are truly peptide independent or can recognize empty MHC (53). We therefore conclude that the high frequency of alloreactive T cells is caused by the diversity of peptides that are bound by the MHC molecules of wild-type mice.

During the last decade it has become clear that all forms of T cell recognition, including the specific recognition of antigen, self-tolerance, intrathymic positive selection (54) and superantigen recognition (55) requires MHC-bound peptides. Earlier studies with CTL and the results presented here indicate that MHC-bound peptides play an equally important role in alloreactivity. This conclusion may be important for future strategies directed at inhibiting allograft responses.


    Acknowledgments
 
We wish to thank Drs Elizabeth Bikoff, Willi Born and Barney Graham for their gifts of various reagents. L. V. K. is an Assistant Investigator of the Howard Hughes Medical Institute.


    Abbreviations
 
APCantigen-presenting cell
B6C57BL/6
CLIPclass II-associated invariant chain peptide
CTLcytotoxic T lymphocytes
Iiinvariant chain

    Notes
 
1 Present address: GeneMedicine, The Woodlands, TX 77381, USA Back

2 Present address: Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322, USA Back

Transmitting editor: E. Simpson

Received 26 August 1998, accepted 9 November 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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