Negative selection imparts peptide specificity to the mature T cell repertoire

Abstract

The T cell αβ receptor (TCR) recognizes foreign peptide antigens bound to proteins encoded in the MHC. The MHC portion of this complex contributes much to the footprint of the TCR on the ligand, yet T cells are usually very specific for individual foreign peptides. Here, we show that the development of peptide-specific T cells is not intrinsic to thymocytes that undergo thymic-positive selection but is an outcome of eliminating, through negative selection, thymocytes bearing TCRs with extensive peptide cross-reactivity. Hence, thymic-negative selection imposes peptide specificity on the mature T cell repertoire.

Mature T cells bearing αβ T cell receptors (TCRs) react with cells expressing host MHC proteins bound to foreign antigens, usually in the form of peptides. In healthy animals, these same T cells do not react productively with host MHC bound to self-peptides (1, 2) although such T cells may detect the host combinations weakly (3). Individual T cells are usually very specific for particular foreign peptides, often reacting only with the immunogen or its close relatives (4, 5). However, occasional T cells have been identified that react with host MHC bound to a variety of peptides that are not closely related to each other (6, 7). It has been argued that this degeneracy for peptide is essential to the ability of T cells to anticipate all conceivable foreign peptides (8). TCRs that can react with two or more unrelated peptides bound to allogeneic MHC proteins have also been described (9, 10). In fact, peptide promiscuity may be more common for TCRs reacting with allogeneic rather than syngeneic MHC (11).

X-ray crystallography has revealed the footprint of TCRs on their MHC/peptide ligands. In most cases, contact between the amino acids of the TCR and the MHC contributes extensively to the binding (12). Thus, the fact that the TCR concerned is usually very specific for the peptide is perhaps surprising. Such specificity could arise for three reasons. First, by their very nature, the genomically encoded segments of TCR α and β chains might not allow sufficiently strong reaction with any MHC protein to drive the response of mature T cells. Additional binding energy may always have to be supplied by interactions between the TCR and the peptide. Second, peptide specificity may be the consequence of positive selection. Positive selection involves low avidity/affinity reactions between the TCRs on thymocytes and MHC/self-peptides in the thymus, and thus gives rise to mature T cells bearing TCRs with only low affinity for MHC (13). Mature T cells, however, need a strong reaction to be triggered to full activation (14–16). Because the MHC protein involved in positive selection of the thymocyte and activation of the mature T cell is usually one and the same, the additional binding energy required to activate the mature T cell must come from the TCR/peptide interaction. Such additional strength of binding may usually demand increased peptide specificity from the T cell. Finally, peptide specificity may be imposed on the TCR repertoire by negative selection. Many different self-peptides can bind to any given MHC protein (17, 18). Developing thymocytes are exposed to most of these combinations (19). Thus, any thymocyte that can recognize both self-peptides and foreign peptides will be eliminated. This phenomenon will lead to the removal of cells bearing both marginally and markedly promiscuous TCRs.

If the germ line-encoded TCR α and β segments confer peptide specificity, then all T cells regardless of the selecting MHC haplotype should be peptide specific. Similarly, if the ability to undergo positive selection requires peptide specificity, then T cells derived from mice expressing self-MHC, regardless of the diversity of peptides displayed, should be reactive to specific peptides in bound to the selecting MHC molecule. However, if negative selection imparts peptide specificity, then the degree of allowable peptide promiscuity of a given TCR repertoire should correlate with the level of negative selection.

The idea that self-tolerance controls the repertoire of TCRs on mature T cells has been examined in the past. We and others showed that a large percentage of thymocytes that can be positively selected are in fact deleted in the thymuses of normal mice (20–23), leading to a considerable reduction in the mature T cell repertoire. Moreover, others showed that the repertoire of T cells for allogeneic MHC could be pared down to a set of cells that is very peptide specific, once tolerance to the foreign MHC/mouse peptides had been achieved (24, 25). However, neither of these types of experiments investigated the specificities of the negatively selected thymocytes. Because thymocytes detect MHC/peptide combinations with lower avidity than mature T cells do (14–16), these cells might have been deleted by reactions with quite low avidity ligands, avidities that would be too low to drive reaction of the cells once they had matured. Thus, the deleted thymocytes may have been quite specific for MHC bound to just a few peptides, self or foreign. On the other hand, the thymocytes deleted in these situations may have included a subset that is very peptide promiscuous.

To find out how peptide specificity is conferred to the mature T cell repertoire, we analyzed T cells from mice with differing levels of MHC-specific tolerance. T cells from animals that were fully tolerant to a particular MHC protein/self-peptide were very specific for peptides against which they were immunized. In contrast, T cells that had not been tolerized for recognition of a particular MHC protein/self-peptide were often very promiscuous for peptide recognition. This result was not affected by positive selection on different H2 haplotypes. Hence it is negative selection that focuses the attention of the mature T cell repertoire on specific peptides. The existence of the extremely peptide cross-reactive TCRs in inadequately tolerized animals suggests that elements of TCRs may indeed be evolutionarily selected to react with MHC proteins with some affinity (26). Searches for the secrets governing the obsession of T cells for MHC proteins might do well to focus on these TCRs.

Materials and Methods

Mice and Generation of Bone Marrow Chimeras. C57BL/6, B10.BR, B10.D2, and C3H × C57BL/6F₁ mice were purchased from The Jackson Laboratory. MHC class II locus-deficient mice were a gift from C. Benoist and D. Mathis (27). IE^k-Hb P5A, IE^k-MCC P5A, and IA^b-Eα single peptide mice have been described and crossed onto the invariant chain (Ii) and MHC class II locus-deficient background (28, 29). IA^b-2W1S single peptide mice were generated in the same way as the IA^b-Eα mice and crossed onto the IA^b and Ii-deficient background (30). Parent and F₁ into parent bone marrow (BM) chimeras were generated by reconstituting lethally irradiated C57BL/6 mice with 5 × 10⁶ BM cells isolated from either C57BL/6 or C57BL/6 × C3H F₁ mice. After 8 to 12 wk, mice were immunized and tested. All mice were maintained in a pathogen-free environment in accordance with institutional guidelines in the Animal Care Facility at the National Jewish Medical and Research Center.

Generation of IAβ^b–/– Ii^–/– C57BL/6 Fibroblast Cell Lines Expressing IE^k and IA^b Linked Peptides. Kidneys from IAβ^b–/– Ii^–/– C57BL/6 mice were diced and digested in 0.07% collagenase/0.001% DNase for 4 h at 37°C, followed by 48 h adherence to plastic. Nonadherent cells were then washed away. After 1 wk in culture, cells were immortalized by infection with a retrovirus expressing human papilloma virus (HPV) E6 and E7 genes and subcloned twice (31). Intercellular adhesion molecule-1 and CD80 were introduced to the fibroblast cell line by retroviral transduction (32). The transformed cells were then transfected with a series of mouse stem cell virus (MSCV)-based retroviruses (33) expressing the cDNAs for IEα^k and peptide-linked IEβ^k or IAα^b and peptide-linked IAβ^b. Cells were subcloned and selected for equivalent expression of IE^k or IA^b.

Immunization of Mice and Generation of T Cell Hybridomas. In vitro-activated IE^k allogeneic T cell hybridomas were derived from CD4⁺ T cells purified from the spleens and lymph nodes of C57BL/6 (H-2^b) or B10.D2 (H-2^d) mice by passage over Cellect columns (Cytovax Biotechnologies, Alberta, Canada) and incubated for 4 days with mitomycin C-treated B10.BR spleen cells in the presence of 20 μg/ml anti-IA^k antibody 11-5-2 to prevent activation of T cells reacting with IA^k. T cell blasts were then purified on Ficoll step gradients, incubated for 3 days with saturating amounts of IL-2, and fused to BW cells (34). Hybridomas were screened for reactivity with IE^k by using fibroblasts expressing wild-type IE^k MHC molecules. T cell hybridomas prepared from IE^k-expressing mice immunized with various foreign peptides were collected from the laboratories that made them: 2B4, AD10 (35, 36); ST4 (37); KMAC 126, 5KC-73.8 (38); and KH8.3 (39). KC-99A 4.18, 2KC-99A-12.3, KH10.1, and KALA-7.13 were generated from peptide-immunized mice by using standard techniques (34).

IE^k-Hb P5A and IE^k-MCC P5A-specific T cell hybridomas were derived from the various mice immunized with dendritic cells (DC) derived from IE^k-Hb P5A and IE^k-MCC P5A mice (39). DC were grown from BM in medium containing granulocyte–macrophage colony-stimulating factor, 1% normal mouse serum for 7 days (40). Mice were immunized by i.v. injection of 10⁷ DC. After 7 days, CD4⁺ T cells were purified from the immunized spleens and restimulated in vitro, again in 1% normal mouse serum, with spleen cells from IE^k-MCC P5A or IE^k-Hb P5A single peptide mice, and converted to T cell hybridomas (34).

IA^b-3K-specific T cell hybridomas were derived from mice immunized with IAβ^b–/– Ii^–/– BM-derived DC transduced with an IA^b-3K-expressing retrovirus (41, 42). After 7 days, CD4⁺ T cells were purified from immunized spleen and restimulated in vitro with DC transfected with an IA^b-3K-expressing retrovirus and converted to T cell hybridomas (34).

IE^k-Linked Peptide Monomer Production. Soluble IE^k bound to different peptides was prepared in insect cells and purified as described (43). The proteins at 200 μM were coated onto individual wells of Immulon 1B 96-well plates (Thermo Lab-systems, Franklin, MA) overnight at 4°C and then washed to remove unbound protein. Wells were then blocked with media containing serum for 2 hr at room temperature, followed by the addition of hybridomas for analysis.

Hybridoma Stimulation Assay. For all T cell hybridomas/single-peptide antigen-presenting cell (SP-APC) stimulation experiments, 10⁵ T cell hybridomas per well were incubated with 3 × 10⁴ of each of the SP-APCs for 24 h. For hybridomas/plate-bound IE^k monomer, 10⁵ T cell hybridomas were incubated on plate-bound IE^k monomer for 24 h. After overnight incubation at 37°C, supernatants were analyzed for IL-2 production by using a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (34). All T cell hybridoma data were generated from a minimum of three independent experiments.

Results

Characterization of SP-APC Library. In these studies, we have concentrated on T cells specific for the MHC protein IE^k bound to various peptides. To avoid contributions from unknown self-peptides bound to IE^k in our APCs, we constructed a collection of APCs, each of which express IE^k bound to a single, covalently linked peptide (SP-APC). The peptide sequences involved are listed in Fig. 1A. Because the linker in MHC molecules covalently bound to peptides is sensitive to proteases in an Ii-dependent manner, and many non-APCs express Ii, we prepared a fibroblast cell line from mice deficient in Ii as well as MHC class II to use as the parental cell line (described in Materials and Methods). SP-APC clones were selected for similar expression of IE^k (Fig. 5, which is published as supporting information on the PNAS web site, www.pnas.org). All of the SP-APC we could test were able to present IE^k/their linked peptide to T cells specific for that combination (data not shown). To determine the extent to which the linked peptide prevented IE^k from presenting other peptides, we measured the ability of this collection of SP-APC to present a peptide from mouse Hb or moth cytochrome c (MCC) to six T cell hybridomas specific for IE^k bound to either of these peptides. The SP-APC were unable to present exogenously added peptides at any concentration to four of the T cell hybridomas we tested (data not shown) and were able to present exogenously added peptide only very inefficiently (<10^–4 to 10^–5 of the activity of wild-type IE^k) to the two other T cell hybridomas tested (Fig. 1 B and C). We concluded that almost all of the IE^k on the SP-APC was occupied irreversibly with its covalently bound peptide.

Fig. 1.

Generation and analysis of APC-expressing IE^k/linked peptides. The protein sources and sequences of peptides bound to IE^k used in these studies are shown. Colored residues indicate the predicted TCR contact residues of each peptide; underlined residues indicate the MHC anchor positions. The sequences of peptides derived from MCC or mouse Hb but with substitutions at the P3, P5, or P6 positions of the peptides are not shown but can be inferred from their names, which indicate the position and nature of the change in amino acid sequence from the parent peptide. Nearly all of the IE^k on SP-APC is occupied by the covalently linked peptide. SP-APC were incubated with various concentrations of Hb peptide and the IE^k/Hb-specific T cell hybridoma KH-8.3 (B), or MCC peptide and the IE^k/MCC-specific T cell hybridoma AD10 (C). Responses were measured by production of IL-2 by the T cell hybridomas.

Allogeneic T Cells Can Be Highly Peptide-Degenerate. To isolate IE^k-restricted T cells from mice that have not undergone negative selection on IE^k/self-peptides, CD4⁺ T cells from IA^b-expressing C57BL/6 or IA^d/IE^d-expressing B10.D2 mice were stimulated with IE^k/self-expressing B10.BR splenocytes in vitro in a one-way mixed lymphocyte reaction. Responses to the other potential target in this reaction, IA^k, were blocked with an anti-IA^k antibody. The IE^k-reactive T cells were then converted to hybridomas for ease of analysis. These T cell hybridomas were, therefore, picked for their ability to recognize IE^k/self-peptides normally occupying IE^k in B10.BR mice. However, when probed for peptide specificity by using the SP-APC, three distinct groups of cross-reactivity were found. The most degenerate T cells (11/36 T cells) reacted with IE^k bound to many different peptides, whereas T cells in the second group (5/36 T cells) were able to react with IE^k bound to some structurally related peptides as well as the immunizing IE^k MHC molecules presenting wild-type self-peptides (Fig. 2 A and C), and summarized for many (Fig. 3 A and B). The third group (20/36) responded only to the immunizing IE^k MHC molecules presenting wild-type self-peptides (Fig. 3 A and B).

Fig. 2.

IE^k-reactive T cells from mice lacking IE^k can be highly peptide-degenerate. Individual T cell hybridomas were tested for their ability to react with IE^k bound to different peptides. Data shown are the titers of IL-2 produced by two such hybridomas in assays in which the hybridomas were challenged with IE^k bound to various single peptides on SP-APC fibroblasts (A and C) or as purified protein prepared in insect cells (B and D). The 17-5 T cell hybridoma from H2^b mice reacts with IE^k bound to many different peptides on SP-APC (A), as well as with purified, plate-bound IE^k linked to many different peptides (B). The 17-37 T cell hybridoma from H-2^b mice is differentially activated by all SP-APC tested (C) and responds similarly to purified, plate-bound IE^k-linked peptide monomers and SP-APC (D).

Fig. 3.

The extent of IE^k-restricted peptide specificity correlates with the level of IE^k-specific T cell tolerance. The hybridomas were tested for their ability to react with IE^k/various peptides presented on SP-APC. T cell hybridomas from H2^d mice (A) or H2^b (B), raised in one-way mixed lymphocyte reactions against IE^k, are very peptide promiscuous. (C) IE^k-restricted peptide degeneracy or self-reactivity is not observed in T cell hybridomas derived from mice expressing wild-type IE^k. (D and G) T cell hybridomas derived from H2^b animals immunized with DCs bearing IE^k bound to a single peptide are very peptide promiscuous. Immunizations were with DCs bearing Hb P5-A (D) or MCC P5-A (G). (E and H) Self-reactivity and some identifiable peptide cross-reactivity is observed from T cell hybridomas derived from IE^k-MCC P5-A (E) and IE^k-Hb P5-A (H) single peptide mice immunized with DCs expressing IE^k-Hb P5-A or IE^k-MCC P5-A, respectively. (F and I) IE^k-restricted peptide degeneracy or self-reactivity is not observed in T cell hybridomas derived from IE^k-expressing wild-type mice immunized with DCs expressing IE^k-Hb P5-A (F) or IE^k-MCC P5-A (I).

To check that the cross-reactivities were not due to presentation, by the SP-APC, of contaminating peptides from the mouse proteins of the APC or the FBS of the culture medium, we tested the ability of the hybridomas to respond to a panel of soluble IE^k-SP proteins that had been produced in insect cells, purified, and then coated on plastic (43). The results for two of the hybridomas are shown in Fig. 2 B and D. The spectrum of peptides recognized by the hybridomas on the insect cell-produced IE^k was similar to that bound to the SP-APC although the response to the plate-bound IE^k-SP was usually smaller. Some low-affinity responses were absent in the plate-bound soluble IE^k experiments as compared with the SP-APC experiments, likely due to a lack of adhesion or costimulatory molecules. Therefore, the hybridoma responses to the SP-APC were indeed against IE^k/covalently linked peptide. This ability of allogeneic T cells to react with IE^k bound to many peptides was not dependent on positive or negative selection on a particular MHC class II protein because it was common to T cells derived from either IA^b-expressing or IA^d and IE^d-expressing animals. Additionally, TCR sequencing has shown that this peptide cross-reactivity is not due to expression of multiple TCR α or β chains by the hybridomas concerned because four of the five hybridomas so analyzed express only one TCR α and β chain (data not shown).

Over the years, a large number of T cell hybridomas have been made from mice expressing IE^k that have been primed with various antigens (35–39). We tested some of these hybridomas for their ability to react with our collection of SP-APC. As shown in Fig. 3C, they were each specific for their immunizing peptide, or its close relatives, and did not react with APC expressing other peptides. Thus, the peptide promiscuity we found for T cells responding to allogeneic IE^k is in sharp contrast to the peptide specificity of T cells isolated from IE^k wild-type mice immunized with peptide or protein antigen.

In contrast to some reports of peptide-independent MHC recognition (44, 45), none of the allogeneic IE^k-reactive T cells isolated were completely ignorant of the bound peptide because in no case did a T cell react with all of the SP-APC equivalently. Furthermore, the extent of IE^k reactivity did not correlate with minor differences in IE^k expression level on the SP-APC (Fig. 5). The cross-reactivity of the T cells did not necessarily correlate with the nature of the potential TCR-contacting amino acids of the peptide, (compare cross-reactivities in Figs. 2 and 3 with the peptide sequences in Fig. 1). Additionally, the cross-reactivity, or lack of it, of the T cells was not necessarily due to bulky side chains of the peptide masking critical TCR binding residues on the MHC molecule because many of these allogeneic T cells failed to recognize IE^k with a peptide expressing alanines at all TCR contact residues (Figs. 2 and 3). Therefore, although some of the cross-reactivities are easily interpretable [for example the cross-reaction by many of the hybridomas between alanine and leucine at position P5 of the MCC peptide (Fig. 3)], many of them are not. Thus, some of these cross-reactivities may be due to subtle conformational changes in amino acids of the TCR or MHC when bound to different peptides (46, 47). Further understanding of this problem awaits more extensive structural studies.

Hb and MCC-Specific T Cells Derived from C57BL/6 Mice Can Be Highly Peptide-Degenerate. We wondered whether the marked MHC/peptide cross-reactivity of some T cells generated in the mixed lymphocyte reactions was simply a consequence of the way the T cells had been generated. To find out whether such T cells would also appear if they were generated in response to MHC bound to a single peptide, C57BL/6 mice were primed with DCs bearing a single allogeneic MHC protein, IE^k, bound to a single peptide, MCC P5A or Hb P5A (Fig. 3 D and G). As controls, C57BL/6 × C3H F₁ animals were similarly immunized. Some of the T cells in these F₁ animals have, like those in C57BL/6 mice, been positively selected on IA^b. However, all of the T cells in the F₁ mice, unlike those in C57BL/6 mice, have been tolerized to IE^k/mouse peptides.

As expected, T cell hybridomas generated from the F₁ mice expressing wild-type IE^k MHC molecules and immunized with IE^k/antigenic peptide were always highly specific for their immunizing antigen. None reacted with IE^k bound to self-peptides, and none of the T cells raised in response to Hb P5A reacted with any of the MCC-related peptides or vice versa (Fig. 3 F and I). In contrast, many of the T cells isolated from C57BL/6 mice immunized with IE^k bound to one of the two single peptides were again found to be degenerate in their recognition of other peptides (Fig. 3 D and G). Although some were highly specific for the immunizing peptide, others were reactive with many other peptides in addition to the immunogen.

Foreign Antigen-Specific T Cells Derived from MHC-SP Mice Are Often Self-Reactive. The fact that T cells derived from mice expressing wild-type IE^k MHC molecules react with IE^k in a very peptide-specific fashion may be a function of positive selection on IE^k or negative selection on IE^k/the many self-peptides to which it is bound in wild-type IE^k-expressing animals. Alternatively, peptide specificity may be due to tolerance to the overall conformational property of IE^k, which is common to all IE^k molecules regardless of the peptide to which they are bound. To distinguish these possibilities, we checked the peptide cross-reactivity of T cells that had been positively and negatively selected on IE^k bound almost entirely to a single peptide, by using mice expressing IE^k covalently bound to one peptide (IE^k-SP mice) (39). T cells isolated from immunized IE^k-SP mice reacted with IE^k/immunizing antigen (Fig. 3 E and H). They were also to some degree peptide-degenerate because many of them reacted with IE^k bound to some unknown self-peptides in an Ii-dependent and/or -independent manner (12/19 T cells from IE^k-MCC P5A SP mice, 18/23 T cells from IE^k-Hb P5A SP mice). They were, however, less peptide-degenerate than their counterparts from IE^k-negative animals because none of them showed extensive cross-reactivity on the SP-APC panel.

Foreign antigen-specific T cells derived from two different IA^b-SP mice show a similar frequency of cross-reaction with wild-type IA^b-expressing cells (10/11 T cells from IA^b-Eα SP mice, 4/4 T cells from IA^b-2W1S SP mice) (Fig. 6, which is published as supporting information on the PNAS web site). These results indicate that a single peptide bound to MHC is not sufficient to eliminate all broadly reactive T cells, but that the process may require the many conformations offered by the normal collection of self-peptides bound to MHC.

BM-Derived Cells Eliminate Peptide-Degenerate T Cells. Our results suggest that the presence of wild-type IE^k/many peptides during negative selection is sufficient to remove broadly peptide-reactive T cells regardless of the thymic MHC alleles involved in positive selection. As a final test of this idea, we prepared BM chimeric mice in which the positively selecting thymic MHC was IA^b, but the negatively selecting BM-derived cells did ([H2^b × H2^k]F₁ → H2^b) or did not (H2^b → H2^b) express IE^k. T cells from these mice were primed with DCs bearing IE^k/Hb P5A and analyzed for their cross-reactivity with other peptides. Many of the T cells derived from animals that had undergone positive and negative selection on only IA^b MHC molecules were highly peptide-degenerate (Fig. 4A). In contrast, T cells that had been positively selected on IA^b but negatively selected in the presence of IE^k were very peptide specific (Fig. 4B). Thus, the phenotype of T cells derived from the allogeneic, single peptide, wild-type, and chimeric mice indicates that the extent of allowable peptide degeneracy is controlled by MHC-specific tolerance.

Fig. 4.

Negative selection eliminates peptide-degenerate T cells from the mature T cell repertoire. Irradiated BM-reconstituted mice, in which positive selection could occur on IA^b but the T cells were or were not tolerant to IE^k, were prepared as described in Materials and Methods. The mice were immunized with DCs expressing IE^k-Hb P5-A. T cell hybridomas specific for IE^k-Hb P5-A were prepared from the animals and tested for their ability to react with APC-expressing IE^k/various peptides. IE^k-Hb P5-A-reactive T cell hybridomas derived from IA^b-expressing C57BL/6 mice reconstituted with BM from IA^b-expressing C57BL/6 mice can be highly peptide-degenerate (A) whereas IE^k-Hb P5-A-specific T cell hybridomas derived from C57BL/6 mice reconstituted with IA^b/IE^k-expressing (C57BL/6 × C3H) F₁ BM are highly peptide specific (B).

Discussion

The individual contains a limited number of mature T cells, yet must be able to respond to a vast number of potential foreign antigens. This feat could be achieved efficiently if individual TCRs could recognize many different peptide antigens. However, the increase in the breadth of the immune response that could be so occasioned has to be balanced with the fact that broadly cross-reactive T cells are likely to react with self-MHC bound to the many self-peptides. Thus, the requirement that T cells cover the waterfront of potential foreign antigens has to be balanced with the need that they not react with self.

A number of people have thought about this problem and concluded that T cells must be somewhat, but not overly, peptide cross-reactive (8, 11, 48, 49). Experimental evidence suggests that the peripheral TCR repertoire is such that the TCRs on individual mature T cells are able to recognize only a limited number of peptides bound to self-MHC molecules (4, 5). How this balance of peptide degeneracy is established and whether each TCR in the repertoire is similarly cross-reactive had yet to be determined.

To ensure self-tolerance, thymic negative selection eliminates self-reactive T cells. Estimates have suggested that this process eliminates 50–75% of the positively selected T cells (20–23). There are perhaps 10¹⁰ possible combinations of TCRs; of these, ≈1–5% are thought to allow positive selection on any given haplotype set of MHC proteins (50–52). Thus, if the estimates are right, MHC proteins bound to a couple of thousand self-peptides are able to delete in the thymus thymocytes bearing >10⁸ different TCRs. If TCRs have only limited peptide cross-reactivity, how is this possible? In part the paradox is resolved by the fact that thymocytes are more sensitive to negative selection than mature T cells are to activation (14–16). Thus MHC/peptides that could not productively stimulate peripheral T cells can nevertheless participate in thymocyte deletion.

The experiments described here, however, provide an additional explanation. In the absence of negative selection, the repertoire of TCRs is a continuum of peptide degeneracy. During negative selection, a significant percentage of thymocytes that are deleted suffer this fate because they bear very cross-reactive TCRs, TCRs that can react with MHC/many peptides with affinities that are high enough to drive responses by mature T cells bearing them. Thus, negative selection refines the potential TCR repertoire into one focused on self-MHC bound to specific peptides.

The repertoire of T cells specific for IE^k/peptide, isolated from mice expressing allogeneic class II protein, includes TCRs that are much more peptide promiscuous than those isolated from IE^k-SP mice, reflecting, probably, more efficient removal of IE^k-reactive T cells in the latter animals. Previous estimates of the percentages of T cells removed by negative selection were based on the numbers of T cells that escape negative selection in class II-SP mice. However, the results with allogeneic animals reported here indicate that a reasonable percentage of potentially MHC-reactive T cells (those that are extremely cross-reactive) are deleted in the class II-SP animals. This percentage amounts to about half of the IE^k-reactive T cells in both C57BL/6 and B10.D2 mice (26/50 for all allogeneic T cells analyzed in this study). Thus, the actual percentage of T cells able to undergo positive selection, which are deleted by negative selection, could be as high as 90%.

Our data offer an answer to the long-standing quandary about the nature of the high frequency of allo-MHC-specific T cells. The two, not necessarily exclusive, models proposed suggest either that the high frequency of allo-reactivity derives from T cells recognizing unique features of the self-peptides presented by the allogeneic MHC molecule (53), or that T cells recognize amino acid residue polymorphisms of the allogeneic MHC molecules with little or no regard for the bound peptide (46, 54). The data presented here indicate that mice lacking IE^k-specific negative selection have IE^k peptide-dependent, as well as peptide-independent, T cells in relatively equivalent numbers. When the T cell repertoire undergoes negative selection on IE^k/single peptide, the IE^k peptide-independent T cells are eliminated. The remaining foreign antigen-specific T cells in these mice, however, have a high degree of reactivity toward IE^k/wild-type self-peptides. Whether the nature of this foreign and self-reactivity is due to an increase in peptide promiscuity of the T cells or is due to the T cells recognizing shared antigenic features between the foreign and self-peptides requires further investigation. The finding that the majority of foreign antigen-specific T cells are also self-reactive suggests that the inherent MHC bias of the germ line TCR repertoire may be even stronger than previously proposed and best examined in T cells that have been positively, but not negatively, selected (52, 55).

Thus, we conclude that it is primarily negative selection that reduces an intrinsically peptide-degenerate T cell repertoire to a collection of TCRs whose specificity is highly focused on the MHC-bound peptide. This process allows the body to balance the benefits of T cell recognition efficiency due to peptide promiscuity with the requirements of self-tolerance.