Public T cell receptor beta-chains are not advantaged during positive selection

Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.     

Antiretroviral therapy reduces the magnitude and T cell receptor repertoire diversity of HIV-specific T cell responses without changing T cell clonotype dominance

After initiation of antiretroviral therapy (ART), HIV loads and frequencies of HIV epitope-specific immune responses decrease. A diverse virus-specific T cell receptor (TCR) repertoire allows the host to respond to viral epitope diversity, but the effect of antigen reduction as a result of ART on the TCR repertoire of epitope-specific CD8(+) T cell populations has not been well defined. We determined the TCR repertoires of 14 HIV-specific CD8(+) T cell responses from 8 HIV-positive individuals before and after initiation of ART. We used multiparameter flow cytometry to measure the distribution of memory T cell subsets and the surface expression of PD-1 on T cell populations and T cell clonotypes within epitope-specific responses from these individuals. Post-ART, we noted decreases in the frequency of circulating epitope-specific T cells (P = 0.02), decreases in the number of T-cell clonotypes found within epitope-specific T cell receptor repertoires (P = 0.024), and an overall reduction in the amino acid diversity within these responses (P < 0.0001). Despite this narrowing of the T cell response to HIV, the overall hierarchy of dominant T cell receptor clonotypes remained stable compared to that pre-ART. CD8(+) T cells underwent redistributions in memory phenotypes and a reduction in CD38 and PD-1 expression post-ART. Despite extensive remodeling at the structural and phenotypic levels, PD-1 was expressed at higher levels on dominant clonotypes within epitope-specific responses before and after initiation of ART. These data suggest that the antigen burden may maintain TCR diversity and that dominant clonotypes are sensitive to antigen even after dramatic reductions after initiation of ART.     

Immunodominance is altered in T cell receptor (beta-chain) transgenic mice without the generation of a hole in the repertoire.

Despite the tremendous plasticity of the TCR repertoire, T cells recognize a limited number of antigenic sites (frequently a single site, or immunodominant epitope) on a complex protein Ag. Current models suggest that the immunodominant epitope of a complex protein is the processed peptide that binds to the MHC molecule with the highest affinity. Conversely, the inability of the T cell population to recognize a specific epitope, termed a "hole" in the repertoire, can prevent the immunodominance of a peptide despite efficient processing and MHC binding of the peptide. The role of specific TCR alpha- or beta-chains in determining MHC restriction and recognizing specific epitopes is complex and incompletely understood. To evaluate the contribution of each TCR chain to the functional diversity of the T cell repertoire, we investigated in vivo the T cell response to phage lambda-repressor protein in transgenic mice expressing a single rearranged beta-chain gene (C57L beta mice) in association with the complete germline alpha-chain repertoire. Our results demonstrate that expression of the TCR beta-chain transgene alters the immunodominant epitope recognized by T cells. However, after immunization with the appropriate peptide the transgenic mice can also respond to the nonimmunodominant epitope; thus, the expression of the TCR beta-chain transgene does not create a hole in the repertoire. These data indicate that the primary site, or immunodominant epitope, of an Ag recognized by T cells can be altered by the preimmune TCR repertoire independent of antigen processing and MHC affinity.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

Selection of T cell clones expressing high-affinity public TCRs within Human cytomegalovirus-specific CD8 T cell responses

Assessment of clonal diversity of T cell responses against human CMV (HCMV), a major cause of morbidity in immunodepressed patients, provides important insights into the molecular basis of T cell immunodominance, and has also clinical implications for the immunomonitoring and immunotherapy of HCMV infections. We performed an in-depth molecular and functional characterization of CD8 T cells directed against an immunodominant HLA-A2-restricted epitope derived from HCMV protein pp65 (NLV/A2) in steady state and pathological situations associated with HCMV reactivation. NLV/A2-specific T cells in healthy HCMV-seropositive donors showed limited clonal diversity and usage of a restricted set of TCR Vbeta regions. Although TCRbeta-chain junctional sequences were highly diverse, a large fraction of NLV/A2-specific T cells derived from distinct individuals showed several recurrent (so-called "public") TCR features associated in some cases with full conservation of the TCRalpha chain junctional region. A dramatic clonal focusing of NLV/A2-specific T cells was observed in situations of HCMV reactivation and/or chronic inflammation, which resulted in selection of a single clonotype displaying similar public TCR features in several patients. In most instances the NLV/A2-specific dominant clonotypes showed higher affinity for their Ag than subdominant ones, thus suggesting that TCR affinity/avidity is the primary driving force underlying repertoire focusing along chronic antigenic stimulation.     

Distinct CD8+ T cell repertoires primed with agonist and native peptides derived from a tumor-associated antigen

Heteroclitic peptides are used to enhance the immunogenicity of tumor-associated Ags to break T cell tolerance to these self-proteins. One such altered peptide ligand (Cap1-6D) has been derived from an epitope in human carcinoembryonic Ag, CEA(605-613) (Cap1). Clinical responses have been seen in colon cancer patients receiving a tumor vaccine comprised of this altered peptide. Whether Cap1-6D serves as a T cell agonist for Cap1-specific T cells or induces different T cells is unknown. We, therefore, examined the T cell repertoires elicited by Cap1-6D and Cap1. Human CTL lines and clones were generated with either Cap1-6D peptide (6D-CTLs) or Cap1 peptide (Cap1-CTLs). The TCR Vbeta usage and functional avidity of the T cells induced in parallel against these target peptides were assessed. The predominant CTL repertoire induced by agonist Cap1-6D is limited to TCR Vbeta1-J2 with homogenous CDR3 lengths. In contrast, the majority of Cap1-CTLs use different Vbeta1 genes and also had diverse CDR3 lengths. 6D-CTLs produce IFN-gamma in response to Cap1-6D peptide with high avidity, but respond with lower avidity to the native Cap1 peptide when compared with the Cap1-CTLs. Nevertheless, 6D-CTLs could still lyse targets bearing the native epitope. Consistent with these functional results, 6D-CTLs possess TCRs that bind Cap-1 peptide/MHC tetramer with higher intensity than Cap1-CTLs but form less stable interactions with peptide/MHC as measured by tetramer decay. These results demonstrate that priming with this CEA-derived altered peptide ligand can induce distinct carcinoembryonic Ag-reactive T cells with different functional capacities.     

Very diverse CD8 T cell clonotypic responses after virus infections

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-gamma capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vbeta chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1-2 orders of magnitude than predicted previously.     

Selection and long-term persistence of reactive CTL clones during an EBV chronic response are determined by avidity, CD8 variable contribution compensating for differences in TCR affinities.

Recent studies have suggested that the diversity of TCR repertoire after primary immunization is conserved in memory T cells and that a progressive narrowing of this repertoire may take place during recall infections. It now remains to be investigated which parameters determine the repertoire of the memory response and possibly restrict its diversity after subsequent antigenic challenges. To address this question, we took advantage of a panel of CD8+ T cell clones from the joint of a rheumatoid arthritis patient and selected for their reactivity against a single MHC/peptide complex. Characterization of both TCR chains documented a great diversity among those clones and the persistence of clonotypes over a 2-yr period. Strikingly, despite the observed repertoire heterogeneity, all clones displayed a narrow range of MHC/peptide density requirements in cytotoxicity assays (ED50 between 9 and 36 nM). TCR affinities were then indirectly estimated by blocking CD8 interaction with an anti-CD8 mAb. We found a wide range of TCR affinities among the different clonotypes that segregated with Vbeta usage. We thus propose that during an in vivo chronic response, a narrow range of avidity of the TCR-CD8 complex conditions long-term clonotype persistence, and that the level of CD8 contribution is adjusted to keep clonotypes with variable TCR affinities within this avidity window.     

Despite biased TRBV gene usage against a dominant HLA B57-restricted epitope, TCR diversity can provide recognition of circulating epitope variants

The role of epitope-specific TCR repertoire diversity in the control of HIV-1 viremia is unknown. Further analysis at the clonotype level is important for understanding the structural aspects of the HIV-1 specific repertoire that directly relate to CTL function and ability to suppress viral replication. In this study, we performed in-depth analysis of T cell clonotypes directed against a dominantly recognized HLA B57-restricted epitope (KAFSPEVIPMF; KF11) and identified common usage of the TCR beta-chain TRBV7 in eight of nine HLA B57 subjects examined, regardless of HLA B57 subtype. Despite this convergent TCR gene usage, structural and functional assays demonstrated no substantial difference in functional or structural avidity between TRBV7 and non-TRBV7 clonotypes and this epitopic peptide. In a subject where TRBV7-usage did not confer cross-reactivity against the dominant autologous sequence variant, another circulating TCR clonotype was able to preferentially recognize the variant peptide. These data demonstrate that despite selective recruitment of TCR for a conserved epitope over the course of chronic HIV-1 infection, TCR repertoire diversity may benefit the host through the ability to recognize circulating epitope variants.     

Identical beta cell-specific CD8(+) T cell clonotypes typically reside in both peripheral blood lymphocyte and pancreatic islets

A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring beta cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8(+) T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3alpha and CDR3beta sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. beta cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.     

Persistence of dominant T cell clones in accepted solid organ transplants

Donor/recipient MHC class II matching is beneficial to the survival of allogeneic kidneys in humans and swine. In the latter, tolerance to class I-disparate grafts can be induced by a short course of immunosuppression, a peripheral mechanism that implicates regulatory T cells. Absence of treatment will lead to prompt rejection. Rejected grafts are infiltrated by dominant alloaggressive T cells, whereas there is still speculation on the specificity and function of T cells invading accepted tissues. To characterize the TCR repertoire of graft-infiltrating T cells (GITC) in accepted kidneys, we have used the RT-PCR-based spectratyping technique to assess the length polymorphism of the porcine TCRbeta chain complementary-determining region 3 (CDR3). Results show that T cells infiltrating accepted kidneys (n = 5) express a restricted polymorphism of the CDR3 length, whereas PBL from the same animal have the polymorphic distribution of CDR3 lengths found in naive animals; that the skewed Vbeta repertoire in accepted grafts involved distinct Vbeta subfamilies in otherwise MHC-identical recipient animals; that GITC clonal dominance is not caused by immunosuppression because a second kidney, accepted without drug treatment, exhibits the same TCR Vbeta CDR3 profiles than those detected in the first graft; and that intragraft clonal dominance intensifies with time, indicating progressive preeminence of nonaggressive GITC clones. Collectively, these data represent the first example, in a preclinical model, of the emergence of nonaggressive intragraft clones, which may be involved in the induction/maintenance of local tolerance to allogeneic tissues.     

Common intra-articular T cell expansions in patients with reactive arthritis: identical beta-chain junctional sequences and cytotoxicity toward HLA-B27

Spondyloarthropathies constitute a group of autoimmune diseases of special interest because of their tight association with the MHC class I molecule HLA-B27 and the bacterial triggering of some clinical forms called reactive arthritis (ReA). One current hypothesis is the presentation by HLA-B27 of a so-called arthritogenic peptide to T cells. To better focus on the relevant T cell populations within the joint, we performed an extensive beta-chain T cell repertoire analysis of synovial fluid compared with PBL in seven patients, four of whom were characterized as having ReA triggered by Yersinia enterocolitica, Chlamydia trachomatis, or Shigella sonnei. Analysis of the size diversity of the beta-chain complementarity-determining region 3 (CDR3) allowed us to evaluate the degree of T cell clonality in the samples. Oligoclonal T cell expansions were frequently observed in the joint. In one patient, CDR3 amino acid sequences of major expansions using two different BV genes were identical. One dominant T cell expansion and several CDR3 amino acid sequences were identical in two different patients. Furthermore, one sequence was identical with a sequence reported independently in a Salmonella-induced ReA patient. Together, these data indicate a surprisingly high degree of conservation in the T cell responses in recent-onset ReA triggered by different micro-organisms. A CD8+ synovial line expressing shared clonotypes was established and reacted toward several B*2705 lymphoblastoid cell lines, therefore supporting a molecular mimicry phenomenon at the T cell level in the disease mechanism.     

Changing patterns of dominant TCR usage with maturation of an EBV-specific cytotoxic T cell response

Infection with EBV provides a unique opportunity to follow the human CD8(+) T cell response to a persistent, genetically stable agent from the primary phase, as seen in infectious mononucleosis (IM) patients, into long-term memory. This study focuses on the response to an immunodominant HLA-A2.01-restricted epitope, GLCTLVAML, from the EBV-lytic cycle Ag BMLF1. TCR analysis of the highly amplified primary response to this epitope revealed markedly oligoclonal receptor usage among in vitro-derived clones, with similar clonotypes dominant in all three IM patients studied. Direct staining of IM T cell preparations with the A2.01/GLCTLVAML tetramer linked this oligoclonal epitope-specific response with appropriate Vbeta subset expansions in the patients' blood. These patients were studied again >2 years later, at which time TCR analysis of in vitro-reactivated clones suggested that rare clonotypes within the primary response had now come to dominate memory. Five additional A2. 01-positive IM patients were studied prospectively for Vbeta subset representation within primary and memory epitope-specific populations as identified by tetramer staining. In each case, the primary response contained large Vbeta2, Vbeta16, or Vbeta22 components, and in three of five cases the originally dominant Vbeta was represented very poorly, if at all, in memory. We conclude 1) that an EBV epitope-specific primary response large enough to account for up to 10% CD8(+) T cells in IM blood may nevertheless be dominated by just a few highly expanded clonotypes, and 2) that with persistent viral challenge such dominant T cell clonotypes may be lost and replaced by others in memory.     

Rapid CD8+ T cell repertoire focusing and selection of high-affinity clones into memory following primary infection with a persistent human virus: human cytomegalovirus

To investigate the mechanism of selection of individual human CD8+ T cell clones into long-term memory following primary infection with a persistent human virus (human CMV (HCMV)), we undertook a longitudinal analysis of the diversity of T cell clones directed toward an immunodominant viral epitope: we followed this longitudinally from early T cell expansion through the contraction phase and selection into the memory pool. We show that following initial HCMV infection, the early primary response against a defined epitope was composed of diverse clones possessing many different TCR Vbeta segments. Longitudinal analysis showed that this usage rapidly focused predominantly on a single TCR Vbeta segment within which dominant clones frequently had public TCR usage, in contrast to subdominant or contracted clones. Longitudinal clonotypic analysis showed evidence of disproportionate contraction of certain clones that were abundant in the primary response, and late expansion of clones that were subdominant in the primary response. All dominant clones selected into memory showed similar high functional avidity of their TCR, whereas two clones that greatly contracted showed substantially lower avidity. Expression of the IL-7R is required for survival of murine effector CD8+ T cells into memory, but in primary HCMV infection IL-7R was not detected on circulating Ag-specific cells until memory had been established. Thus, the oligoclonal T cell repertoire against an immunodominant persistent viral epitope is established early in primary infection by the rapid selection of public clonotypes, rather than being a stochastic process.     

Antigen-driven selection of TCR In vivo: related TCR alpha-chains pair with diverse TCR beta-chains

Ag-driven selection mediates effective T cell help and the development of Th cell memory in vivo. To analyze the dynamics of interclonal competition during the selection process in vivo, we use the I-Ek-restricted murine response to pigeon cytochrome c (PCC). The dominant PCC-specific clonotype expresses Valpha11Vbeta3 V regions with preferred sequence features in the third hypervariable regions (CDR3). In the current study we define and quantitatively monitor four subdominant PCC-specific clonotypes that express Valpha11 paired with non-Vbeta3 TCR beta-chains (Vbeta6, Vbeta8.1/8. 2, Vbeta8.3, and Vbeta14). The subdominant clonotypes emerge with similar dynamics to the dominant clonotype and together amount to similar numbers as the dominant clonotype in vivo. These subdominant clonotypes do not efficiently enter germinal centers, although they enter the memory compartment and rapidly re-emerge upon secondary challenge. Analysis of CDR3 diversity in the TCR alpha-chains identifies many preferred sequence features expressed by the dominant clonotype. These studies quantitatively demonstrate selection for diverse Th cells in vivo and highlight TCR alpha-chain dominance in Ag-driven selection for best fit.     

The lymphocytic infiltration in calcific aortic stenosis predominantly consists of clonally expanded T cells

Valve lesions in degenerative calcific aortic stenosis (CAS), a disorder affecting 3% of those older than 75 years, are infiltrated by T lymphocytes. We sought to determine whether the alphabeta TCR repertoire of these valve-infiltrating lymphocytes exhibited features either of a polyclonal nonselective response to inflammation or contained expanded clones suggesting a more specific immune process. TCR beta-chain CDR3-length distribution analysis using PCR primers specific for 23 Vbeta families performed in eight individuals with CAS affecting tri- or bileaflet aortic valves revealed considerable oligoclonal T cell expansion. In five cases, beta-chain nucleotide sequencing in five selected Vbeta families showed that an average of 92% of the valve-infiltrating T cell repertoire consisted of expanded T cell clones, differing markedly in composition from the relatively more polyclonal peripheral CD8 or CD4 T cell subsets found even in this elderly population. Twenty-four of the valve-infiltrating T cell clones also had the same clone identified in blood, some of which were highly expanded. Interestingly, 22 of these 24 shared clones were CD8 in lineage (p = 1.5 x 10(-12)), suggesting a possible relationship to the expanded CD8(+)CD28(-) T cell clones frequently present in the elderly. Additionally, the sequences of several TCR beta-chain CDR3 regions were homologous to TCR beta-chains identified previously in allograft arteriosclerosis. We infer that these findings are inconsistent with a nonselective secondary response of T cells to inflammation and instead suggest that clonally expanded alphabeta T cells are implicated in mediating a component of the valvular injury responsible for CAS.     

CTL recognition of a protective immunodominant influenza A virus nucleoprotein epitope utilizes a highly restricted Vbeta but diverse Valpha repertoire: functional and structural implications

To investigate protective immunity conferred by CTL against viral pathogens, we have analyzed CD8(+) T cell responses to the immunodominant nucleoprotein epitope (NP(366-374)) of influenza A virus in B6 mice during primary and secondary infections in vivo. Unlike the highly biased TCR Vbeta repertoire, the associated Valpha repertoire specific for the NP(366-374)/D(b) ligand is quite diverse. Nonetheless, certain public and conserved CDR3alpha clonotypes with distinct molecular signatures were identified. Pairing of public Valpha and Vbeta domains creates an alphabeta TCR heterodimer that binds efficiently to the NP(366-374)/D(b) ligand and stimulates T cell activation. In contrast, private TCRs, each comprising a distinct alpha chain paired with the same public beta chain, interact very differently. Molecular dynamics simulation reveals that the conformation and mobility of the shared Vbeta CDR loops are governed largely by the associated Valpha domains. These results provide insight into molecular principles regarding public versus private TCRs linked to immune surveillance after infection with influenza A virus.     

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.     

Modeling of the TCR-MHC-peptide complex

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.