T-cell Receptor Vα Research Articles

The Feature of Distribution and Clonality of TCR Vα and Vβ Repertoire in Cord Blood.

Analysis of T cell receptor (TCR) Vα and Vβ repertoire is one of the sensitive methods to understand the distribution and clonality of T cells from different samples, which is used to identify clonal expansion T cells that response to tumor associated antigens. Cord blood has been used successfully as a source of hematopoietic stem cells for transplantation. Recently, stem cells transplantation was demonstrated more important as immunotherapy against malignance cells. And cord blood T cells are resourceful for production the specific CTL to use in leukemia immunotherapy. But the feature of distribution and clonality of TCR Vα and Vβ subfamily T cells in cord blood is not yet clearly defined. In the present study, the CDR3 of 29 TCR Vα and 24 Vβ subfamily genes were analyzed in T cells from 10 cases of cord blood, which obtained at delivery from full-term healthy pregnancies, using RT-PCR and genescan technique. Peripheral blood of nine cases healthy adult volunteers served as controls. The results showed that 15–19 of 29 Vα and 9–15 of Vβ subfamily T cells could be identified in the most cases, whereas only 2–4 of Vα subfamilies expressed was found in 3 cases. The most frequently expressed Vα subfamilies were Vα 3 and Vα 10 (100%), Vα 4, 5, 6, 8, 12, 15, 17 and Vα 21 (70%), Vα1, 13, 25, and Vα26 (60%), with a lower expression rate found in Vα16, 24 and Vα 28 (10%). Vα 9 and Vα 29 were not detected in both CB and healthy adults. In analysis of TCR Vβ repertoire, Vβ3, 5, 8 and Vβ9 genes could be identified in all samples, Vβ7 and Vβ13 could be found in 90% of samples, whereas Vβ4, 6, 11, 12, 18, 23 and Vβ24 were absent in all samples, which could be detected in normal peripheral blood samples. Genescan analysis showed that all PCR products of TCR Vα and Vβ subfamilies from cord blood displayed a Gaussian distribution of CDR3 lengths (multi-peaks), which are corresponding to polyclonal rearrangement pattern, except for tow cases, which displayed oligoclonal peak in Vα 24 or Vα28 respectively. In contrast, some Vα subfamily products from 8 of 9 cases of healthy adult volunteers contained at least an oligoclonal peak in different Vα subfamilies respectively, however, multi-peaks were found in all PCR products of TCR Vβ from normal peripheral blood. In conclusion, more than 20% of TCR Vα or Vβ subfamily T cells were absent in cord blood. The majority of TCR Vα and Vβ subfamily T cells in cord blood displayed polyclonality. Occasional oligoclonal peaks are identified in some cases.

Analysis of the T-Cell Receptor Vα Gene Repertoire and Clonal Expansion in the Benzene-Exposed Group.

Benzene is a potent human leukemogen, but its mechanism of hematotoxicity is uncertain. It is well know that benzene inhibit T cells proliferation. Several reports revealed that clonal expansion TCR Vβ T cells could be found in workers exposed to benzene. In this study we observe the distribution of TCR Vα gene repertoire and clonal expansion in peripheral blood mononuclear cells from 9 donors and 16 workers exposed to benzene. Complementarity determining region 3 (CDR3) of TCR Vα subfamily genes were amplified using RT-PCR. The PCR products were further analyzed by genescan to evaluating clonality of T cells. 29 Vα subfamily could be detected in 9 donors.1~11 Vα subfamilies were identified in all but one of the workers studied. The most frequently expressed Vα subfamily were Vα3, Vα12 and Vα19 (68.8%), Vα14(56.3%), with a lower expression rate found in Vα5,Vα15, Vα16, Vα 22, Vα 23 and Vα 24 (6.3%). Clonal expansion T cells in one or more Vα subfamily were found in 12 out of all workers studied, including oligoclonal, oligocolonal trend and bioclonal patterns. The frequency of clonal expansion T cells in Vα12, Vα14 and Vα19 subfamilies were higher than others. In conclusion, skewed distribution and clonal expansion of TCR Vα subfamily T cells could be found in workers exposed to benzene. Vα12, Vα14 and Vα19 subfamilies may be highly sensitive to benzene exposed. This is the first report of clonal expansion TCR Vα T cells in the benzene-exposed group. The bias pattern of TCR Vα T cells may be due to the immune cytotocicity from benzene. However, whether the oligoclonality in some Vα subfamilies reflect the phenomenon of clone absense or may be a response clone to benzene-related impairment during exposed to benzene, remains an open question.

T-Cell Receptor Vα Spectratype Analysis of a CD4-Mediated T-Cell Response against Minor Histocompatibility Antigens Involved in Severe Graft-versus-Host Disease

Although CD4 + T cells can have an important role in mediating lethal graft-versus-host disease (GVHD) directed to multiple minor histocompatibility antigens (miHA) after bone marrow transplantation, their precise characterization and effector function remains elusive. In this regard, T cell receptor (TCR) Vβ spectratype analysis has been a powerful tool for identifying donor CD4 + T cell populations expanding to host miHA after bone marrow transplantation in the major histocompatibility complex–matched C57BL/6 (B6) → C.B10-H2 b (BALB.B) model of lethal GVHD. Removal of all of the Vβ + T cell families containing these responding cells from the donor inoculum has proven to be an effective means of preventing the development of GVHD. Previous studies have also found that of the 11 miHA-responsive B6 CD4 + Vβ + T cell families, transplantation of Vβ2 + and Vβ11 + T cells together into lethally irradiated BALB.B mice appeared to be primarily responsible for the severity of resultant GVHD. Further focusing on these critical CD4 responses, in this study we demonstrate that B6 CD4 +Vβ11 + T cells alone can induce lethal GVHD in BALB.B recipients. In addition, immunohistochemical staining of host lingual and intestinal epithelial tissues supported the capacity of Vβ11 + T cells to infiltrate typical GVHD-associated target areas. To further characterize the specific CD4 +Vβ11 + T cells involved in this anti-miHA response, TCR Vα spectratype analysis was performed and indicated that 6 Vα chains were used by this reactive population. These results provide further evidence that a restricted repertoire of T cell specificities, presumably recognizing a correspondingly low number of miHA, is sufficient for the induction of severe GVHD.

T cells and the thymus in developing zebrafish

The expression of genes encoding T cell receptor (TCR) α was used to follow the development of the thymus and to analyze the distribution of T cells in zebrafish. In the thymus, expression was first detected, by in situ hybridization, at four days post fertilization. In RNA extracted from whole fish, TCRα transcripts were also detected at four days and reached adult levels at three weeks. At six weeks, TCRα was expressed throughout the thymus, whereas rag1 expression was localized to the peripheral regions. Expression of TCRα outside the thymus was detected at nine days. In adult peripheral organs, the greatest expression was in the pronephros, mesonephros and intestine; expression in the spleen became greater as fish age. Three new, relatively highly expressed, TCR Vα families were identified.

T cell receptor (TCR) repertoire and function of human epidermal T cells: restricted TCR V alpha-V beta genes are utilized by T cells residing in the lesional epidermis in fixed drug eruption.

Human epidermis contains a phenotypically heterogeneous population of T cells. No information however, is available regarding the TCR repertoire of these T cells and their relevant physiologic and pathologic functions in vivo. To this end, T cells were prepared from the lesional epidermis in two patients with fixed drug eruption (FDE) and their phenotype, function and TCR repertoire were examined in parallel. Both epidermal T cells, termed FDE-1 and -2 cells, respectively, expressed alpha beta TCR, but displayed some phenotypic heterogeneity. These T cells were induced to display cytolytic activity by ligation of the CD3/TCR-alpha beta complex. Comparative analyses of TCR V alpha and V beta expression in the epidermal T cells and the paired peripheral blood lymphocytes (PBL) by quantitative polymerase chain reaction (PCR) demonstrated that the epidermal T cells, but not the paired PBL, utilized a very limited range of V alpha and V beta genes. These results indicate that some expansion or preferential migration of epidermal T cells that recognize a restricted set of antigens expressed within the epidermis could occur in situ following ingestion of the causative drug. The persistence of these epidermal T cells in FDE lesions suggests their pathologic role in a drug-induced flare.

Regulated T-cell development: a victim of multiple conspiracies.

Over about 400 million years there has been a conserved subdivision of T cells into those bearing a γδ T-cell receptor (TCR) and those bearing an αβ TCR. No one is sure why this is, but the two cell types have some interesting distinguishing characteristics, particularly their antigen specificities and their anatomical homing.1 In addition, the development of γδ cells seems always to precede that of αβ T cells. This is a good thing for γδ cells, because the TCRδ locus lies embedded within the TCRα locus, and when TCR Vα and Jα gene segments rearrange, they delete the TCRδ genes. Unchecked, this would substantially reduce the chances of making γδ cells.

Modeling of the TCR-MHC-peptide complex

The methodology for generating a homology model of the T1 TCR-PbCS-Kd 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 α1/α2 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 Vβ chain, the 1934.4 TCR Vα chain, and the H-2 Kb-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 CDR3α, CDR1β, CDR2β and CDR3β 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.

Allogeneic responses to the class Ib antigen Qa1: limited T-cell receptor Valpha but not Vbeta chain usage.

Allogeneic responses to the class Ib antigen Qa1: limited T-cell receptor Valpha but not Vbeta chain usage.

B7.1 costimulation increases T-cell proliferation and cytotoxicity via selective expansion of specific variable alpha and beta genes of the T-cell receptor

Background: Optimal T-cell activation requires not only ligation of the T-cell receptor (TcR) but also delivery of costimulatory signals by various accessory molecules. The interaction of the costimulatory molecule B7.1 (CD80) with its receptor CD28 provides a strong positive signal to T cells. Methods: The B7.1 gene was transduced into cultured human ovarian, breast, and pancreatic tumor cells by using a retroviral vector. Autologous as well as allogeneic naive T-cells were stimulated with either wild-type or B7.1-transduced tumor cells in a mixed lymphocyte tumor cell culture (MLTC). In addition to cytolytic activity, T-cell proliferation, T-cell subset composition, and the frequencies of TcR variable (V) alpha and beta genes were compared in T cells from both types of MLTC. Results: Introduction of the B7.1 gene into tumor cells was successful in all tumors to a varying degree. Those tumors expressing high levels of B7.1 induced significantly higher levels of T-cell proliferation than wild-type tumor cells. T-cell subset composition did not markedly differ between T cells stimulated with wild-type tumor cells or B7.1-expressing tumor cells. However, T cells stimulated with B7.1-expressing tumor cells showed a significantly increased cytolytic potential. The increased cytotoxic T lymphocyte activity was associated with a higher frequency of specific TcR Vα and Vβ genes. In addition, B7.1 costimulation promoted oligoclonality among the responding T cells. Conclusions: These data suggest that costimulation through B7.1 promotes T-cell proliferation and cytotoxic activity through clonal expansions of T cells bearing antigen-specific TcR Vα and Vβ genes and through promotion of oligoclonality. The data also suggest that promoting B7.1-mediated costimulation is an important aspect of immune therapies. (Surgery 2000;127:342-50.)

The X-ray crystal structure of a Vα2.6Jα38 mouse T cell receptor domain at 2.5 å resolution: alternate modes of dimerization and crystal packing

We describe here the structure of a murine T cell receptor (TCR) Vα2.6Jα38 (TCRAV2S6J38) domain, derived from a T cell hybridoma with specificity for the H-2Dd major histocompatibility complex class I molecule bound to a decamer peptide, P18-I10, from the HIV envelope glycoprotein gp120, determined by X-ray crystallography at 2.5 Å resolution. Unlike other TCR Vα domains that have been studied in isolation, this one does not dimerize in solution at concentrations below 1 mM, and the crystal fails to show dimer contacts that are likely to be physiological. In comparison to other Vα domains, this Vα2.6 shows great similarity in the packing of its core residues, and exhibits the same immunoglobulin-like fold characteristic of other TCR Vα domains. There is good electron density in all three complementarity-determining regions (CDRs), where the differences between this Vα domain and others are most pronounced, in particular in CDR3. Examination of crystal contacts reveals an association of Vα domains distinct from those previously seen. Comparison with other Vα domain structures reveals variability in all loop regions, as well as in the first β strand where placement and configuration of a proline residue at position 6, 7, 8, or 9 affects the backbone structure. The great variation in CDR3 conformations among TCR structures is consistent with an evolving view that CDR3 of TCR plays a plastic role in the interaction of the TCR with the MHC/peptide complex as well as with CDR3 of the paired TCR chain.

Structural basis of T cell recognition.

Exciting breakthroughs in the last two years have begun to elucidate the structural basis of cellular immune recognition. Crystal structures have been determined for full-length and truncated forms of alpha beta T cell receptor (TCR) heterodimers, both alone and in complex with their peptide-MHC (pMHC) ligands or with anti-TCR antibodies. In addition, a truncated CD8 coreceptor has been visualized with a pMHC. Aided in large part by the substantial body of knowledge accumulated over the last 25 years on antibody structure, a number of general conclusions about TCR structure and its recognition of antigen can already be derived from the relatively few TCR structures that have been determined. Small, but important, variations between TCR and antibody structures bear on their functional differences as well as on their specific antigen recognition requirements. As observed in antibodies, canonical CDR loop structures are already emerging for some of the TCR CDR loops. Highly similar docking orientations of the TCR V alpha domains in the TCR/pMHC complex appear to play a primary role in dictating orientation, but the V beta positions diverge widely. Similar TCR contact positions, but whose exact amino acid content can vary, coupled with relatively poor interface shape complementarity, may explain the flexibility and short half-lives of many TCR interactions with pMHC. Here we summarize the current state of this field, and suggest that the knowledge gap between the three-dimensional structure and the signaling function of the TCR can be bridged through a synthesis of molecular biological and biophysical techniques.

Somatic hypermutation of the T cell receptor Vβ gene in microdissected splenic white pulps from HIV-1-positive patients

Somatic mutation of rearranged immunoglobulin V genes occurs in germinal centers (GC), resulting in affinity maturation of the immune response. Rearranged T cell receptor (TCR) genes were thought to be excluded from this process despite similarities in their gene structure. Somatic mutations were found among TCR Vα (TCRAV) chains of antigen-specific T cells localized in GC of mice. Here, somatically mutated TCR Vβ (TCRBV) chains are identified among microdissected splenic white pulps from HIV-positive individuals. Both the frequency and the nature of the base substitutions were found to be similar to those of mutated immunoglobulin VH genes. This was true for intrinsic mutations in the TCR framework regions as well as for mutations underlying selective pressures in the TCRBV5 gene segment. The concentration of mutations and a preference for replacement mutations in complementarity determining regions of expanded clones were indicative of a positive selection process.

Linkage Analysis of Candidate Genes in Autoimmune Thyroid Disease: 1. Selected Immunoregulatory Genes

Graves’ and Hashimoto’s diseases are autoimmune thyroid diseases in which the genetic contribution is complex. For this reason, identification of necessary susceptibility genes has been difficult. However, a number of immunoregulatory genes have been implicated by association studies, including: CTLA-4, a recently described protein involved in antigen presentation, located on chromosome 2q33; the T-cell receptor Vα and Vβ gene complexes, located on 14q11 and 7q35, respectively; and the Ig gene complex (IgH), located on 15q11. We used polymorphic microsatellite markers located within these genes, or gene complexes, to test for linkage (rather than association), to each of these candidates. Using markers within the loci allowed us to assume a fixed recombination fraction of 0.01 in the tested model. Three hundred eight subjects from 48 multiplex families were studied, with 142 affected subjects. Using this set of families, we have previously shown evidence of linkage with a major susceptibility locus for Gr...

Conserved motifs in T-cell receptor CDR1 and CDR2: implications for ligand and CD8 co-receptor binding

Recent X-ray crystallographic structures of the T-cell receptor (TCR) α and β chains, as well as their trimolecular complexes with peptide—MHC ligand, have established their structural similarity with the immunoglobulin molucules. The complementarity-determining region (CDR1) and CDR2 encoded within the TCR germline variable (V) sequence genes are well conserved across different TCR Vα and Vβ subfamilies. Muliple sequence alignments have been made based on structural information; they indicate that there will be only a limited number of canonical conformations for the first and second CDR loops. The limited diversity shown by CDRs 1 and 2 contrasts with the extreme junctional CDR3 diversity. Furthermore, CDR2 alignments have revealed coservation of a positive net charge in Vα subfamilies. A model has been proposed for a direct interaction of the lateral part of CDR2α with the negatively charged membrane-proximal ‘stalk’ region of the CD8 molecule.

Dominant TCR α-chain clonotypes and interferon-γ are expressed in the pancreas of patients with recent-onset insulin-dependent diabetes mellitus

In order to clarify the nature of T lymphocytes infiltrating the pancreatic islets of patients with insulin-dependent diabetes mellitus (IDDM), we analysed T cell receptor (TCR) gene transcripts expressed in pancreatic biopsy specimens of patients with recent-onset IDDM. We also investigated the expression of cytokines (interferon-γ: IFN-γ; tumour necrosis factor-α: TNF-α; interleukin-4: IL-4; interleukin-6: IL-6) in the same specimens. The TCR Vβ repertoire was not restricted either in the pancreas or the peripheral lymphocytes of IDDM patients. In contrast, the TCR Vα repertoire was restricted in the pancreas, but not in the peripheral blood lymphocytes, of IDDM patients. The sequence analysis of the complementarity-determining region 3 (CDR3) of the TCRα revealed the presence of dominant clonality in α chains of T cells in the patients. IFN-γ mRNA was highly expressed in the pancreas of IDDM patients, while IL-4 mRNA was deficient. A lower level of expression of IL-6 mRNA was detected in the IDDM pancreas than in the control tissue. These results indicate that T cells bearing a distinct TCRα chain are selectively retained and activated within the pancreas of recent-onset IDDM.

Restricted Usage of T-Cell Receptor Vα-Vβ Genes in Infiltrating Cells in Aortic Tissue of Patients With Takayasu’s Arteritis

Infiltration by perforin-secreting killer lymphocytes, such as T cells and natural killer cells, has been shown to be involved in the pathogenesis of vascular cell damage in Takayasu's arteritis. To investigate the immunological mechanisms involved, especially the nature of T-cell infiltration in Takayasu's arteritis as well as atherosclerosis, we analyzed the expression of T-cell receptor (TCR) Valpha and Vbeta genes in infiltrating cells in the aortic tissue of patients with Takayasu's arteritis and the atherosclerotic aortic aneurysm by polymerase chain reaction (PCR). We also analyzed the expression of cytokine genes by PCR. We found that the repertoires of TCR Valpha as well as Vbeta gene transcripts in Takayasu's arteritis were restricted. The infiltrating cells expressing Valpha2, Valpha16, Valpha17, Vbeta7, and Vbeta13.1 were found in 3 of 4 patients. In contrast, TCR Valpha-Vbeta repertoires in atherosclerotic aortic aneurysm were polyclonal. There was no significant difference in the pattern of cytokine gene expression between the two diseases. The restricted usage of TCR Valpha as well as Vbeta genes by infiltrating T cells in Takayasu's arteritis may indicate that a specific antigen in the aortic tissue was targeted. Our findings provide the evidence that distinct immunological mechanisms are involved in the pathogenesis of Takayasu's arteritis and atherosclerotic aortic aneurysm.

RESTRICTED USAGE OF T‐CELL RECEPTOR Vα GENES IN INFILTRATING CELLS IN MURINE HEARTS WITH ACUTE MYOCARDITIS CAUSED BY COXSACKIE VIRUS B3

In murine myocarditis, it has been shown that natural killer cells first infiltrate the heart, followed by activated T-cells, which play an important role in the pathogenesis of the myocardial damage. In the same model of acute myocarditis, the repertoire of T-cell receptor (TCR) Vα genes in infiltrating cells in the heart has also been shown to be restricted. To study the nature of T-cell infiltration in more detail, the expression of TCR Vα genes in infiltrating cells in the heart has been analysed by the polymerase chain reaction (PCR), confirmed by Southern blot hybridization with a Cα cDNA probe. In contrast to spleen lymphocytes, the repertoire of Vα gene transcripts in the heart was restricted. Infiltrating cells expressing Vα10 were found in five of eight hearts of mice with acute myocarditis and infiltrating cells expressing Vα7 and Vα3 were found in two of eight and one of eight hearts, respectively. Restricted TCR Vα as well as Vβ repertoires indicate that a specific antigen, in the heart was targeted, presented at the groove of major histocompatibility complex molecules. These findings raise the possibility of specific immunotherapy with synthetic TCR Vα or Vβ peptides to prevent T-cell-mediated myocardial damage in patients with viral myocarditis.

Limited heterogeneity of rearranged T cell receptor V alpha and V beta transcripts in synovial fluid T cells in early stages of rheumatoid arthritis.

The identification of activated T cells in synovial fluid and synovium, and the association of rheumatoid arthritis (RA) with specific HLA-DR restriction elements, strongly suggest that these T cells play a critical role in the etiology and pathogenesis of RA. Analysis of the T cell receptor (TCR) repertoire in the early stages of RA might be an approach to identify those T cells involved in the initiation and/or perpetuation of the disease. TCR V alpha and V beta transcripts of synovial T cells, sampled at the early stages of RA, were amplified by reverse transcriptase-polymerase chain reaction. HLA-DR subtyping was determined by serologic analysis and dot-blot hybridization of polymerase chain reaction amplification products using digoxigenin-labeled, sequence-specific oligonucleotide probes. Our findings showed a limited heterogeneity of V alpha and V beta TCRs in synovial fluid T cells, and a preferential usage of TCR V alpha 17 in early RA. In contrast, in the later stages of RA, a more polyclonal TCR V alpha and V beta gene usage was observed. Our results support the view that induction of RA is driven by an oligoclonal immune response to an unknown antigen. These findings also suggest a pathogenetic role for V alpha 17 T cells in the early stages of RA.

Derivation of T-cell receptor alpha-chain double expresser lines from normal murine mature T cells.

Because the T-cell receptor (TCR) alpha-chain locus is known to lack allelic exclusion of rearrangements, and as a recent report revealed the existence of alpha-chain double expressers among normal human peripheral blood lymphocytes (PBL), the possible existence of TCR alpha-chain double expressers among mature murine T cells was examined. Although two-colour staining analysis of normal T-cell populations did not immediately reveal recognizable clusters of V alpha double expressers, alternative in vitro stimulations of normal murine T cells with antibodies to two different TCR V alpha chains reproducibly induced TCR alpha-chain double-expresser lines. TCR complexes with different alpha-chains on such T cells were both shown to be functional. The cell lines were heterogeneous with respect to V beta usage and the ratio of the expressed amounts of the two alpha-chains on the surface. The ratio of the two expressed alpha-chains was found to be very stable over a long period of time. These results are consistent with the earlier report on alpha-chain double expressers among human T cells and also show normal occurrence of TCR alpha-chain double expressers in murine T-cell populations.

Plasticity of the T Cell Receptor Repertoire in TCR β-Chain Transgenic Mice

The potential αβ T cell receptor (TCR) repertoire in normal mice is extremely large and estimated by M. M. Davis and P. J. Bjorkman (Nature334, 395, 1988) to include 5.2 × 1018different receptor molecules. This tremendous diversity provides the basis for T cell recognition of the universe of antigens including bacterial, viral, and allogeneic epitopes. Expression of a single TCR β-chain transgene should alter the repertoire by limiting the available diversity, therefore, creating holes in the repertoire or producing TCR with decreased affinity. To determine the effect of drastically decreasing the size of the repertoire, we investigated T cell responses in TCR β-chain transgenic mice expressing the Vβ8.2 transgene. Previous results showed that >98% of T cells in these mice express the transgene; thus, the TCR repertoire is reduced by orders of magnitude. We tested the T cell responses of the transgenic mice and nontransgenic littermates to nine different MHC haplotypes in mixed lymphocyte reactions, five protein antigens, and eight immunogenic peptides. Surprisingly, the transgenic mice responded to all antigenic stimuli tested indicating the lack of a hole in the TCR repertoire. Interestingly, however, the response in every case was quantitatively lower than the response by the nontransgenic littermates. In contrast, transgenic and nontransgenic T cells responded equivalently to stimulation with mitogens or to stimulation with immobilized α-TCR mAb indicating that the transgenic T cells had a normal capacity to respond. To differentiate between decreased TCR affinity and decreased precursor frequency, we performed a limiting dilution analysis to the peptide antigens CI:NP and OVA324–339. The results showed approximately a three- to eight-fold decrease in the frequency of transgenic T cells responding to the peptide compared to nontransgenic littermates. We previously showed that the response to cI84–98and PLP could be blocked with anti-Vβ8 mAb indicating that Vβ8.2-bearing T cells are capable of responding to peptide antigen. Analysis of TCR Vα chain expression by PCR and flow cytometry showed similar Vα expression in both the transgenic and the nontransgenic mice. These results demonstrate tremendous plasticity in the TCR repertoire permitting T cell responses by the transgenic mice to all antigens tested. However, the decreased magnitude of the responses may impair the capacity to defend against natural pathogens. Therefore, although the large TCR repertoire of normal mice may not be necessary to producein vitroresponses to many experimental antigens, it may confer survival benefits in natural environments.