The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B¹), and none by I-A^d of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.     

Prevention and treatment of Lewis rat experimental allergic encephalomyelitis with a monoclonal antibody to the T cell receptor Vβ8.2 segment

The predominance of T cell receptor (TCR) Vβ8.2 utilization by encephalitogenic T cells induced in Lewis rats by immunization with myelin basic protein (MBP) is controversial. Thus, both an almost exclusive usage of Vβ8.2 [Burns, F. R., Li, X., Shen, N., Offner, H., Chou, Y. K., Vandenbark, A. A. and Heber-Katz, E., J. Exp. Med. 1989. 169: 27; Chluba, J., Steeg, C., Becker, A., Wekerle, H. and Epplen, J. T., Eur. J. Immunol. 1989. 19: 279] and a quite diverse Vβ composition of CD4 T cells causing experimental autoimmune encephalomyelitis (EAE) [Sun, D., Gold, P. D., Smith, L., Brostoff, S. and Coleclough, C., Eur. J. Immunol. 1992. 22: 591; Sun, D., Le, J. and Coleclough, C., Eur. J. Immunol. 1993. 23: 494] have been reported. Using a recently developed monoclonal antibody (mAb) specific for TCR Vβ8.2, we show that postnatal treatment effectively eliminates Vβ8.2-bearing cells and prevents MBP-induced EAE in the majority of Lewis rats. Moreover, treatment of adult Lewis rats with Vβ8.2-specific mAb as late as on day 12 after MBP immunization suppressed the development of neurological symptoms. Thus, Vβ8.2-bearing cells do play a decisive role in Lewis rat EAE, and suppression of the small (5%) Vβ8.2-expressing T cell subset provides an effective therapeutic strategy.     

Analysis of TCR β chains in Lewis rats with experimental allergic encephalomyelitis. II. Vβ8.2+ T cells with limited CDR3 N region additions derive from the adult thymus

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68 – 86. The TCR β chains of these disease-causing T cells show three distinct features: they are almost exclusively Vβ8.2, they use AspSer as the first two amino acid residues of the third complementarity-determining region (CDR3) and these junctional region sequences show few if any non-germline N-region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68 – 86-reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery – i.e., that such β chains are conformationally the most appropriate for triggering by an epitope of 68 – 86 complexed to class II RT1.B^l MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post-irradiation adult thymus. These disease-causing T cells are heavily Vβ8.2⁺ , CDR3 AspSer⁺ and use few N region additions. We conclude that T cells with these TCR β chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.     

Predominant usage of V beta 8.3 T cell receptor in a T cell line that induces experimental autoimmune uveoretinitis (EAU).

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease induced in animals by immunization with retinal proteins (or synthetic fragments derived from them) in adjuvant, and it is considered a model of human autoimmune diseases of the eye. To study the T cell clonotypes that may be involved in EAU, we analyzed the T cell repertoire of three related T cell lines: the pathogenic line LR16, specific to the major uveitogenic epitope of IRBP; its pathogenic subline J; and its nonpathogenic subline A. We examined the expression of the genes coding for the variable regions of the 20 known Lewis rat T cell antigen receptor (TCR) V beta families. The nonpathogenic subline was found to contain mostly T cells expressing V beta 5, V beta 8.2, and V beta 19 while the pathogenic subline consisted mainly of cells expressing V beta 8.3 TCRs. Genomic Southern blot analysis of DNA from the pathogenic subline showed that V beta 8.3-expressing T cells were the dominant clonotype, and DNA sequence analyses of V beta 8.3 cDNAs revealed that two V beta 8.3 TCRs were expressed in the pathogenic subline. One of the V beta 8.3 cDNAs encoded a variable region gene segment identical to previously reported rat V beta 8.3 TCR while the other differed by two amino acids in the second complementarity determining region (CDR2). Taken together with previous data showing overrepresentation of V beta 8-expression in T cell lines that induce EAU, but not in nonuveitogenic T cell lines, our results suggest that V beta 8.3-expressing T cells represent a pathogenic clonotype in IRBP-induced EAU.     

Encephalitogenic,myelin basic protein-specific T cells from naive rat thymus: preferential use of the T cell receptor gene Vβ8.2 and expression of the CD4− CD8− phenotype

Using a primary limiting dilution approach to generate T cell lines, we compared myelin basic protein (MBP)-specific T cell clones from naive unprimed Lewis rat thymuses with the corresponding T cell repertoire of primed rats. We found that in the naive thymus repertoire MBP-specific, encephalitogenic T cell clones preferentially use T cell receptor Vβ8.2 genes, along with CDR3 sequences typical for the primed Lewis anti-MBP response. In contrast to T cells from primed immune organs, which all display the CD4⁺ CD8^? phenotype, the majority of naive thymus-derived T cell clones expressed reduced levels of the CD4 co-receptor. Some clones were completely CD4^?CD8^?, while others included CD4^? CD8^? subpopulations along with CD4⁺CD8^? T cells. In the one mixed population examined in detail, the CD4^?CD8^? and CD4⁺CD8^? T cell subpopulations used a T cell receptor with identical β chain sequence. The data suggest that in the Lewis rat the biased T cell receptor gene usage by encephalitogenic T cells is a property of the natural thymic T cell repertoire, possibly as a consequence of positive selection. The unusually low expression of CD4 in the major histocompatibility complex class II-restricted autoreactive T cells could be related to their escape from negative selection within the thymus.     

Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256 – 270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Vα2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256 – 270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256 – 270) glycosylated with a monosaccharide (β-D -galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.     

T cell receptor beta chain usage in myelin basic protein-specific rat T lymphocytes

Myelin basic protein (MBP)-specific T cell lines and clones have been established from rats of the major histocompatibility complex (MHC)-compatible Lewis and BS strains. All lines and clones are MHC class II restricted and share the CD4+ phenotype. The cells proliferate specifically in response to either a peptide representing amino acids #68-88 of guinea pig MBP, to residues #47-67 or to an unidentified myelin antigen which is distinct from MBP. All lines and clones specific for MBP express the same T cell receptor (TcR) variable (V) beta chain element, which is homologous to the mouse V beta 8.2 gene segment. Three lines/clones with the same antigen fine specificity have identical V beta D beta J beta junctions on the protein level, a region which represents part of the potential antigen-binding portion of the TcR; two of the lines express members of the V alpha 2 family. These results suggest biased usage of TcR V beta elements in rat T cells specific for MBP. Our findings broaden the basis for a rational therapeutic strategy to specifically intervene in the rodent model system of experimental allergic encephalomyelitis.     

T cell development and repertoire of mice expressing a single T cell receptor α chain

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) α chain derived from the H-2D^b -lymphocytic choriomeningitis virus (LCMV)-specific cytolytic T lymphocyte (CTL) clone P14. To generate these α P14 mice, mice transgenic for the P14 TCR α chain were backcrossed to TCR α-deficient mice. Thymi from α P14 mice exhibited a marked decrease of mature CD4⁺8^? and CD8⁺4^? single-positive thymocytes comparable to thymi from TCR α-deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from α P14 mice generated a primary anti-LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with Vβ-specific antibodies showed a diverse endogenous TCR Vβ repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30-fold) allogeneic and the absence of a vesicular stomatitis virus-specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR α chain.     

Characterization of rat encephalitogenic T cells bearing non-V beta 8 T cell receptors.

In this study, we demonstrate that T cell lines specific for a synthetic peptide representing sequence 87 to 99 of myelin basic protein (MBP) are encephalitogenic in Lewis rats. However, unlike syngeneic T cells specific for MBP residues 68 to 88 which exclusively use V beta 8 in their antigen receptors, these cells do not. None of the 10 T cell lines and T hybridomas specific for MBP (87-99) used V beta 8 in their T cell receptors. Our results document for the first time that rat encephalitogenic T cells do not exclusively use V beta 8 in T cell receptors that rat encephalitogenic T cells specific for MBP (87-99) are heterogeneous and that MBP (87-99) contains at least two epitopes for rat T cells.     

Soluble antigen therapy induces apoptosis of autoreactive T cells preferentially in the target organ rather than in the peripheral lymphoid organs

The administration of soluble myelin proteins is an effective way of down-regulating the inflammation in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. To shed more light on the mechanism of this antigen-specific therapy, we determined the effect of the intraperitoneal (i.p.) injection of soluble myelin basic protein (MBP) on T cell apoptosis in the CNS and peripheral lymphoid organs of Lewis rats with EAE induced by inoculation with MBP and complete Freund's adjuvant. In particular we assessed the level of apoptosis of Vβ8.2⁺ T cells, which constitute the predominant encephalitogenic MBP-reactive T cell population in the Lewis rat. The daily i.p. injection of MBP for 3 days from the onset of neurological signs inhibited the further development of neurological signs of EAE. Using two-color flow cytometry we found that a single i.p. injection of MBP increased the level of apoptosis of the Vβ8.2⁺ T cell population in the CNS to 26.2 % compared to 7.4 % in saline-treated rats and 7.6 % in ovalbumin-treated rats. In contrast, treatment with MBP did not increase the level of apoptosis of the Vβ8.2⁺ population in the popliteal lymph node draining the inoculation site (1.4 %) or in the spleen (1.6 %) above that occurring in saline-treated rats (1.6 % and 1.1 %, respectively). Limiting dilution analysis revealed that the frequency of T cells reactive to the major encephalitogenic epitope, MBP_{72 – 89}, was decreased in the CNS but not in the popliteal lymphnode by this treatment. Three-color flow cytometry in MBP-treated rats demonstrated that CNS Vβ8.2⁺ T cells expressing Fas (CD95) and Fas ligand were highly vulnerable to apoptosis compared to Vβ8.2⁺ T cells not expressing these proteins. We conclude that the i.p. injection of MBP increases the spontaneously occurring Fas-mediated activation-induced apoptosis of auto reactive T cells in the CNS in EAE and that this contributes to the therapeutic effect of the injection.     

The homogeneity of the TCRδ repertoire expressed by the Thy-1dull γ δ T cell population is due to cellular selection

Thy-1^dull γ δ T cells are an unusual subset of mature TCRγ δ T cells characterized by their highly restricted TCR repertoire. In DBA/2 mice, they predominantly express the product of the Vγ1 gene together with that of a member of the Vδ6 subfamily (the Vδ6.4 gene) and their junctional sequences show very little diversity. To address the mechanisms underlying the expression of the restricted TCRγ δ repertoire, we have cloned all Vδ6 subfamily members present in DBA/2 mice and studied their frequency of expression in Thy-1^dull and Thy-1^bright γ δ thymocyte populations. Furthermore, we have also cloned non-functional Vδ6DδJδ1 rearrangements present in the Thy-1^dull γ δ T cell population and compared their Vδ6 gene utilization and their junctional sequences with those expressed by this population. Our results indicate that the restricted TCRδ repertoire expressed by the Thy-1^dull γ δ thymocytes results from cellular selection, rather than molecular constraints suggesting the existence of a limited set of self-ligands. Finally, phenotypic, functional and TCRγ δ repertoire analysis of Thy-1^dull γ δ T cells in β2 -microglobulin (β₂m)-deficient mice indicated that these putative ligands are not β₂m-dependent major histocompatibility complex class I or class I-like molecules.     

Selection of the cutaneous intraepithelial gammadelta+ T cell repertoire by a thymic stromal determinant

Intraepithelial lymphocytes constitute a group of T cells that express mainly monospecific or oligoclonal T cell receptors (TCRs). Like adaptive TCR alphabeta+ T cells, intraepithelial lymphocytes, a subset enriched in TCR gammadelta+ T cells, are proposed to be positively selected by thymically expressed self agonists, yet no direct evidence for this exists at present. Mouse dendritic epidermal T cells are prototypic intraepithelial lymphocytes, displaying an almost monoclonal TCR gammadelta+ repertoire. Here we describe an FVB substrain of mice in which this repertoire was uniquely depleted, resulting in cutaneous pathology. This phenotype was due to failure of dendritic epidermal T cell progenitors to mature because of a heritable defect in a dominant gene used by the thymic stroma to 'educate' the natural, skin-associated intraepithelial lymphocyte repertoire to be of physiological use.     

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL

We selected three peptides from the germ-line sequence of the Vβ8.2 and Jβ2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative K^d- and L^d-restricted epitopes. Immunization of BALB/c (H-2^d) mice with the Vβ8.2(67–90) 23-mer peptide 1 as well as the 15-mer Vβ8.2(95–108)-peptide 2 efficiently primed specific CD8⁺ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-Vβ8.2 epitopes. Vβ8.2⁺ T cells were not deleted in TCR peptide-immunized mice because the fractions of Vβ8.2⁺ CD4⁺ and Vβ8.2⁺ CD8⁺ T cells in spleen and lymph nodes were not altered. The proliferative response of Vβ8.2⁺ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60–80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the Jβ2.3-derived peptide 3 stimulated a CD8⁺ CTL response against a class I-restricted epitope within this Jβ segment that was also generated during natural “endogenous” processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8⁺ CTL recognize class I-restricted, natural Vβ/Jβ-TCR epitopes. Such anti-TCR CTL may, thus, operate in Vβ-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.     

Rat stem cells developing in irradiated SCID mice fail to become tolerized and cause lethal graft-versus-host disease.

Graft-versus-host disease (GVHD) is prominent in irradiated hosts given whole allogeneic bone marrow cells but is generally undetectable when T-depleted stem cells are transferred; under these conditions, the mature T cells arising from the donor stem cells become tolerant to host antigens and fall to cause GVHD. We show here that a radically different situation can occur when hosts are reconstituted with xenogeneic stem cells. When lightly irradiated, adult C.B-17 SCID mice injected with Lewis rat fetal liver (FL) cells show near-total repopulation with rat-derived lymphohemopoietic cells, including T and B cells. However, in marked contrast to chimeras prepared with allogeneic mouse FL cells, rat FL-->SCID chimeras develop severe and often lethal chronic GVHD. In these rat-->mouse chimeras, the rat T cells show limited tolerance to host mouse antigens as determined by various parameters including mixed lymphocyte reaction and cytotoxic T lymphocyte assays in vitro, adoptive transfer of T cells to secondary SCID hosts, and the lack of V beta deletion to endogenous host mtv antigens. GVHD in irradiated rat-->SCID chimeras is most prominent with Lewis FL but also applies to Fisher 344 and Wistar Furth FL cells. The failure of newly formed rat T cells in rat-->SCID chimeras to become fully tolerant to host mouse antigens appears to be due to depletion of host antigen-presenting cells by irradiation. Thus, rat-->SCID chimeras generated by transplanting rat FL cells into unirradiated neonatal SCID mice fail to develop GVHD, and the rat T cells display self-tolerance. As allogeneic H-2-different mouse FL-->irradiated SCID chimeras display strong self-tolerance, presumably through recognition of host antigens on thymic epithelial cells, the implication is that mouse thymic epithelial cells are tolerogenic only for mouse and not for rat immature T cells.     

Control of TCR V alpha-mediated positive repertoire selection and alloreactivity by differential J alpha usage and CDR3 alpha composition

In rats expressing the f allele of the rat MHC (RT1f), CD8 T cells utilizing the V alpha 8.2 segment are 10-fold overselected during thymic development, resulting in V alpha 8.2 expression by 14% of mature CD8 T cells as compared to 1-2% in MHC congenic strains. In the alloreactive responses of CD8 T cells from RT1f-negative rats against RT1f, V alpha 8.2+ CD8 T cells are also preferentially expanded. Neither overselection nor alloreactivity of V alpha 8.2+ TCR require selective V beta pairing. However, RT1f alloreactive V alpha 8.2+ TCR preferentially use a related set of J alpha segments which contribute short homogeneous CDR3 alpha loops, with features suggesting peptide promiscuity, and little N additions. In contrast, only few overselected V alpha 8.2+ CD8 T cells showed an imprint of positive selection on J usage or CDR3 composition. The results demonstrate that a single V alpha segment can promote both MHC allele-specific positive selection and alloreactivity, and that the latter is more dependent on an additional contribution of CDR3 alpha, possibly by promoting reactivity with a diverse set of MHC-bound peptides or by providing additional MHC contacts.      

A self-reactive T cell population that is not subject to negative selection.

In male mice expressing a transgenic alpha beta TCR which recognizes a male antigen (HY), T cells which do not express normal levels of CD8 escape thymic deletion and appear in the periphery. These consist of two distinct populations, one which lacks expression of both CD4 and CD8, and one with low levels of CD8. Neither population has anti-HY reactivity, consistent with the known requirement of this TCR for CD8. We now describe the consequences of expression of both the anti-HY TCR transgene and a constitutive CD8.1 transgene on T cells of male mice. Peripheral T cells in these male 'double transgenic' mice express both the anti-HY TCR and normal levels of CD8, and can proliferate to male antigen in vitro. These cells do not express the endogenous allele of CD8 (CD8.2), suggesting that the increase in CD8 levels due to the CD8.1 transgene leads to the deletion of the CD8.2low population. In contrast, the CD8.1 transgene does not lead to the deletion of the CD8.2- population. This implies that, unlike the majority of alpha beta T cells, TCR+CD4-CD8- cells in TCR transgenic mice are not subject to deletion.     

Rat thymic epithelium positively selects mouse T cells with specificity for rat MHC class II antigens but fails to induce detectable tolerance in the mouse T cells to the rat MHC antigens.

BALB/c (H-2d) nude mice were grafted with allogeneic AKR/J (H-2k) or xenogeneic (ACI-N rat, RT1av1) fetal thymuses which were depleted of hemopoietic cells by incubating with 2'-deoxyguanosine (2'dGuo) in vitro prior to grafting. The nylon-wool-passed LN T cells from nude mice grafted with 2'dGuo-treated AKR/J thymus showed a poor proliferative response to B10BR (H-2k) stimulator cells, confirming that mouse thymic epithelium has the capacity to induce tolerance against the mouse MHC antigens on the thymic epithelium. On the other hand, the nylon-wool-passed LN T cells from nude mice grafted with untreated or 2'dGuo-treated ACI/N rat thymus showed significant proliferative responses to ACI/N, which can be blocked by anti-rat MHC class II mAb, whereas the nylon-wool-passed LN T cells from nude mice grafted with syngeneic thymus hardly responded to the xenogeneic stimulator cells. These results suggest that rat thymic stromal cells including thymic epithelium can not induce detectable tolerance in mouse T cells to rat MHC antigens; but rat thymic epithelium may positively select mouse T cells with specificity for rat MHC class II antigens, resulting in a mouse T cell repertoire with strong xeno-reactivity.     

Preferential association of alpha and beta chains of the T cell antigen receptor.

The cytotoxic T lymphocyte (CTL) clone B6.2.16 expresses a V beta 8.2/J beta 2.3/C beta 2-encoded T cell receptor (TcR) beta chain and an alpha chain that is encoded by a novel V alpha gene segment, J alpha 27 and C alpha. While expression of V alpha B6.2.16 and J alpha 27 is not detectable in lymph node cells of normal C57BL/6 mice, expression of these gene segments was readily seen in transgenic mice expressing the rearranged beta chain gene of the B6.2.16 T cell clone. This finding indicates that only a limited number of alpha chains can associate with the B6.2.16 beta chain and strongly suggests that the size of the TcR repertoire of mature T cells is not only limited by TcR ligand-mediated thymic selection but also by restrictions in alpha-beta combinatorial chain association.     

Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.