Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin-derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition.
ABSTRACT: To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin-derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR-specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of a common V element, V beta 2.1, is that this V region plays a dominant role in the recognition of the tt830-844/DR complex.
Project description:We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all.
Project description:We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.
Project description:Dominant expression of T-cell receptor (TCR) alpha or beta chain variable region (V alpha or V beta) gene families has been observed in the T-cell response to some conventional peptide antigens. Current models for the interaction of TCR V region elements with different determinants of a major histocompatibility complex (MHC)-peptide complex, the normal TCR ligand, suggest that the TCR V-J junctional region (CDR3, where J is joining) is the primary contact with a peptide epitope and that other TCR V region segments may interact directly with neighboring MHC determinants. This suggests that V alpha or V beta dominance in a specific response can be MHC-selected. In this case, if related peptides bind to an MHC molecule in a similar orientation, they could select for identical V alpha or V beta dominance even if they are noncrossreactive at the level of T-cell activation. We have screened for this possibility by introducing minimal conservative substitutions in a synthetic peptide, YYEELLKYYEELLK, that is presented to T cells in association with an uncommon A beta E alpha d mixed Ia isotype. We report here that the peptide variant FFEELLKFFEELLK is noncrossreactive with YYEELLKYYEELLK but appears to preserve the same MHC binding motif since T-cell responses are restricted to the same mixed A beta E alpha isotype. Although the two peptides are noncrossreactive in either direction, the same members of the V alpha 4 gene family are dominantly expressed in T cells specific for either peptide. We conclude that the similar topography of the two MHC-peptide complexes gives functional significance to a unique A beta E alpha determinant that selects for V alpha 4 dominance.
Project description:T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-alpha and -beta chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471-94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-alpha chain even when it paired with a TCR-beta chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-alpha chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-alpha chains of human lupus Th clones. The lupus TCR-alpha chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-alphabeta+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes "Pantigens" (for promiscuous antigens).
Project description:The alpha beta T-cell receptor (TCR) recognizes antigenic peptides bound to major histocompatibility complex (MHC) molecules. In contrast to the antibody combining site, for which the antigen contact or complementarity-determining residues (CDRs) have been precisely defined, the location and function of the corresponding CDR regions of the alpha and beta TCR chains are not known. To develop a model system for systematic analysis of the CDRs of the alpha beta TCR, we isolated a panel of murine T-cell clones that recognize a lysozyme peptide containing residues 74-88 bound to either Ab or Abm-12 MHC class II molecules. Although these two MHC molecules differ by only three amino acid residues within the A beta chain, each of the T-cell clones was specific for peptide bound to the self-MHC molecule and did not recognize the same peptide bound to the other MHC molecule. The structural basis for this exquisite ligand specificity of the TCRs was analyzed by isolation and characterization of alpha and beta chain genes from five closely related T-cell clones. Comparison of predicted amino acid sequences mapped the ligand specificity differences to residues present within the alpha chain variable region segment and the alpha and beta chain variable-joining region junction regions. Thus with current models of TCR-ligand interactions, the results suggest that residues 26-30 of the alpha chain variable region may constitute one of the CDR regions of the TCR.
Project description:Restricted use of T cell receptor (TCR) gene segments is characteristic of several induced autoimmune disease models. TCR sequences have previously been unavailable for pathogenic T cells which react with a defined autoantigen in a spontaneous autoimmune disease. The majority of T cell clones, derived from islets of NOD mice which spontaneously develop type I diabetes, react with insulin peptide B-(9-23). We have sequenced the alpha and beta chains of TCRs from these B-(9-23)-reactive T cell clones. No TCR beta chain restriction was found. In contrast, the clones (10 of 13) used V alpha13 coupled with one of two homologous J alpha segments (J alpha45 or J alpha34 in 8 of 13 clones). Furthermore, 9 of 10 of the V alpha13 segments are a novel NOD sequence that we have tentatively termed V alpha13.3. This dramatic alpha chain restriction, similar to the beta chain restriction of other autoimmune models, provides a target for diagnostics and immunomodulatory therapy.
Project description:In order to determine the factors that influence the glycosylation of an integral membrane protein, we investigated the N-glycosylation of a molecule of the human major histocompatibility complex (MHC) class II, the HLA-DR antigen. This glycoprotein was studied in a human Epstein-Barr-virus-transformed B cell line and in a mouse fibroblastic cell line co-transfected with DR alpha and DR beta genes. We observed that the HLA-DR-antigen glycosylation pattern depends on the cell line in which processing takes place and is closely related to the glycosylation pattern of the overall cellular glycoproteins. Furthermore, when comparing the glycosylation of the separated alpha- and beta-chains, differences were noticed within the same molecule, showing the importance of the individual peptide backbone for the glycosylation process.
Project description:Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseases, ranging from asthma to rhinitis and dermatitis. We have studied the T-cell receptor (TCR) usage of house-dust-mite-specific CD4+ T-cell clones isolated from an atopic individual, by using the anchored polymerase chain reaction, and have analyzed the peripheral TCR repertoire of the same individual. Several T-cell clones had identified TCRs at the sequence level, despite the fact that they had been independently isolated, in some cases, in different years. These data suggest the presence in vivo of long-lived T-cell clones. We have also shown that junctional sequences identical to these clones are present in peripheral blood T cells taken 6 years after the isolation of the T-cell clones. The analysis of TCR genes used by the panel of clones reveals oligoclonality, with the variable (V) region gene segments V alpha 8 and V beta 3 being dominant, although there is minimal conservation of junctional sequences. The results have implications for understanding the TCR recognition of an environmental aeroallergen and the life span of T-cell clones in vivo during a chronic immune response.
Project description:Class II major histocompatibility complex (MHCII) molecules present antigens to CD4(+) T cells. In addition to the most commonly studied human MHCII isotype, HLA-DR, whose beta chain is encoded by the HLA-DRB1 locus, several other isotypes that use the same alpha chain but have beta chains encoded by other genes. These other DR molecules also are expressed in antigen-presenting cells and are known to participate in peptide presentation to T cells and to be recognized as alloantigens by other T cells. Like some of the HLA-DRB1 alleles, several of these alternate DR molecules have been associated with specific autoimmune diseases and T cell hypersensitivity. Here we present the structure of an HLA-DR molecule (DR52c) containing one of these alternate beta chains (HLA-DRB3*0301) bound to a self-peptide derived from the Tu elongation factor. The molecule shares structurally conserved elements with other MHC class II molecules but has some unique features in the peptide-binding groove. Comparison of the three major HLA-DBR3 alleles (DR52a, b, and c) suggests that they were derived from one another by recombination events that scrambled the four major peptide-binding pockets at peptide positions 1, 4, 6, and 9 but left virtually no polymorphisms elsewhere in the molecules.
Project description:Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.