V beta gene polymorphism and a major polyclonal T cell receptor idiotype

Genetic polymorphism in the beta variable gene pool (V beta) is responsible for the strain-specific distribution of the KJ16 T cell receptor (TcR) marker on 20% of peripheral T cells. KJ16+ strains carry two homologous V beta genes that are absent in KJ16- strains. All functional KJ16+ T cell hybrids tested express a member of this V beta subset. mRNA hybridizing to this variable-region probe can be easily detected in total splenic T cells of a prototype KJ16+ strain. Thus, 20% of the TcR from the cytotoxic and helper T cell population, with various MHC restrictions and antigen reactivities, can be generated from two V beta genes. However, their deletion appears to have no effect on the functionality of the T cell repertoire.     

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.     

Assessing the role of the T cell receptor beta gene enhancer in regulating coding joint formation during V(D)J recombination

To assess the role of the T cell receptor (TCR) beta gene enhancer (Ebeta) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta-deleted (DeltaEbeta) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with DeltaEbeta, DeltaEbeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta-Jbeta and Vbeta14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus.     

Abnormal V(D)J recombination of T cell receptor beta locus in SMAR1 transgenic mice

Scaffold/matrix-associated region-1-binding protein (SMAR1) specifically interacts with the MARbeta sequence, which is located 400-bp upstream of the murine TCRbeta enhancer and is highly expressed during the DP stage of thymocyte development. To further analyze the functions of SMAR1, transgenic mice were generated that express SMAR1 in a tissue-independent manner. SMAR1-overexpressing mice exhibit severely altered frequency of the T cells expressing commonly used Vbetas (Vbeta5.1/5.2 and Vbeta8.1/8.2/8.3). The rearrangements of Vbeta5.1/5.2, Vbeta8.1/8.2/8.3 loci are also reduced in SMAR1 transgenic mice. The T cells in SMAR1 transgenic mice exhibit a mild perturbation at the early DN stage. SMAR1 transgenic mice exhibit hypercellular lymph nodes and spleen accompanied with prominent architectural defects in these organs. These results indicate that SMAR1 plays an important role in the regulation of T cell development as well as V(D)J recombination besides maintaining the architecture of the lymphoid organs.     

T cell receptor gene segment usage in a panel of hen-egg white lysozyme specific, I-Ak-restricted T helper hybridomas

We have studied the relationship between MHC-restricted, Ag-specific recognition and TCR structure in a panel of seven Th-hybridomas specific for the foreign protein Ag, hen egg-white lysozyme, and the I-Ak class II MHC molecule. The fine specificity of these Th cells had been determined previously by their reactivity to a panel of APC lines bearing mutant I-Ak molecules and to proteolytic fragments of HEL. TCR gene segment composition was determined by cDNA cloning and DNA sequencing. A heterogeneous, yet repetitive usage of gene segments was observed within the panel. The same V alpha C10-J alpha MD13 rearrangement was used in three of the hybridomas, two with identical Ag and MHC-restriction fine specificities. The prevalent usage of the V beta 14 gene segment and members of J beta 2 cluster was noted. Inasmuch as gene segment usage did not correlate with MHC-restriction or Ag fine specificity alone, these results favor an interactive Ag model of T-cell recognition, in which Ag and MHC are recognized as a bimolecular complex.     

Preferential use of a T cell receptor V beta gene by acetylcholine receptor reactive T cells from myasthenia gravis-susceptible mice.

Experimental autoimmune myasthenia gravis (EAMG) is an important model for testing current concepts in autoimmunity and novel immunotherapies for autoimmune diseases. The EAMG autoantigen, acethylcholine receptor (AChR), is structurally and immunologically complex, a potential obstacle to the application of therapeutic strategies aimed at oligoclonal T cell populations. Inasmuch as we had previously shown that the clonal heterogeneity of T cell epitope recognition in EAMG was unexpectedly limited, we examined TCR V beta expression. AChR primed lymph node T cells and established AChR reactive T cell clones from EAMG-susceptible C57BL/6 (B6; H-2b, Mls-1b) mice showed preferential utilization of the TCR V beta 6 segment of the TCR. After in vivo priming and in vitro restimulation for 7 days with AChR or a synthetic peptide bearing an immunodominant epitope, V beta 6 expressing lymph node cells (LNC) were expanded several-fold, accounting for up to 75% of recovered viable CD4+ cells. The LNC of B6.C-H-2bm12 (bm12; H-2bm12, Mls-1b) mice, which proliferated in response to AChR but not to the B6 immunodominant peptide, failed to expand V beta 6+ cells. Inasmuch as nonimmune bm12 and B6 animals had similar numbers of V beta 6+ LNC (4-5%), this suggested that structural requirements for TCR recognition of Ag/MHC complexes dictated V beta usage. Results concerning peptide reactivity and V beta 6 expression among T cells from (B6 x bm12)F1 animals also suggested that structure-function relationships, rather than negative selection or tolerance, accounted for the strain differences between B6 and bm12. To examine the potential effects of thymic negative selection of V beta 6+ cells on the T cell response to AChR, CB6F1 (H-2bxd, Mls-1b; V beta 6-expressing) and B6D2F1 (H-2bxd, Mls-1axb; V beta 6-deleting) strains were analyzed for AChR and peptide reactivity and V beta 6 expression. Both F1 strains responded well to AChR but the response of B6D2F1 mice to peptide was significantly reduced compared to CB6F1. Short and long term cultures of peptide-reactive B6D2F1 LNC showed no expansion of residual V beta 6+ cells, although similar cultures of CB6F1 LNC were composed of more than 60% V beta 6+ cells. The results from the F1 strains further indicated that the T cell repertoire for peptide was highly constrained and that non-V beta 6 expressing cells could only partially overcome Mls-mediated negative selection of V beta 6+ TCR capable of recognizing peptide.(ABSTRACT TRUNCATED AT 400 WORDS)     

A T cell receptor V beta segment that imparts reactivity to a class II major histocompatibility complex product

We have identified in mice an allele of a new T cell receptor V beta gene, V beta 17a, whose product is bound by the monoclonal antibody KJ23a. Over 90% of T cell hybridomas prepared from V beta 17a+ T cells of SWR mice respond to allogeneic forms of the IE class II MHC protein, indicating that V beta 17a has an appreciable affinity for IE regardless of the other components of the T cell receptor. These results suggest a bias in the germ-line T cell receptor repertoire toward recognition of MHC proteins and indicate that the V beta portion of the receptor may form the most important contact points with MHC ligands.     

Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.     

T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining

We have identified circular DNA containing T cell receptor (TCR) beta gene sequences in mouse thymocytes, thereby providing direct evidence for the intramolecular DNA deletion model of V-D-J joining in TCR beta genes. Two types of excision products of V-D-J joining have been identified. Type I, a circular reciprocal recombinant of normal V-D or D-J joining, contains a 7mer-7mer head-to-head structure expected from an excised product of normal V-D or D-J joining. Type II contains a D beta 2-J beta 1 structure on the circular DNA; the recombination event producing this molecule occurs between an upstream J and a downstream D segment, probably leaving the reciprocal 7mer-7mer structure on the chromosome. Some type I molecules seem to represent excision products of secondary joining after formation of the first D-J or V-D-J structure. The recombination mechanism that generates the circular DNA is discussed.     

Alpha beta lineage-specific expression of the alpha T cell receptor gene by nearby silencers

T cells expressing either the alpha beta or gamma delta antigen receptor (TCR) are distinct cell lineages. The single locus encoding the TCR alpha and delta genes requires special regulation to avoid alpha gene expression in gamma delta T cells. We show here that the minimal alpha enhancer is active in the gamma delta T cell lineage but gains alpha beta lineage specificity through negative cis-acting elements 3' of the C alpha gene that silence the enhancer in gamma delta T cells. The negative elements at the C alpha locus consist of several silencers that work in an orientation- and distance-independent fashion. These silencers also act on a retroviral enhancer that is normally ubiquitously expressed, restricting its activity to alpha beta cells. The alpha silencers are active in non-T cell lines, suggesting that the decision of a cell to differentiate into the alpha beta T cell lineage may involve specific relief from these silencers. Silencers are likely to be as important as enhancers in establishing lineage-specific gene expression in many systems.     

The T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate

The murine T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate, as indicated by several independent lines of evidence. First, three out of four arsonate-reactive T cell clones tested (two I-Ad-and one I-Ak-restricted) utilized V alpha 3. Second, bulk splenic cultures enriched for arsonate/H-2d and arsonate/H-2k responsive T cells showed increased expression of V alpha 3 mRNA. Third, a V alpha 3-containing alpha chain (Ar-5: arsonate/I-Ad) transferred arsonate responsiveness to an appropriate recipient T cell (O3: ovalbumin/I-Ad). Fourth, an independently derived V alpha 3-expressing T cell clone (2C: alloreactive to Ld) showed a response to arsonate/Ld. Thus, a V alpha 3 gene segment, in conjunction with at least two different J alpha segments (J alpha 20'(Ar-5) and J alpha pHDS58(2C)) and at least three different beta chains (V beta 2(Ar-5), V beta 6(O3), and V beta 8(2C], confers reactivity to arsonate in association with at least three different MHC proteins (I-Ad, I-Ak, and Ld). We suggest that V alpha 3 encodes a protein sequence with a binding site for the arsonate hapten.     

Mouse T lymphocyte response to acetylcholine receptor determined by T cell receptor for antigen V beta gene products recognizing Mls-1a.

Mice of strain B6, but not AKR/J, respond to immunization with Torpedo acetylcholine receptor (AChR) by manifesting in vitro an Ag-specific T lymphocyte proliferative response. Our analysis of (AKR x B6)F1 mice reveals that the T cell unresponsiveness of AKR/J is inherited as a dominant trait, possibly associated with expression of the Mls-1a allele. Mice derived from backcrossing (AKR x B6)F1 x B6 were selected for H-2b homozygosity and were classified as Mls-1a or Mls-1b according to the relative numbers of peripheral blood T cells that expressed the TCR V beta 6 gene product. After challenge by injection with AChR in CFA, lymph node cells from mice classified as having less than 2% of V beta 6+ peripheral T cells had low responsiveness to AChR, whereas mice with greater than 7% V beta 6+ peripheral T cells had high T cell responsiveness to AChR. These results are consistent with the notion that regulation of the T cell repertoire by Mls loci may be a determinant of susceptibility to autoimmunity.     

Rearrangement and expression of T-cell antigen receptor genes in human T-lymphocyte tumor lines and normal human T-cell clones: evidence for allelic exclusion of Ti beta gene expression and preferential use of a J beta 2 gene segment.

The gene encoding the beta chain of the human T-cell receptor for antigen is composed of variable (V), diversity (D), joining (J), and constant (C) gene segments which undergo specific rearrangements during T-lymphocyte ontogeny. Southern blot analyses of seven human T-cell tumor lines and normal human T-lymphocyte clones revealed that most of these T-cell lines rearrange their Ti beta genes differently. The T-cell tumor line HPB-MLT rearranges and transcribes both of its Ti beta genes. Cloning and sequencing of the Ti beta cDNAs corresponding to these rearrangements revealed that one of the rearranged Ti beta genes is defective, while the other is functional and corresponds to the Ti beta protein expressed on the surface of these cells. Thus, this cell line displays a pattern of allelic exclusion of Ti beta gene expression. A comparison of four C beta 2-containing Ti beta cDNAs from three different cell lines revealed that three of the four utilize the same J beta 2.5 gene segment joined to different D beta and V beta genes, suggesting that there may be preferential use of this J gene during J beta 2 rearrangements. Hybridization analyses with probes for the alpha and beta genes of the T-cell receptor and the T-cell-specific T gamma gene revealed that HPB-MLT cells appear to express approximately equivalent amounts of RNA corresponding to each of the rearranged Ti alpha and Ti beta genes. However, they express a much lower level of T gamma RNA.     

The extent of the human germline T-cell receptor V beta gene segment repertoire

An assessment of the size of the human TCRBV gene segment repertoire based on the identification of TCRBV gene segments in genomic DNA was undertaken. PCR amplification from cloned and uncloned genomic DNA sources, nucleotide sequencing, Southern blot hybridization, and cosmid cloning were used to identify TCRBV gene segments in multiple unrelated individuals. The key advantages to this approach were: (1) TCRBV gene segments which are expressed only at very low levels in cDNA libraries were still detectable, and (2) it was possible to discriminate between alleles at the same locus vs products of different loci. A total of 63 unique TCRBV gene segments were identified and sequenced. Six of these TCRBV gene segments had not been previously described. Thirty-four cosmid clones containing 51 of the 63 identified TCRBV gene segments were isolated and screened for the presence of additional novel TCRBV subfamily members. These results, obtained by a variety of complementary approaches, indicate that the human TCRBV gene segments of which 52 are functional. The availability of the majority of these TCRBV gene segments on cosmid clones should facilitate further investigation of germline TCRBV gene segment polymorphism and putative disease associations.