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.     

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.     

The T cell receptor V beta 6 domain imparts reactivity to the Mls-1a antigen

A monoclonal antibody secreting hybridoma was established by fusing spleen cells from a rat immunized with a murine T cell clone, OI11, which has I-Ab restricted specificity for the male H-Y antigen and unrestricted specificity for the minor lymphocyte stimulating antigen, Mls-1a, to the mouse myeloma P3X63AG8.653 and screening for the capacity of the hybridoma supernatants to stimulate the OI11 T cell clone. An antibody (RR4-7) was found to be specific not only for the immunizing T cell clone but virtually for all T cells using the V beta 6 TCR gene product as part of their surface antigen receptor. When the expression of the V beta 6 gene in various strains of mice was analyzed, it was found that strains expressing the Mls-1a antigen contained few T cells expressing V beta 6-encoded TCRs. The majority of T cell hybridomas which expressed V beta 6-encoded TCRs were found to be reactive to the Mls-1a antigen. These data confirm the finding of H. R. MacDonald et al. (Nature (London) 332, 40, 1988) that most TCRs encoded by the V beta 6 gene have a biased specificity for the Mls-1a antigen.     

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.     

Genomic organization of the mouse T cell receptor V alpha family.

Based on the analysis of V alpha gene segment deletions in a panel of T lymphomas, we have constructed a map of the mouse T cell receptor alpha/delta region and assigned the relative position of 72 distinct V gene segments. Three major observations have emerged from such studies. First, members of a given V alpha subfamily are not organized in discrete units along the chromosome but largely interspersed with members of other V alpha subfamilies. Second, analysis of the deletion map suggests the existence of repetitive patterns (V alpha clusters) in the chromosomal distribution of the V alpha gene segments. Third, the present-day organization of the V alpha/delta region may be readily explained by a series of sequential duplications involving three ancestral V alpha clusters. Direct evidence for the existence of these unique structural features has been gained by cloning approximately 370 kb of DNA and positioning 26 distinct V alpha gene segments belonging to six different subfamilies. Finally, the relationships existing between the V alpha/delta gene segment organization and usage are discussed in terms of position-dependent models.     

Labile intracellular zinc is associated with 3T3 cell growth

Increasing evidence shows that labile intracellular zinc is metabolically important. Depletion of labile intracellular zinc using chelators suppresses DNA synthesis. In this study, we tested the hypothesis that labile intracellular zinc could be modulated via varying zinc nutrition. This could result in an altered availability of labile intracellular zinc, which, in turn, could influence zinc-dependent cellular events involved in cell proliferation and ultimately suppress growth. Labile intracellular zinc was detected by using N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), a membrane-permeable fluorescence probe. After 48 h culture in a zinc-depleted medium, labile intracellular zinc in 3T3 cells was diminished along with a suppressed DNA synthesis and cell proliferation. In contrast, supplementation of zinc to the zinc-depleted medium increased the labile intracellular zinc and promoted DNA synthesis and cell proliferation. Furthermore, growth factor-dependent stimulation of DNA synthesis and cell proliferation was also accompanied by increased labile intracellular zinc. Together, our data showed an association between the labile intracellular zinc, detected using TSQ, and 3T3 cell growth, suggesting that labile intracellular zinc could be an important cellular link between zinc nutrition and growth.     

T cell receptor alpha-chain influences reactivity to Mls-1 in V beta 8.1 transgenic mice.

Most, but not all, V beta 8.1+ T cells respond to M1s-1 and are clonally deleted in the thymus of M1s-1-expressing animals. To formally examine the role of the TCR alpha-chain in reactivity and tolerance to M1s-1, we have analyzed M1s-1 reactivity in a large panel of CD4+ hybridomas generated from TCR V beta 8.1 transgenic mice, that express an identical, potentially M1s-1-reactive beta-chain. The data show that the alpha-chain strongly influences the M1s-1 reactivity of the hybridomas and that the differences in reactivity had relevance for tolerance. Thus, V alpha 11+ hybridiomas were biased toward M1s-1 reactivity and V alpha 11+ T cells were correspondingly absent from the peripheral repertoire of M1s-1-expressing transgenic mice. V alpha 2+ hybridomas, on the other hand, were biased against M1s-1 reactivity, and V alpha 2+ T cells were correspondingly amplified in the M1s-1-expressing transgenic mice. Structural analysis of the alpha-chains revealed that the M1s-1 reactivity of the V alpha 11+ hybridomas segregated precisely with family member, such that V alpha 11.1+ hybridomas were M1s-1-reactive and V alpha 11.3+ hybridomas were not M1s-1-reactive. On the other hand, there was not a clear correlation between family member and M1s-1 reactivity in the V alpha 2+ hybridomas. The hybridomas also showed striking variation in their reactivity to staphylococcal enterotoxin B (SEB), and the SEB reactivity of the V alpha 11+ hybridomas correlated precisely with family member and with M1s-1 reactivity. In contrast, there was not a clear correlation with V alpha 2+ alpha-chain structure and SEB reactivity. Also, there was no correlation between M1s-1 reactivity and SEB reactivity in individual V alpha 2+ hybridomas, suggesting that the recognition of the two superantigens by the same TCR is not equivalent. Taken together, these data define a role for the TCR alpha-chain in superantigen reactivity and T cell tolerance, and provide a structural explanation for the different fates of M1s-1-reactive T cells in normal and transgenic mice.     

Restricted usage of T cell receptor V alpha/J alpha gene segments with different nucleotide but identical amino acid sequences in HLA-DR3+ sarcoidosis patients.

BACKGROUND: Sarcoidosis is a granulomatous disease characterized by the accumulation of activated T cells in the lungs. We previously showed that sarcoidosis patients expressing the HLA haplotype DR3(17),DQ2 had increased numbers of lung CD4+ T cells using the T cell receptor (TCR) variable region (V) alpha 2.3 gene segment product. In the present study, the composition of both the TCR alpha- and beta-chains of the expanded CD4+ lung T cells from four DR3(17),DQ2+ sarcoidosis patients was examined. MATERIALS AND METHODS: TCR alpha-chains were analyzed by cDNA cloning and nucleotide sequencing. TCR beta-chains were analyzed for V beta usage by flow cytometry using TCR V-specific monoclonal antibodies or by the polymerase chain reaction (PCR) using V beta- and C beta-specific primers. J beta usage was analyzed by Southern blotting of PCR products and subsequent hybridization with radiolabeled J beta-specific probes. RESULTS: Evidence of biased J alpha gene segment usage by the alpha-chains of V alpha 2.3+ CD4+ lung T cells was found in four out of four patients. Both different alpha-chain nucleotide sequences coding for identical amino acid sequences and a number of identically repeated alpha-chain sequences were identified. In contrast, the TCR beta-chains of FACS-sorted V alpha 2.3+ CD4+ lung T cells were found, with one exception, to have a nonrestricted TCR V beta usage. CONCLUSIONS: The finding of V alpha 2.3+ CD4+ lung T cells with identical TCR alpha-chain amino acid sequences but with different nucleotide sequences strongly suggests that different T cell clones have been selected to interact with a specific sarcoidosis associated antigen(s). The identification of T cells with restricted TCR usage, which may play an important role in the development of sarcoidosis, and the possibility of selectively manipulating these cells should have important implications for the treatment of the disease.     

First T cell receptor alpha gene rearrangements during T cell ontogeny skew to the 5' region of the J alpha locus

Fetal, neonatal, and early postnatal thymi were assessed for TCR J alpha gene rearrangements. Gene probes spanning the distance from 5' to 3' regions of the J alpha locus were used to determine the approximate location of gene rearrangements within hybridomas representing each of the early thymocyte populations. The predominant location of rearrangements was within the 5' region of the J alpha locus. Among the several cells in which rearrangements were found on only one chromosome, the one rearrangements was always in the 5' region. When two rearrangements were found, the rearrangements on homologous chromosomes were usually in the same region. The overall pattern among thymocytes was in great contrast to that previously observed among hybridomas derived from stimulated adult spleen cells within which rearrangements fell mostly to the 3' side of the alpha-locus. Results reveal the nonrandom nature of the TCR-alpha gene rearrangement event and may reflect an incidence of multiple V-J alpha joining events on each chromosome during T cell development in vivo. Due to the fact that most mature cells bear two J alpha joins, the allelic exclusion of alpha-chains cannot be explained by a mechanism whereby a functional rearrangement on one chromosome inhibits subsequent rearrangement on the second. Instead allelic exclusion may rely on a low frequency of productive vs nonproductive rearrangement events and an incompatibility between multiple alpha- and beta-protein pairs.     

Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.     

The human T cell antigen receptor is encoded by variable, diversity, and joining gene segments that rearrange to generate a complete V gene

A cDNA clone YT35 , synthesized from poly(A)+ RNA of the human T cell tumor Molt 3, exhibits homology to the variable (V), joining (J), and constant (C) regions of immunoglobulin genes. We have isolated and sequenced the germ-line V and J gene segment counterparts to YT35 from a human cosmid library, and these failed to encode 14 nucleotides of the cDNA clone between the V and J regions. We postulate that these 14 nucleotides are encoded by a third gene segment analogous to the diversity (D) gene segments of immunoglobulin heavy chain genes. This T cell antigen receptor V gene appears to be assembled from three gene segments, V, D, and J, and accordingly most closely resembles immunoglobulin heavy chain V genes.     

V75R576 IL-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4R alpha in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4R alpha allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4R alpha with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4R alpha allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4R alpha haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4R alpha requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.     

Restriction fragment length polymorphism of the human T cell receptor alpha gene

Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3 joining (J) exon and the most 5 constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3 untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.Abbreviations used in this paper RFLP restriction fragment length polymorphism - Ti-alpha alpha gene of the T-cell receptor for antigen     

The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele-specific antibodies.

Allelic polymorphism in TCR loci may play an important role in shaping the T cell repertoire and in disease susceptibility. We have used a combination of antibody and sequence analysis to investigate polymorphism in the murine V alpha 11 family. Two different antibodies have been analyzed that recognize particular V alpha 11 family members of the V alpha b and V alpha d haplotypes. One antibody shows J alpha dependency, suggesting a conformational element to the epitope. Investigation of the anti-V alpha 11 staining pattern on different mouse strains indicates that there is a marked influence of MHC haplotype on V alpha 11 selection and that V alpha 11 is preferentially expressed on CD4+ cells. Sequence analysis of V alpha 11 genes from the V alpha a, V alpha b, and V alpha d haplotypes shows two potential regions for the haplotype-specific epitopes. The relatedness of the different V alpha 11 family members from different haplotypes suggests that the V alpha 11.1/11.2 gene duplication is relatively recent, but that V alpha 11.3 separated much earlier. Differences between V alpha 11.3 and V alpha 11.1/11.2 are concentrated in the putative complementarity determining regions (CDR), whereas differences between alleles are not clearly clustered. However, the V alpha 11.1a and V alpha 11.1d alleles differ from V alpha 11.1b and V alpha 11.2b in CDR1. A V alpha 11.2-expressing anti-cytochrome c T cell has the same V-J junction as a V alpha 11.1-bearing cell with a similar fine specificity, indicating that V alpha 11.1b and V alpha 11.2b do not contribute different Ag specificities.     

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.     

CD3+4-8- alpha beta T cell population with biased T cell receptor V gene usage. Presence in bone marrow and possible involvement of IL-3 for their extrathymic development.

Analysis of TCR of a series of CD4-8- (double negative; DN) alpha beta T cell lines induced with IL-3 revealed that their V gene usage was biased for V alpha 4 and V beta 2. This has been confirmed in the primary short-term cultures. Thus, IL-3 induced the generation of DN alpha beta T cells with predominant V beta 2 gene expression from the CD4+/CD8+ T cell-depleted spleen or bone marrow (BM) cells of both normal and nude BALB/c mice within 10 days. It was further indicated that the V beta 2+ beta-chain genes contained few junctional N regions in both IL-3-induced primary DN alpha beta T cells and continuous lines. Search for the in vivo counterpart of in vitro IL-3-induced DN alpha beta T cells revealed that BM, but not spleens, of normal BALB/c and B6 mice did contain a significant proportion of DN alpha beta T cells, and that the majority of them expressed V beta 2+ beta-chain genes with few junctional N regions. The presence of V beta 2+ DN alpha beta T cells was similarly observed in the BM of BALB/c nude mice, but their proportion varied markedly among various strains of mice, which was not linked to H-2 haplotypes. The results indicated that V beta 2+ DN alpha beta T cells in the BM represented one of the thymus-independent T cell populations, whose development was under the major histocompatibility Ag complex-unlinked genetic control. TCR of these T cells were shown to be functional as judged by the proliferative response to anti-V beta 2 antibody. Taken together, present results suggested that IL-3 could induce differentiation and/or proliferation of DN alpha beta T cells with uniquely limited repertoire, which existed preferentially in BM in vivo, and implied the possible involvement of extrathymic endogenous ligands as a positive selection force.     

Up-regulation of gene related to anergy in lymphocytes is associated with Notch-mediated human T cell suppression

A growing body of literature indicates that the Notch pathway can influence the activation and differentiation of peripheral murine T cells, though comparatively little is known about the effects of Notch signaling in human T cells. In the present report we demonstrate that Jagged-1-induced Notch signaling (using immobilized Jagged-1 fusion protein) during stimulation of purified human CD4+ and CD8+ T cells potently inhibits T cell proliferation and effector function, including both Th1- and Th2-associated cytokines. Inhibition of T cell activation is not due to apoptosis or disruption of proximal TCR signaling, but is associated with up-regulation of GRAIL (gene related to anergy in lymphocytes) in CD4+ T cells, with modest effects on other E3 ubiquitin ligases such as c-Cbl and Itch. When evaluated for its effects on CD4+ T cell differentiation, Jagged-1-mediated signaling inhibits T cell cytokine secretion with no significant effect on proliferative responses. Collectively, these data demonstrate that Notch signaling in human T cells induced by Jagged-1 promotes a novel form of T cell hyporesponsiveness that differs from anergy, whereby primary T cell proliferation and cytokine secretion are potently inhibited, and effector function but not proliferative capacity are ameliorated upon secondary stimulation.     

The bovine T cell receptor alpha/delta locus contains over 400 V genes and encodes V genes without CDR2

αβ T cells and γδ T cells perform nonoverlapping immune functions. In mammalian species with a high percentage of very diverse γδ T cells, like ruminants and pigs, it is often assumed that αβ T cells are less diverse than γδ T cells. Based on the bovine genome, we have created a map of the bovine TRA/TRD locus and show that, in cattle, in addition to the anticipated >100 TRDV genes, there are also >300 TRAV or TRAV/DV genes. Among the V genes in the TRA/TRD locus, there are several genes that lack a CDR2 and are functionally rearranged and transcribed and, in some cases, have an extended CDR1. The number of bovine V genes is a multiple of the number in mice and humans and may encode T cell receptors that use a novel way of interacting with antigen. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.