Diversity and structure of human T-cell receptor beta-chain variable region genes.

The nucleotide sequences of 27 T-cell receptor beta cDNA clones isolated from a human peripheral lymphocyte library were determined and compared to five additional published sequences. These cDNA clones represent 22 distinct T-cell receptor beta-chain variable region (V beta) gene segment sequences, which fall into 15 different V beta gene subfamilies, each containing six or fewer members. From this analysis, we estimate that the repertoire of expressed human V beta genes is less than 59, apparently much smaller than the immunoglobulin heavy chain and light chain variable region (VH and VL) repertoires. Variability plots comparing these human V beta regions with each other and with published mouse V beta regions provide evidence for only four hypervariable regions homologous to those seen in comparisons of immunoglobulin V regions. Somatic hypermutation appears to be used infrequently, if at all, in these V beta genes.     

Sequence and evolution of the human T-cell antigen receptor beta-chain genes.

We present the nucleotide sequences of the two genomic constant (C)-region gene segments, C beta 1 and C beta 2, encoding the beta chain of the human T-cell antigen receptor. The two C beta genes are organized identically to each other and to the corresponding mouse genes, both having four exons, whose boundaries were confirmed from the sequence of a C beta 2 cDNA clone from the T-cell line MOLT-4. The predicted amino acid sequences of human C beta 1 and C beta 2 differ at only five positions, which suggests that the proteins have very similar functions. This similarity is the result of strong nucleotide-sequence conservation in protein-coding regions, which extends to silent positions. A quantitative analysis of an alignment of the nucleotide sequences of the two human genes shows that whereas the 5' ends (including the first exon) are extremely homologous, the 3' ends are widely divergent, with other regions having intermediate levels of homology. Analysis of published data [Gascoigne, N.R.J., Chien, Y., Becker, D.M., Kavaler, J. & Davis, M.M. (1984) Nature (London) 310, 387-391] shows that the mouse C beta 1 and C beta 2 genes are also virtually identical in their first exons but more divergent in the remaining coding regions. Therefore, partial gene conversion events may have occurred during the evolution of both human and mouse C beta genes.     

Rearrangement of T-cell receptor beta-chain genes during T-cell development.

The kinetics and order of rearrangements in the gene complex encoding T-cell-receptor beta chains were studied by Southern blot hybridization in a collection of hybridomas derived from fetal thymocytes at various stages of ontogeny (day 14 to day 17). Our results show a steady increase in the frequency of rearranged beta complexes during this period and suggest that these rearrangements occur within the thymus. beta-chain diversity region (D beta) to beta-chain joining region (J beta) joining preceded other types of rearrangements. More complex hybridization patterns consistent with fully rearranged functional beta-chain genes did not begin to accumulate until day 16, 1 day prior to significant surface expression of the receptor protein.     

T-cell receptor V beta expression in normal human skin.

The skin-associated immune system is the first line of defense against pathogenic attack from the environment and is simultaneously tolerant to localized autoantigens and to antigens of the normal microbial flora. The T-cell receptor (TCR) repertoire of skin lymphocytes may therefore be influenced by the skin microenvironment. We studied the expression of TCR beta-chain variable region (V beta) genes in normal skin by a polymerase chain reaction-based comparative method. When comparing the amplification of V beta genes in peripheral blood and normal skin, we found that TCR V beta 1, -7, -14, and -16 were often highly expressed in skin relative to peripheral blood, whereas V beta 5.1 was often highly expressed in peripheral blood mononuclear cells but not in skin. These results demonstrate that the TCR repertoire of skin lymphocytes is not determined by random sampling of peripheral blood mononuclear cells but may be molded by the interaction with self antigens and/or the normal microbial flora in the microenvironment of the skin.     

Reduced expression of a human V beta 6.1 T-cell receptor allele.

We have previously described an allelic polymorphism in the V beta 6.1 T-cell receptor gene. The V beta 6.1B allele is associated with disease in a subgroup of patients with juvenile rheumatoid arthritis. Limited sequence data demonstrated nucleotide differences that resulted in two amino acid changes between the two alleles in positions predicted to be important in major histocompatibility complex/antigen recognition. The present study demonstrates substantially reduced expression of mRNA from the disease-associated allele (V beta 6.1B) in peripheral blood and thymic tissue. The complete genomic sequence of both alleles revealed two additional amino acid changes in the V beta 6.1B gene as well as nucleotide differences in the promoter and intron. A cysteine-to-arginine substitution at position 92 in the disease-associated allele makes this a non-functional beta chain, since this conserved cysteine is involved with disulfide bonding to cysteine-23 to form an immunoglobulin-like domain structure, thus resulting in a potential hole in the T-cell receptor repertoire.     

Murine T-cell receptor mutants with deletions of beta-chain variable region genes.

Genomic Southern blots of DNA from eight strains of mice were examined for restriction fragment length polymorphisms in their loci encoding the variable region of the T-cell receptor beta chain (V beta), using 16 different V beta-specific probes. Mouse strains BALB/c, C57BL/6, C3H, and PL were identical, while strains SJL, C57BR, C57L, and SWR shared several polymorphisms with respect to the other four strains. In addition, SJL, C57L, C57BR, and SWR DNAs were missing 50% of the hybridizing bands visualized in BALB/c DNA. A cDNA library from concanavalin A-stimulated SJL spleen blasts was constructed and examined for V beta gene usage. Ten genes were found to account for all V beta-containing clones isolated, including three newly identified V beta genes. All 10 of these genes were found to be present in BALB/c mice. We conclude that SJL, C57L, C57BR, and SWR mice represent V beta deletion mutants of the BALB/c genotype.     

Organization and sequences of the diversity, joining, and constant region genes of the human T-cell receptor beta chain.

The organization and sequences of the human beta-chain T-cell receptor diversity, joining, and constant region segments are described. The beta chain of the human T-cell receptor, analogous to the mouse counterpart, consists of two distinct constant region genes approximately equal to 10 kilobases apart. The two constant region genes, C beta 1 and C beta 2, are very similar not only in sequence but also in genomic organization. The coding sequences of each of these C beta constant region genes are divided into four exons. The first two exons encode most of the extracellular constant domain. The third exon encodes a major part of the presumed transmembrane portion, and the last exon contains the cytoplasmic coding sequence as well as 3' untranslated sequences. Except for a stretch of approximately equal to 95 highly conserved nucleotides extending 3' of the first exon of the C region genes, little homology can be found between the intron sequences of C beta 1 and C beta 2. A small cluster of joining region (J beta) gene segments is located approximately equal to 5 kilobases upstream of each of these two constant regions. The first cluster, J beta 1, contains six functional J gene segments while the second, J beta 2, contains seven functional J gene segments. In addition, diversity region (D beta) gene segments are located approximately equal to 600 base pairs upstream of each J beta. Recombinational signals containing highly conserved heptamer and nonamer sequences separated by 12 or 23 bases are found adjacent to all of these D beta and J beta gene segments. These signal sequences are thought to be involved in the somatic recombination processes. These results indicate that what appears to be a gene duplication event giving rise to these two distinct regions must have arisen a long time ago in the evolution of this gene locus.     

Two monoclonal rat antibodies with specificity for the beta-chain variable region V beta 6 of the murine T-cell receptor.

Two rat monoclonal antibodies (mAbs), 44-22-1 and 46-6B5, which recognize an alloreactive cytotoxic clone, 3F9, have been further tested on a panel of T hybridomas and cytotoxic T-cell clones for binding and functional activities. The mAbs recognized only those cells sharing the expression of the T-cell receptor beta-chain variable region gene V beta 6 with 3F9. All V beta 6+ cells were activated by these mAbs under cross-linking conditions and their antigen-specific activation was blocked by soluble mAb. Furthermore, depletion of 46-6B5+ normal lymph node T cells eliminated all cells expressing the epitope recognized by 44-22-1 and V beta 6 mRNA.     

Organization of the T-cell antigen-receptor beta-chain locus in mice.

We used pulsed-field gel electrophoresis to determine the organization of the beta-chain gene of the T-cell receptor for antigen in normal and mutant inbred strains of mice. In normal mice, the variable (V)- and constant (C)- region elements of this locus span 700-800 kilobases of chromosomal DNA. All but one of the V beta gene segments analyzed lie 5' of the J beta C beta locus (J beta represents the joining region), with the closest being 280-360 kilobases away. The mutant mouse strain SJL has an internal V beta-region gene deletion that compacts the V beta region by 100-200 kilobases. Taken together with other data, these results indicate that the beta-chain locus can use either a looping-out/deletion or an inversion mechanism to appose V beta to DJ beta gene segments (D is the diversity region) and can accomplish the former (at least) over very large distances.     

CD8+ T cells in psoriatic lesions preferentially use T-cell receptor V beta 3 and/or V beta 13.1 genes.

Psoriasis is an inflammatory skin disorder characterized by epidermal keratinocyte hyperproliferation in association with a cellular infiltrate. There is evidence that activated T cells play a role in psoriatic plaque formation. We examined the T-cell receptor beta-chain variable gene segment (V beta) use of epidermal T cells in shave biopsies of psoriatic lesions. Our results show increased expression of V beta 3 and/or V beta 13.1 messages in the CD8+, but not CD4+, T cells in the lesions of a majority of patients studied. Sequence analysis of complementarity-determining region 3 (CDR3) of these two V beta genes from the skin demonstrated monoclonality or marked oligoclonality. A second biopsy from the same or different lesions, performed 3.5-8 months later in four patients, again revealed increased V beta 3 and/or V beta 13.1 expression and clonality. Moreover, in three of the four patients, the same V beta CDR3 rearrangement was found in both biopsies, although there was no V beta CDR3 homology between patients. In two patients in which V beta 3 and/or V beta 13.1 was not increased, an increase in V beta 17 gene use and clonality was found. The clonality of V beta sequence data indicates these cells are recruited and expanded in situ. The persistence of V beta 3-and/or V beta 13.1-bearing CD8+ T cells in lesions that did not undergo resolution suggests their role as effector cells rather than as regulatory cells.     

Organization of human cardiovascular-expressed genes on chromosomes 21 and 22

The recent availability of the sequenced and annotated DNA sequences of chromosomes 21 and 22 has initiated the next phase in the human genome project: the application of this resource. One facet of these data is that they provide a list of ordered genes along the chromosome that can be capitalized upon to determine gene position effects. Specifically, the physical position and distribution of genes along the chromosomes may be related to gene expression in specific organs or organ systems. In this report we index the subset of genes constituting the human "cardiovascular genome" on chromosomes 21q and 22q as well as report the identification of several "cardiovascular gene" clusters. These gene clusters are suggestive of a higher order of tissue-specific gene regulation at the chromosomal level.     

CD3+ leukemic large granular lymphocytes utilize diverse T-cell receptor V beta genes

CD3+ large granular lymphocyte (LGL) leukemia is a disease of unknown etiology characterized by clonal proliferation of T cells that usually express T-cell receptor (TCR) alpha beta heterodimers. The purpose of this study was to identify the variable (V), joining (J), and diversity (D) region TCR beta-chain genes expressed by CD3+ LGL leukemic cells in an attempt to gain insights into the etiology of this disorder. Twelve patients with LGL leukemia were studied, including seven with both LGL leukemia and rheumatoid arthritis (RA). RA is also a disease of unknown etiology that occurs frequently in patients with LGL leukemia. Clonally expanded T cells that express specific TCR V beta genes have been identified in fluid and tissue specimens from the joints of patients with RA. In this study, V beta expression was determined by PCR using a panel of 22 unique V beta primers to amplify cDNA prepared from peripheral blood mononuclear cells (PBMC). A dominant V beta gene product was readily apparent in all patients. To confirm that the dominant V beta gene originated from a clonal expansion, DNA fragments corresponding to the dominant V beta genes were subcloned into plasmids and independently isolated recombinants were sequenced. V-D-J region sequences that occurred repeatedly indicated clonality. The V beta and J beta genes expressed by the leukemic cells showed a pattern of distribution that followed the frequency with which these genes are represented in the peripheral blood. The residues corresponding to the third complementarity-determining region of the TCR beta chain were different in all cases. A specific pattern of VDJ usage was not identified for those patients with both LGL leukemia and RA; however, utilization of V beta-6 by LGL clones (N = 3) was observed only in the setting of RA. These data suggest that leukemic CD3+ LGL cells have been clonally transformed in a random fashion with respect to the TCR beta chain.     

Inversion of chromosome 7 in ataxia telangiectasia is generated by a rearrangement between T-cell receptor beta and T-cell receptor gamma genes

Specific and recurrent chromosomal rearrangements are often observed in the karyotypes of phytohemagglutinin-stimulated lymphocytes. The percentage of cells demonstrating these rearrangements is dramatically increased in the genetic disease ataxia telangiectasia. Inversion of chromosome 7 represents approximately half of the chromosomal rearrangements in this disease. Because the chromosomal locations of the inv(7) breakpoints coincide precisely with those of the T-cell antigen receptor (TCR) beta and gamma genes, it has been hypothesized that this rearrangement may occur by recombination between those two loci. Here, we present direct evidence that inversion of chromosome 7 in ataxia telangiectasia is generated by site-specific recombination between a TCR gamma variable segment and a TCR beta joining segment.     

A method for production of antibodies to human T-cell receptor beta-chain variable regions.

Mouse T-cell hybridomas bearing human V beta elements were produced by transfection of human/mouse hybrid T-cell receptor beta-chain genes into a mouse T-cell hybridoma lacking an endogenous beta-chain gene. These hybridomas were entirely mouse in origin except for the human V beta region. These cells were used to immunize mice against human V beta elements. Mouse monoclonal antibodies have thus been generated against human V beta 13.1 and -13.2. We expect that the method outlined in this paper will be useful in the production of monoclonal antibodies specific for other human V beta or V alpha elements.     

Low frequency of somatic mutation in beta-chain variable region genes of human T-cell receptors.

We have cloned three pairs of rearranged and germ-line variable region (V beta) genes of the beta chain of the human T-cell receptor from the cell lines ATL2, ATL12, and MT-1 of patients with adult T-cell leukemia (ATL). The pairs were derived from the same (for ATL2 and ATL12) and different (for MT-1) individuals. Comparison of the nucleotide sequences showed no somatic mutation in V beta X ATL2 and beta X ATL12-2. Although one nucleotide change was found in V beta X MT1-1, the possibility of polymorphism was not excluded. These results indicate that the frequency of somatic mutation in the V beta gene of the T-cell receptor is 1/10th or less than that in the immunoglobulin gene. Both alleles of the rearranged T-cell receptor gene were analyzed for ATL12 and MT-1. In both, only one of the two rearranged J beta alleles was an active variable-diversity-joining (V-D-J) complex. The results suggest that allelic exclusion occurs in the T-cell receptor gene.     

Restricted T-cell receptor V beta gene usage by myelin basic protein-specific T-cell clones in multiple sclerosis: predominant genes vary in individuals.

Recent studies in experimental autoimmune encephalomyelitis as a model for multiple sclerosis (MS) have demonstrated limited heterogeneity in T-cell antigen receptors (TCR) specific for myelin basic protein (MBP). To investigate restricted beta-chain variable-region (V beta) gene usage in humans, we analyzed TCR gene rearrangements in two lines and 34 MBP-specific T-cell clones that were isolated from five MS patients and two healthy subjects. The T cells were characterized for their specificity to MBP epitopes and HLA-restricting molecules. We demonstrate here that MBP-specific T-cell clones from these different MS patients and healthy individuals, in contrast to T cells from rodents, display a more diverse V beta gene usage as evidenced by their TCR V beta gene rearrangements. However, the different MBP-specific T-cell clones isolated from each individual MS patient showed a common V beta gene usage, suggesting individual-specific TCR restriction. Out of 16 MBP-specific clones derived from a single MS patient, 12 clones (75%) utilized the V beta 15 gene for their TCR gene rearrangement. MBP-specific clones isolated from four other MS patients also showed a consistent tendency for a predominant, but different, TCR V beta gene rearrangement. These results suggest a TCR heterogeneity among MBP-specific T-cell clones from different individuals but a limited TCR V beta gene usage among MBP-specific T-cell clones of the same individual. The predominant V beta gene used by the MBP-specific T-cell clones studied here was not found to correlate with the epitope specificity of T cells or with their restricting HLA molecule. These findings may support the possibility of intervention with monoclonal antibodies to specific V beta gene products as an approach to immune therapy of MS but also imply the necessity for an individual-specific immunotherapeutic approach.