A novel mechanism of somatic rearrangement predicted by a human T-cell antigen receptor beta-chain complementary DNA

The T-cell antigen receptor is a cell surface molecule vital in mediating the cellular immune response. The arrangement and rearrangement of the gene segments encoding the beta-chain polypeptide of the receptor are similar to those of immunoglobulin gene segments. The two constant region genes of the human T-cell antigen receptor are 8 kilobases apart with a cluster of joining segments located 5' of each constant region gene. Although most beta-chain gene rearrangements involve the variable, diversity, and joining segments, analysis of a beta-chain complementary DNA clone suggests the occasional occurrence of another type of rearrangement.     

A large deletion within the T-cell receptor beta-chain gene complex in New Zealand white mice

The T-cell receptor beta-chain gene complex contains a duplication of D beta, J beta, and C beta gene segments in mice and man. When DNA from many inbred strains of mice was screened an unusual allele of the beta locus was identified in New Zealand White (NZW) mice. This allele is distinguished by the deletion of an 8.8-kilobase segment of DNA containing C beta 1, D beta 2 and the J beta 2 cluster. Despite the fact that all NZW T-cell receptors must be derived from a single set of beta-chain gene segments, this strain has functional T cells and is phenotypically normal. This deletion of T-cell receptor beta-chain segments occurs in a strain known to contribute to lupus-like autoimmune disease.     

RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination

The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding and 3' untranslated sequences are contained in single exons. RAG-1 and RAG-2 might activate the expression of the V(D)J recombinase but, more likely, they directly participate in the recombination reaction.     

Isotypic exclusion of gamma delta T cell receptors in transgenic mice bearing a rearranged beta-chain gene

The rearrangement of T cell antigen receptor beta- and gamma-chain gene segments was studied in transgenic mice that bear a functional beta-chain gene. Virtually all CD3-positive T cells derived from transgenic mice express beta chains containing the transgene-encoded V beta 8.2 variable region on their surfaces and do not express endogenous beta-chain variable regions. Expression of endogenous V beta genes is inhibited at the level of somatic recombination during thymic ontogeny. Furthermore, rearrangements of the TCR gamma-chain genes are also markedly inhibited in these transgenic animals. Hence expression of the TCR beta transgene has led to allelic exclusion of alpha beta receptors and isotypic exclusion of gamma delta T cell receptors.     

Structure and diversity of the human T-cell receptor beta-chain variable region genes

In order to characterize the variability of the expressed human T-cell receptor (TCR) beta-chain repertoire and contrast this variability to the known murine beta-chain repertoire, 15 independent complementary DNA (cDNA) clones containing TCR beta-chain variable region (V beta) genes were isolated from a human tonsil cDNA library. The nucleotide and derived amino acid sequences of these 15 V beta genes were analyzed together with 7 previously defined sequences. Fifteen different human V beta genes could be identified from 22 independent sequences. By means of DNA hybridization and sequence homology comparisons, it was possible to group these 15 genes into ten distinct V beta subfamilies, each containing from one to seven members. Minimal polymorphism was noted between individuals, except in multimember subfamilies. The amino acid sequences of these genes contain conserved amino acids that are also shared by murine TCR V beta genes and immunoglobulins; no features were found that distinguish human V beta genes from their murine counterparts. Evaluation of secondary structure showed that maximum variability coincides with generally hydrophilic portions of the amino acid sequence, while specific hydrophobic regions were conserved in all V beta genes examined.     

T-cell receptor beta-chain expression: dependence on relatively few variable region genes

Fifteen independently isolated complementary DNA clones that contain T-cell receptor (TCR) V beta genes were sequenced and found to represent 11 different V beta genes. When compared with known sequences, 14 different V beta genes could be defined from a total of 25 complementary DNA's; 11 clones therefore involved repeated usage of previously identified V beta's. Based on these data, we calculate a maximum likelihood estimate of the number of expressed germline V beta genes to be 18 with an upper 95 percent confidence bound of 30 genes. Southern blot analysis has shown that most of these genes belong to single element subfamilies which show very limited interstrain polymorphism. The TCR beta-chain diversity appears to be generated from a limited V beta gene pool primarily by extensive variability at the variable-diversity-joining (V-D-J) junctional site, with no evidence for the involvement of somatic hypermutation.     

Influence of the major histocompatibility complex on positive thymic selection of V beta 17a+ T cells

A monoclonal antibody was used to show directly positive thymic selection of the T cell repertoire in mouse strains expressing the 17a beta-chain variable domain (V beta 17a) of the T cell receptor. In the absence of the potent tolerizing class II major histocompatibility complex (MHC) molecule, I-E, peripheral expression of V beta 17a+ T cell receptors varied with the MHC haplotype of the mouse strain. In the most extreme case, H-2q mice expressed high peripheral levels of CD4+ V beta 17a+ T cells (14 to 19 percent), whereas H-2b mice expressed low levels (3 to 4 percent). Analysis of (b x q)F1 mice and chimeric mice showed that these differences were determined by positive thymic selection and implicated the thymic epithelium as the controlling cell type.     

Failure of T cell receptor V beta negative selection in an athymic environment

The mature T cell receptor (TCR) repertoire is the result of selection events during T cell development. Previous assessment of TCR beta-chain selection with serologic and molecular probes demonstrated both positive and negative selection. Although this work suggested a critical role for the thymus, no direct assessment has been made of the requirement for a thymus in TCR V beta selection. A comparison of TCR V beta expression in four different congenic pairs of normal and nu/nu (athymic) mice indicated that the normal V beta deletions associated with tolerance to self minor lymphocyte stimulating (Mlsc) antigens or to self major histocompatibility complex (MHC)-encoded E alpha E beta products did not occur in most athymic mice. Thus, the thymus has a critical role in mediating self tolerance by negative selection.     

Evidence for the effects of a superantigen in rheumatoid arthritis

While studying the alpha beta T cell receptor repertoire in rheumatoid arthritis (RA) patients, we found that the frequency of V beta 14+ T cells was significantly higher in the synovial fluid of affected joints than in the peripheral blood. In fact, V beta 14+ T cells were virtually undetectable in the peripheral blood of a majority of these RA patients. beta-chain sequences indicated that one or a few clones dominated the V beta 14+ population in the synovial fluid of individual RA patients, whereas oligoclonality was less marked for other V beta's and for V beta 14 in other types of inflammatory arthritis. These results implicate V beta 14-bearing T cells in the pathology of RA. They also suggest that the etiology of RA may involve initial activation of V beta 14+ T cells by a V beta 14-specific superantigen with subsequent recruitment of a few activated autoreactive v beta 14+ T cell clones to the joints while the majority of other V beta 14+ T cells disappear.     

Chromosomal location of human T-cell receptor gene Ti beta

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.     

Genes for beta chain of human T-cell antigen receptor map to regions of chromosomal rearrangement in T cells

The T-cell antigen receptor is a cell-surface molecule that participates in the immune response. In the present experiments the genes encoding the beta chain of the T-cell receptor were found to reside on the long arm of human chromosome 7 at or near band q32. Related sequences were found on the short arm of chromosome 7 in bands p15-21 in some experiments. Chromosomal rearrangements in T-cells from normal individuals and patients with ataxia telangiectasia have previously been observed at and near these map assignments for the beta-chain genes.     

Predominant expression of T cell receptor V alpha 7 in tumor-infiltrating lymphocytes of uveal melanoma

Expression of T cell receptor (TCR) V alpha genes in tumor-infiltrating lymphocytes (TILs) within intraocular melanoma was studied. Primers for 18 different human TCR V alpha families were used to analyze TCR V alpha-C alpha gene rearrangements in TIL in these melanomas obtained at surgery. A limited number of TCR V alpha genes were expressed and rearranged in these tumors, and TILs expressing V alpha 7 were found in seven of eight of these uveal melanomas. TCR gene usage is also restricted in experimental autoimmune disease, in T cells within organs like skin and other epithelial tissues, and in the brain of patients with multiple sclerosis (MS). The restricted usage of TCR genes in TIL may indicate that a specific antigen in these melanomas is targeted.     

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.