Unusual organization and diversity of T-cell receptor alpha-chain genes

T lymphocytes recognize cell-bound antigens in the molecular context of the self major histocompatibility complex (MHC) gene products through the surface T-cell receptor(s). The minimal component of the T-cell receptor is a heterodimer composed of alpha and beta subunits, each of relative molecular mass (Mr) approximately 45,000 (refs 1-3). Recently, complementary DNA clones encoding these subunits have been isolated and characterized along with that of a third subunit of unknown function, termed gamma (refs 4-9). These studies revealed a primary structure for each subunit that was clearly similar to that of immunoglobulin and indicated a somatic rearrangement of corresponding genes that are also immunoglobulin-like. Recently, the analysis of the sequence organization of the T-cell receptor beta-chain and T-cell-specific gamma-chain gene families has been reported. We now present an initial characterization of the murine T-cell receptor alpha-chain gene family, and conclude that although it is clearly related to the gene families encoding immunoglobulins, T-cell receptor beta-chains and also T-cell gamma-chains, it shows unique characteristics. There is only a single constant (C) region gene segment, which is an exceptionally large distance (approximately 20-40 kilobases (kb) in the cases studied here) from joining (J) gene segments. In addition, the J cluster and the variable (V) segment number seen to be very large. Finally, in the case studied here, a complete alpha-chain gene shows no somatic mutation and can be assembled directly from V alpha, J alpha and C alpha segments without inclusion of diversity (D alpha) segments.     

Conserved organization of the human and murine T-cell receptor beta-gene families

Generation of an immune response depends on the interaction of haematopoietic cell types, among which T cells and their receptors are of central importance. The T-cell receptor is a heterodimer consisting of disulphide-linked alpha and beta-chains, each chain divided into variable (V) and constant (C) regions. The beta-chain is encoded by the rearrangement of separate variable (V beta), diversity (D beta) and joining (J beta) gene segments during T-cell differentiation. To examine the mechanisms of somatic DNA rearrangement and evolution of the beta-gene segments, we have constructed a physical map of the human T-cell receptor beta-chain family containing 40 V beta gene segments as well as both C beta gene clusters. A comparison of the published nucleotide sequences of human and murine V beta gene segments reveals 12 examples of gene segments sharing 65% or more interspecies homology. The relative order of these human and murine V beta gene segment homologues is also conserved along the chromosome, apart from more extensive human gene duplication, presumably as a consequence of constraints imposed on evolutionary mechanisms operating to diversify these gene families or of selective pressures operating to maintain order.     

Expression of the T-cell-specific gamma gene is unnecessary in T cells recognizing class II MHC determinants

Subtractive complementary DNA cloning combined with partial protein sequencing has allowed identification of the genes encoding the alpha and beta subunits of T-cell receptors. The subtractive cDNA library prepared from the cytotoxic T lymphocyte (Tc) clone 2C has been found to contain a third type of clone encoding the gamma chain. The gamma gene shares several features with the alpha and beta genes: (1) assembly from gene segments resembling immunoglobulin V, J and C (respectively variable, joining and constant region) DNA segments; (2) rearrangement and expression in T cells and not in B cells; (3) sequences reminiscent of transmembrane and intracytoplasmic regions of integral membrane proteins; (4) a cysteine residue at the position expected for an interchain disulphide bond. The alpha and beta genes are expressed at equivalent levels in both Tc cells and helper T cells (TH). The gamma gene, obtained from 2C, has been found to be expressed in all Tc cells studied. Here we present evidence that strongly suggests that TH cells do not require gamma gene expression.     

The structure, rearrangement and expression of D beta gene segments of the murine T-cell antigen receptor

It has been postulated that the variable region of the beta-polypeptide of the murine T-cell antigen receptor is encoded by three distinct germ-line gene segments--variable (V beta), diversity (D beta) and joining (J beta)--that are rearranged to generate a V beta gene. Germ-line V beta and J beta gene segments have been isolated previously. Here we report the isolation and characterization of two germ-line D beta gene segments that have recognition signals for DNA rearrangement strikingly similar to those found in the three immunoglobulin gene families and in V beta and J beta gene segments. The D beta and J beta segments can join in the absence of V beta gene segment rearrangement and these rearranged sequences are transcribed in some T cells.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

Variability and repertoire size of T-cell receptor V alpha gene segments

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.     

Primary structure of human T-cell receptor alpha-chain

The T-cell receptor has been studied intensely over the past 10 years in an effort to understand the molecular basis for major histocompatibility complex (MHC) restricted antigen recognition. The use of anti-receptor monoclonal antibodies to isolate and characterize the receptor from human and murine T-cell clones has shown that the protein consists of two disulphide-linked glycopeptides, alpha and beta, distinct from known immunoglobulin light and heavy chains. Like immunoglobulin light and heavy chains, however, both the alpha- and beta-chains are composed of variable and constant regions. Molecular cloning has revealed that the beta-chain is evolutionarily related to immunoglobulins, and is encoded in separate V (variable), D (diversity), J (joining) and C (constant) segments that are rearranged in T cells to produce a functional gene. We report here cDNA clones encoding the alpha-chain of the receptor of the human T-cell leukaemia line HPB-MLT. Using these cDNA probes, we find that expression of alpha-chain mRNA and rearrangement of an alpha-chain V-gene segment occur only in T cells. The protein sequence predicted by these cDNAs is homologous to T-cell receptor beta-chains and to immunoglobulin heavy and light chains, particularly in the V and J segments.     

T gamma protein is expressed on murine fetal thymocytes as a disulphide-linked heterodimer

During the search for genes coding for the mouse alpha and beta subunits of the antigen-specific receptor of mouse T cells we encountered a third gene, subsequently designated gamma. This gene has many properties in common with the alpha and beta genes, somatic assembly from gene segments that resemble the gene segments for immunoglobulin variable (V), joining (J) and constant (C) regions; rearrangement and expression in T cells and not in B cells; low but distinct sequence homology to immunoglobulin V, J and C regions; other sequences that are reminiscent of the transmembrane and intracytoplasmic regions of integral membrane proteins; and a cysteine residue at the position expected for a disulphide bond linking two subunits of a dimeric membrane protein. Despite these similarities the gamma gene also shows some interesting unique features. These include a relatively limited repertoire of the germ-line gene segments, more pronounced expression at the RNA level in immature T cells such as fetal thymocytes and an apparent absence of in-frame RNA in some functional, alpha beta heterodimer-bearing T cells or cultured T clones and hybridomas. To understand the function of the putative gamma protein it is essential to define the cell population that expresses this protein. To this end we produced a fusion protein composed of Escherichia coli beta-galactosidase and the gamma-chain (hereafter referred to a beta-gal-gamma) using the phage expression vector lambda gt11 and raised rabbit antisera against the gamma determinants. Using the purified anti-gamma antibody we detected a polypeptide chain of relative molecular mass 35,000 (Mr 35K) on the surface of 16-day old fetal thymocytes. The gamma-chain is linked by a disulphide bridge to another component of 45K. No such heterodimer was detected on the surface of a cytotoxic T lymphocyte (CTL) clone 2C from which an in-phase gamma cDNA clone was originally isolated.     

The gene encoding the T-cell receptor alpha-chain maps close to the Np-2 locus on mouse chromosome 14

Serological and molecular genetic analyses of T-cell clones have shown that the T-cell antigen receptor apparently comprises two glycosylated, disulphide-linked polypeptide chains (alpha and beta), both of which span the cell membrane. Cloning of the genes encoding the two chains from mouse and human DNA has shown that the alpha- and beta-chains are composed of variable (V) and conserved (C) regions in agreement with peptide mapping data. Gene segments encoding variable and conserved domains of the beta-chain have been identified and undergo rearrangements during T-cell differentiation. The genes encoding the alpha-chain, so far described at the level of complementary DNA clones, also identify DNA rearrangements. Thus, the genes encoding the T-cell receptor show the same structure and dynamic behaviour as immunoglobulin genes, indicating that the two gene families belong to the same supergene family; this evolutionary relationship is supported by the fact that the genes encoding the beta-chain of the T-cell receptor are closely linked to immunoglobulin kappa light-chain genes on chromosome 6 in mouse. In man, however, the beta genes map to chromosome 7 (ref. 14) whereas the kappa-chain genes are located on chromosome 2, indicating that linkage between the two gene families is not needed for proper expression. Here we describe genomic clones encoding the constant portion of the T-cell receptor alpha-chain and map the gene to chromosome 14 in mouse, close to the gene for purine nucleoside phosphorylase (Np-2) which, in man, has been associated with T-cell immunodeficiencies.     

Rearrangement and transcription of the beta-chain genes of the T-cell antigen receptor in different types of murine lymphocytes

Rearrangements of T-cell receptor beta-chain genes are usually found on both chromosomal homologues, occurring by both deletional and non-deletional mechanisms. Two constant-region (C beta) genes have been identified previously and at least one is transcribed in every helper or cytotoxic T cell tested, but the choice of C beta gene expression is not correlated with the specialized functions of these T lymphocytes. By contrast, four of five suppressor T-cell hybridomas examined have deleted all known joining (J beta) gene segments and C beta genes and therefore may have antigen receptors encoded by different T-cell receptor gene families.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

Intestinal intraepithelial lymphocytes are a distinct set of gamma delta T cells

Lymphocytes are most reliably subdivided on the basis of their receptors for antigen at the cell surface. Three subtypes of lymphocytes are well defined: B cells that bear surface immunoglobulin and make antibody, CD4+T cells with CD3 alpha beta receptors specific for antigen associated with class II major histocompatibility complex molecules, and CD8+T cells with CD3 alpha beta receptors specific for antigen associated with class I MHC molecules. These T cells are responsible for known forms of cell-mediated immunity. The discovery of a third rearranging T-cell specific gene called gamma (refs 1 and 2) has revealed the presence of a new class of T cells bearing a new receptor type, CD3 gamma delta (refs 3-7). To date, neither the function nor the specificity of cells bearing this receptor has been determined. Because gamma delta T cells are the main lymphocyte of epidermis, it was proposed that such cells could be important in surveillance of all epithelia. We have isolated intraepithelial lymphocytes from murine small intestine, and shown that they predominantly or exclusively express CD3 gamma delta receptors. Unlike the epidermal lymphocytes, these cells also express CD8, and they use a different V lambda gene to form their receptor. This strongly suggests that gamma delta T cells home in a very specific manner to epithelia, where they presumably mediate their function.     

Isolation of an excision product of T-cell receptor alpha-chain gene rearrangements

The genes for the T-cell receptor, like the immunoglobulin genes, are rearranged as DNA. The mechanism of this rearrangement is not clear; unequal crossover between chromosomes and the looping-out and excision of the excess DNA have both been suggested. We isolated small polydisperse circular (spc) DNAs from mouse thymocytes and cloned them into a phage vector. Of the 56 clones we analysed, nine contained sequences homologous to T-cell receptor alpha-chain joining (J alpha) segments. We have characterized one of these clones; it contains one J alpha segment, and the product out of the recombination of a variable region of the alpha-chain gene (V alpha) with a J alpha gene segment. This is the first demonstration of the presence in extrachromosomal DNA of a reciprocal recombination product of any rearranging immunoglobulin or T-cell receptor gene. The finding verifies that V alpha-J alpha joining can occur by the looping-out and excision of chromosomal DNA.     

Diversity of murine gamma genes and expression in fetal and adult T lymphocytes

The search for the genes encoding the T-cell receptor alpha and chains revealed a third gene, T gamma (ref. 1), which shares with t T alpha (refs 2-7) and T beta (refs 8-15) genes a number of structure features, including somatic rearrangement during T-cell development. T gamma gene expression appears to be unnecessary in son mature T cells and is at its greatest in fetal thymocytes encouraging speculation that T gamma has a role in T-cell development and may be involved in the recognition of polymorphic major histocompatibility complex (MHC) products during thymic education. One argument against the participation of T gamma in such a process has been its apparently limited diversity, due to the small number of gene segments available for rearrangement. We here describe the identification of additional T gamma V-gene segments and demonstrate that they can be rearranged to previously identified J- and C-gene segments and are expressed in fetal thymocytes. In addition we describe a variety of patterns of T gamma mRNA processing which may be significant for T gamma gene regulation.     

Organization and sequences of the variable, joining and constant region genes of the human T-cell receptor alpha-chain

An essential property of the immune system is its ability to generate great diversity in antibody and T-cell immune responses. The genetic and molecular mechanisms responsible for the generation of antibody diversity have been investigated during the past several years. The gene for the variable (V) region, which determines antigen specificity, is assembled when one member of each of the dispersed clusters of V gene segments, diversity (D) elements (for heavy chains only) and joining (J) segments are fused by DNA rearrangement. The cloning of the beta-chain of the T-cell antigen receptor revealed that the organization of the beta-chain locus, which is similar to that of immunoglobulin genes, is also composed of noncontiguous segments of V, D, J and constant (C) region genes. The structure of the alpha-chain seems to consist of a V and a C domain connected by a J segment. We report here that the human T-cell receptor alpha-chain gene consists of a number of noncontiguous V and J gene segments and a C region gene. The V region gene segment is interrupted by a single intron, whereas the C region contains four exons. The J segments, situated 5' of the C region gene, are dispersed over a distance of at least 35 kilobases (kb). Signal sequences, which are presumably involved in DNA recombination, are found next to the V and J gene segments.     

Complexity of human T-cell antigen receptor beta-chain constant- and variable-region genes

Immune systems of vertebrates function via two types of effector cells, B and T cells, which are capable of antigen-specific recognition. The immunoglobulins, which serve as antigen receptors on B cells, have been well characterized with respect to gene structure, unlike the T-cell receptors. Recently, cDNA clones thought to correspond to the beta-chain locus of the human and mouse T-cell receptor have been described. The presumptive beta-chain clones detect gene rearrangement specifically in T-cell DNA and show homology with immunoglobulin light chains. The similarity of the T-cell beta-chain gene system to the immunoglobulin genes has been further demonstrated by the recent observation of variable- and constant-region gene segments as well as joining segments and putative diversity segments. We report here the characterization of cDNA and genomic clones encoding human T-cell receptor beta-chain genes. There are two constant-region genes (C beta 1 and C beta 2), each capable of rearrangement and expression as RNA. The gene arrangement, analogous to that of mouse beta-chain genes, shows strong evolutionary conservation of the dual C beta gene system in these two species.     

Distinct antigen receptor repertoires of two classes of murine epithelium-associated T cells

T lymphocytes are found not only as recirculating cells in the lymphoid system, but also as immobile cells in certain epithelia. T-cell antigen receptors (TCR) of both alpha/beta and gamma/delta-heterodimer subtypes can exhibit an extremely high degree of diversity. The diversity of alpha/beta TCRs derives from the use of a large number of variable (V) gene segments, as well as junctional diversity generated during rearrangement of these segments, whereas the diversity of gamma/delta TCRs derives largely from junctional elements, with a smaller contribution from a limited number of V gene segments. Many T cells in the epidermal and intestinal epithelia of mice express TCR composed of gamma/delta heterodimers. We demonstrate here that gamma/delta TCRs of T cells in both these tissues are restricted in V gene usage, with different elements predominating. The TCR junctional diversity of epidermal T cells, however, is extremely limited, whereas that of intestinal T cells is extremely diverse. The distinctive features of these two populations suggest that they develop or are selected differently for particular tissue-specific functions.