T-cell-specific deletion of T-cell receptor transgenes allows functional rearrangement of endogenous alpha- and beta-genes

In B cells the loci encoding immunoglobulin chains usually show allelic exclusion; a given B cell transcribes and translates only one productively rearranged allele of the heavy and light chain loci. This ensures that each B cell expresses only one antigen receptor. The loci encoding T-cell receptor (TCR) alpha- and beta-genes may behave similarly. We have previously reported that the expression of a transgenic TCR beta-chain prevents functional and nonfunctional V beta rearrangements in the endogenous beta-chain loci but not D beta J beta rearrangements. We have also been unable to detect the expression of the TCR gamma-chain locus in thymocytes of these mice (unpublished observations). To study the mechanisms involved in forming a mature T-cell repertoire further, we have constructed mice expressing alpha- and beta-TCR transgenes derived from a cytotoxic T-cell clone that is specific for the male antigen H-Y in the context of H-2Db MHC molecules. Here we show that in these mice rearrangement of endogenous alpha-chain loci is also suppressed, although to a lesser extent than rearrangement of beta-chain loci. In addition, in male alpha beta TCR transgenic mice we observed T-cell clones which had deleted both transgenic alpha- and beta-chain genes and expressed endogenous alpha- and beta-chain TCR genes. These cells are presumably derived from rare thymocytes that leave the male thymus because their TCR no longer recognizes self antigen. The vast majority of CD4+8+ nonmature thymocytes expressing alpha- and beta-transgenes are deleted in the male thymus.     

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.     

A functional T3 molecule associated with a novel heterodimer on the surface of immature human thymocytes

The known T-cell receptors (TCRs) involved in the recognition of antigen and major histocompatibility complex (MHC) molecules are glycoproteins comprised of polymorphic disulphide-linked alpha- and beta-chains. The genes encoding these chains are homologous to immunoglobulin genes and consist of V (variable), J (joining) and C (constant) regions that rearrange during development. TCRs are expressed relatively late in thymocyte development and only in association with an invariant molecular complex of proteins termed T3. Immature thymocytes do not express the TCR-T3 complex but do express messenger RNA encoding a third rearranging T-cell receptor-like gene, termed T gamma. Here we report a clone of normal immature T4-T8- human thymocytes, designated CII, which does not express mature mRNA for T alpha or T beta genes, but does express high levels of T gamma mRNA. This clone also expresses high levels of surface T3, and antibodies to T3 induce immunologically relevant functions in CII cells. Immunoprecipitation of CII surface-labelled proteins with anti-T3 co-precipitates a T3 molecular complex together with two additional and novel peptides of relative molecular mass (Mr), 44,000 (44K) and 62,000 (62K).     

Developmental regulation of T-cell receptor gene expression

In contrast to B cells or their antibody products, T lymphocytes have a dual specificity, for both the eliciting foreign antigen and for polymorphic determinants on cell surface glycoproteins encoded in the major histocompatibility complex (MHC restriction). The recent identification of T-cell receptor glycoproteins as well as the genes encoding T-cell receptor subunits will help to elucidate whether MHC proteins and foreign antigens are recognized by two T-cell receptors or by a single receptor. An important feature of MHC restriction is that it appears to be largely acquired by a differentiating T-cell population under the influence of MHC antigens expressed in the thymus, suggesting that precursor T cells are selected on the basis of their reactivity with MHC determinants expressed in the host thymus. To understand this process of 'thymus education', knowledge of the developmental regulation of T-cell receptor gene expression is necessary. Here we report that whereas messenger RNAs encoding the beta-and gamma-subunits are relatively abundant in immature thymocytes, alpha mRNA levels are very low. Interestingly, whereas alpha mRNA levels increase during further development and beta mRNA levels stay roughly constant, gamma mRNA falls to very low levels in mature T cells, suggesting a role for the gamma gene in T-cell differentiation.     

Transfer of specificity by murine alpha and beta T-cell receptor genes

T-cell receptor alpha- and beta-chain genes were isolated from a class I major histocompatibility complex-restricted cytotoxic T-cell clone and transferred by protoplast fusion into another cytolytic T-cell clone of different specificity. Expression of the transfected alpha and beta genes endowed the recipient cell with the specificity of the donor cell.     

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.     

The human T-cell receptor alpha-chain gene maps to chromosome 14

The T-cell receptor for antigen has been identified as a disulphide-linked heterodimeric glycoprotein of relative molecular mass (Mr) 90,000 comprising an alpha- and a beta-chain. The availability of complementary DNA clones encoding mouse and human beta-chains has allowed a detailed characterization of the genomic organization of the beta-chain gene family and has revealed that functional beta-chain genes in T cells are generated from recombination events involving variable (V), diversity (D), joining (J) and constant (C) gene segments. Recently, cDNA clones encoding mouse and human alpha-chains have been described; the sequences of these clones have indicated that functional alpha-chain genes are also generated from multiple gene segments. It is possible that chromosomal translocations involving T-cell receptor alpha- and beta-chain genes have a role in T-cell neoplasms in much the same way as translocations involving immunoglobulin genes are associated with oncogenic transformation in B cells. In the latter case, the chromosomal localization of the immunoglobulin genes provided one of the first indications of the involvement of such translocations in oncogenic transformation. The chromosomal assignment of the alpha- and beta-chain genes may, therefore, provide equally important clues for T-cell neoplastic transformation. The chromosomal location of the mouse and human beta-chain gene family has been determined: the murine gene lies on chromosome 6 (refs 12, 13) whereas the human gene is located on chromosome 7 (refs 13, 14). Here we use a cDNA clone encoding the human alph-chain to map the corresponding gene to chromosome 14.     

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.     

Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) gamma-gene has recently been found to be expressed on a subset of both peripheral cells and thymocytes. As an initial approach to understanding the role of this gamma-chain of TCR (TCR gamma) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a gamma-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR delta). This TCR gamma delta is first detected approximately two days before the appearance of cell-surface TCR alpha beta heterodimers. We report that N-glycosidase digestions reveal that all of the gamma-protein expressed on fetal thymocytes, as in adult CD4-8-(L3T4-, Lyt2-) thymocytes, bear N-linked carbohydrate side chains. The major gamma-gene transcribed in mature, alpha beta-bearing T cells (V gamma 1.2C gamma 2)encodes no N-linked glycosylation site so these results suggest that the fetal gamma delta receptor defines a distinct T-cell lineage whose development in the thymus precedes classical alpha beta-bearing cells.     

The adult T-cell receptor delta-chain is diverse and distinct from that of fetal thymocytes

T lymphocytes recognize foreign molecules using the T-cell receptor (TCR), a disulphide-linked heterodimer closely associated with the CD3 polypeptide complex on the cell surface. The TCR alpha beta heterodimers seem largely responsible for the recognition properties of both helper (TH) and cytotoxic (TC) T cells. Recently, a second CD3-associated T-cell receptor heterodimer, gamma delta, has been described. Cells bearing the gamma delta receptor appear before those bearing alpha beta during thymic ontogeny and persist as a minor component (1-10%) of mature peripheral T cells. Their function is unknown. As there are a limited number of functional TCR V gamma gene segments, the size and potential diversity of the V delta repertoire is important for the number of different antigens that may be recognized by gamma delta heterodimers. The delta-chain locus is located 75 kilobases (kb) 5' to the TCR C alpha coding region, raising the possibility that the alpha and delta V-region repertoires may overlap. Also, analysis of rearrangements at the delta-chain locus in developing thymocytes shows distinct fetal and adult patterns indicating that there may be differences between the fetal and adult V delta repertoires. To address these questions, we have characterized a large number of delta-containing complementary DNA clones from adult double-negative thymocytes (CD4-8-), an immature population that is enriched for gamma delta-bearing cells. We find that a limited number of V delta sequences are used, showing little overlap with known adult V alpha s and differing significantly from fetal V delta s. But as two D elements may participate simultaneously in V delta gene assembly, and random nucleotides may be added at any one of three junctional points, the potential number of different delta chains that can be made in the adult thymus is very large (approximately 10(13)).     

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.     

Self-tolerance to transgenic gamma delta T cells by intrathymic inactivation

During their intrathymic differentiation, T lymphocytes expressing alpha beta T-cell receptors (TCR) are negatively and positively selected. This selection contributes to the establishment of self-tolerance and ensures that mature CD4+ and CD8+ cell populations are restricted by the self major histocompatibility complex. Little is known, however, about gamma delta T-cell development. To investigate whether selection operates in the establishment of the gamma delta T-cell class, we have generated transgenic mice using gamma- and delta-transgenes encoding a TCR that is specific for a product of a gene in the TL-region of the TLb haplotype. Similar numbers of thymocytes expressing the transgenic TCR were generated in mice of TLb and TLd haplotypes. But gamma delta thymocytes from TLb and TLd transgenic mice differed in cell size, TCR density and in their capacity to respond to TLb stimulator cells or interleukin-2 (IL-2). In contrast to gamma delta T cells from TLd transgenic mice, gamma delta T cells from TLb transgenic mice did not produce IL-2 and did not proliferate in response to TLb stimulator cells, but they did proliferate in the presence of exogenous IL-2. These results indicate that functional inactivation of self-antigen-specific T cells could contribute to the establishment of self-tolerance to thymic determinants.     

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.     

Presence of Ti (WT31) negative T lymphocytes in normal blood and thymus

The antigen receptor expressed on most T lymphocytes is a disulphide-linked heterodimer (Ti) that is composed of alpha-chain and beta-chain subunits. On the surface of human T lymphocytes, Ti is non-covalently associated with three invariant proteins, designated CD3-gamma, -delta, and -epsilon. It has been suggested that Ti is obligatory for CD3 expression. But a T leukaemia cell line, IL-2 (interleukin 2) dependent T-cell clones established from fetal blood and IL-2 dependent cell lines established from immunodeficiency patients with bare lymphocyte syndrome and ectodermal dysplasia syndrome have recently been shown to express CD3, but not Ti (detected due to monoclonal antibody WT31). These lymphocytes may express the product of the T-cell antigen receptor gamma (TCR-gamma) gene, rather than the alpha/beta heterodimer, in association with CD3. Preliminary studies suggested that T cells expressing CD3 but lacking Ti are present in low frequency in normal lymphoid tissues. Here we show that in normal blood and thymus CD3+, WT31-T cells express neither CD4 nor CD8. The low frequency (less than 0.2-0.9% of total thymocytes) of CD3+, WT31- cells in the thymus suggests that this population does not represent a major stage of thymic development and may be a distinct lineage of T cells.     

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.     

A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family

Recent studies have demonstrated that CD3 is expressed on a subset of thymocytes with a CD4-CD8- (double negative) phenotype. At least some of these cells bear the CD3-associated gamma delta T-cell receptor (TCR gamma delta). Here we describe a second subset of double negative thymocytes which expresses CD3-associated alpha beta receptors (TCR alpha beta). Surprisingly, these cells express predominantly the products of a single V beta gene family (V beta 8). These CD4-CD8-, TCR alpha beta+ cells appear relatively late in ontogeny (between birth and day 5 of life) and thus are unlikely to be the precursors to the TCR alpha beta-bearing cells (CD4+CD8- and CD4-CD8+) already present at birth. They can be selectively expanded in vitro by stimulation with a monoclonal antibody to V beta 8 (F23.1) in the presence of interleukin I (IL-1). We propose that this cell type is a unique T-cell population distinguishable from typical TCR alpha beta+ T cells by its CD4-CD8- phenotype and a restricted TCR V beta repertoire. Analysis of the unique phenotype of these cells suggests that they may represent the normal counterpart of the defective CD4-CD8- T cells found in the lpr autoimmune mouse.