Retroviral transduction of T-cell antigen receptor beta-chain and myc genes

Support for multistage models of oncogenesis has been provided by several highly leukaemogenic retrovirus isolates that have transduced more than one host cell gene. Where functional studies have been performed, these retroviral oncogenes show synergy for in vitro transformation and leukaemogenesis. In naturally occurring feline leukaemias associated with feline leukaemia virus (FeLV), retroviral transduction of myc is a frequent oncogenic mechanism. But evidence suggesting that the FeLV v-myc genes might be insufficient for leukaemogenesis was provided by the latency (12 weeks) and clonality of FeLV/v-myc-induced tumours and the absence of demonstrable in vitro transformation by these viruses. In the search for secondary leukaemogenic events in FeLV/v-myc tumours, we have identified a case of FeLV transduction of a T-cell antigen receptor beta-chain gene. The proviruses carrying this gene (which we have named v-tcr) were a separate population from those carrying v-myc. In its normal role, the T-cell receptor beta-chain forms part of a multimeric complex involved in antigen recognition and T-cell activation. We suggest that v-tcr is a novel viral oncogene which assisted v-myc in the genesis of a naturally occurring case of thymic lymphosarcoma.     

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.     

Structure, expression and divergence of T-cell receptor beta-chain variable regions

Analysis of three new T-cell receptor beta-chain variable regions together with those in the literature indicates that they have both remarkable similarities and differences with those of immunoglobulin. Less than 10 V regions appear to predominate in the thymus. V beta sequences are much more heterogeneous at the amino acid level than are immunoglobulin V regions and they appear to diverge between species much more quickly, apparently the result of additional hypervariable regions. Three of these putative new hypervariable regions lie outside of the classical immunoglobulin binding site, an indication that important interactions may be occurring in these regions with polymorphic MHC determinants.     

Preferential expression of a defined T-cell receptor beta-chain gene in hapten-specific cytotoxic T-cell clones

A multitude of different antigens can be recognized by T cells through specific receptors. Both the alpha- and beta-chains of the T-cell receptor contribute to the antigen recognition portion. The repertoire of beta-chain variable region (V beta) gene segments is limited to some 20 elements which seem to be used randomly in different T cells. Diversity at the beta-chain level can be created in several ways: a multiplicity of germline gene segments; combinatorial diversity by rearranging different V, diversity (D), joining (J) and constant (C) region elements; junctional diversity by joining gene segments at different sites; N-region diversity, that is, insertion of random nucleotides at junctional sites; and somatic mutation. However, the major sources and the extent of diversity of the T-cell receptor are unclear. To address this issue, 42 H-2Kb-restricted, 2,4,6-trinitrophenyl (TNP)-specific cytotoxic T-cell (Tc) clones from C57BL/6 mice were characterized with respect to expression of different beta-chain gene segments in messenger RNA using specific oligonucleotide probes. We report here that nearly half of the Tc clones use identical elements for productive beta-chain gene rearrangement. Thus, there is a restriction in the use of beta-chain gene segments in this panel of Tc clones which favours a particular V beta--D beta--J beta--C beta combination with a defined D beta element.     

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.     

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.     

T-cell antigen receptor genes and T-cell recognition

The four distinct T-cell antigen receptor polypeptides (alpha, beta, gamma, delta) form two different heterodimers (alpha:beta and gamma:delta) that are very similar to immunoglobulins in primary sequence, gene organization and modes of rearrangement. Whereas antibodies have both soluble and membrane forms that can bind to antigens alone, T-cell receptors exist only on cell surfaces and recognize antigen fragments only when they are embedded in major histocompatibility complex (MHC) molecules. Patterns of diversity in T-cell receptor genes together with structural features of immunoglobulin and MHC molecules suggest a model for how this recognition might occur. This view of T-cell recognition has implications for how the receptors might be selected in the thymus and how they (and immunoglobulins) may have arisen during evolution.     

Thymocyte subpopulation enriched for progenitors with an unrearranged T-cell receptor beta-chain gene

The development of T cells within the thymus is not well understood. It is known that thymocytes are derived from a progenitor cell in the bone marrow, the prothymocyte, and that cells in the subcapsular area of the thymus can give rise to progeny in both the cortex and the medulla. However, it is not clear whether all medullary thymocytes are necessarily derived from cortical cells. In particular, it has been difficult to distinguish intrathymic progenitor cells. Recently, however, Lesley et al. have defined a thymocyte subpopulation which can be isolated by treatment of the thymus with cytotoxic anti-Thy-1 antibodies and that seems to be enriched for thymocyte progenitors as measured first by its ability to repopulate transiently the thymus of an irradiated host, and second, by its high content of cells bearing Pgp-1 (refs 10, 11), a cell-surface glycoprotein of relative molecular mass 95,000 that is present on most or all prothymocytes of the bone marrow and on fetal thymocytes, but on only a few per cent of cells in the adult thymus. We show here that the gene encoding the beta-chain of the T-cell receptor for antigen, which is rearranged during T-cell ontogeny, is predominantly in the germline configuration in these cells.     

Analysis of specificity for antigen, Mls, and allogenic MHC by transfer of T-cell receptor alpha- and beta-chain genes

The majority of peripheral T lymphocytes bear cell-surface antigen receptors comprised of a disulphide-linked alpha beta dimer. In an immune response, this receptor endows T cells with specificities for foreign antigenic protein fragments bound to cell surface glycoproteins encoded in the major histocompatibility complex (MHC). At a high frequency (greater than 1%), the same population of T lymphocytes responds to allogeneic MHC glycoproteins, or to differences at other genetic loci termed Mls, in conjunction with MHC. The alpha beta-antigen receptor has been implicated in alloreactivity and Mls reactivity. In fact, many monoclonal T-cell lines recognize a foreign protein fragment bound to self-MHC molecules and, in addition, recognize allogeneic MHC glycoproteins, an Mls-encoded determinant, or both. For at least one T-cell clone, a monoclonal antibody directed against the alpha beta antigen receptor has been shown to block activation induced by either antigen-bound self-MHC or by allogeneic MHC. However, it remains to be demonstrated directly that a single alpha beta receptor can mediate antigen specificity, alloreactivity and Mls reactivity, a prerequisite to understanding the structural basis of these high-frequency cross-reactivities. To address this issue we have performed transfers of receptor chain genes from a multiple-reactive T-cell clone into an unrelated host T lymphocyte. We now demonstrate definitively that the genes encoding a single alpha beta-receptor chain pair can transfer the recognition of self-MHC molecules complexed with fragments of antigen, allogeneic MHC molecules, and an Mls-encoded determinant (presumably in conjunction with MHC). In this case the transfer of antigen specificity and alloreactivity requires a specific alpha beta-receptor chain combination, whereas Mls reactivity can be transferred with the beta-chain gene alone into a recipient expressing a randomly selected alpha-chain.     

Predictable acquisition of a new MHC recognition specificity following expression of a transfected T-cell receptor beta-chain gene

Activation of mature T lymphocytes requires specific corecognition of antigen together with membrane-associated glycoprotein products of the major histocompatibility complex (MHC). This dual specificity is determined by a single receptor structure consisting of a clone-specific alpha beta heterodimer. Because both the alpha and beta subunits possess unique combining-site-containing V regions, it remains an open issue as to what contribution each of the two chains of the receptor makes to the antigen versus MHC recognition specificities of the complete dimer present on any given T cell or in the T-cell pool as a whole. In the present work, we have used DNA-mediated gene transfer to express a new alpha or beta chain in a recipient murine T-cell hybridoma possessing a related antigen but distinct MHC specificity compared to the receptor-gene donor. Our results demonstrate that a beta-gene transfected hybridoma expresses new receptors with a predictable hybrid specificity, establishing that the beta chain has the predominant role in MHC molecule recognition in this model.     

Breakpoints in the human T-cell antigen receptor alpha-chain locus in two T-cell leukaemia patients with chromosomal translocations

Specific chromosomal translocations have been observed in several human and animal tumours and are believed to be important in tumorigenesis. In many of these translocations the breakpoints lie near cellular homologues of transforming genes, suggesting that tumour development is partly due to the activation of these genes. The best-characterized example of such a translocation occurs in mouse plasmacytoma and human B-cell lymphoma, where c-myc, the cellular homologue of the viral oncogene myc, is brought into close proximity with either the light- or heavy-chain genes of the immunoglobulin loci, resulting in a change in the regulation of the myc gene. T-cell malignancies also have characteristic chromosomal abnormalities, many of which seem to involve the 14q11-14q13 region. This region has recently been found to contain the alpha-chain genes of the human T-cell antigen receptor. Here we determine more precisely the chromosome breakpoints in two patients whose leukaemic T cells contain reciprocal translocations between 11p13 and 14q13. Segregation analysis of somatic cell hybrids demonstrates that in both patients the breakpoints occur between the variable (V) and constant (C) region genes of the T-cell receptor alpha-chain locus, resulting in the translocation of the C-region gene from chromosome 14 to chromosome 11. As the 11p13 locus has been implicated in the development of Wilms' tumour, it is possible that either the Wilms' tumour gene or a yet unidentified gene in this region is involved in tumorigenesis and is altered as a result of its translocation into the T-cell receptor alpha-chain locus.     

Correlations between T-cell specificity and the structure of the antigen receptor

The derived amino-acid sequences of the heterodimeric antigen receptors expressed by a series of murine T-cell clones are presented. A comparison of the receptor sequences indicates that several mechanisms for generating receptor diversity can influence T-cell specificity, including junctional diversity, combinatorial joining, and combinatorial chain associations.     

Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen

The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.