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.     

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.     

Inversion of chromosome 14 marks human T-cell chronic lymphocytic leukaemia

Rare cases of chronic lymphocytic leukaemia (CLL) in man stem from the malignant proliferation of T cells. The disease is usually more aggressive clinically than B-cell-derived CLL. Various haematological tumours are associated with specific chromosome aberrations (for example, refs 1, 2). Only limited numbers of T-cell CLL patients have so far been studied cytogenetically and, whereas chromosome 12 seems particularly to be involved in B-cell CLL, several markers have been found in T-cell tumours. Recently, by stimulating malignant clones with different mitogens, novel chromosome abnormalities have been detected in T-cell CLL. Using the same approach for additional cases of T-cell CLL, we now report that the most consistent chromosome change is an inversion of the long arm of chromosome 14, inv(14)(q11 q32), in four of five patients. Another remarkable chromosome aberration is trisomy for the long arm of chromosome 8, found in three of five patients.     

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.     

Identification of reciprocal translocation sites within the c-myc oncogene and immunoglobulin mu locus in a Burkitt lymphoma

The association between certain human tumours and characteristic chromosomal abnormalities has led to the hypothesis that specific cellular oncogenes may be involved and consequently 'activated' in these genetic recombinations. This hypothesis has found strong support in the recent findings that some cellular homologues of retroviral onc genes are located in chromosomal segments which are affected by specific tumour-related abnormalities (see ref. 4 for review). In the case of human undifferentiated B-cell lymphoma (UBL) and mouse plasmacytomas, cytogenetic and chromosomal mapping data have identified characteristic chromosomal recombinations directly involving different immunoglobulin genes and the c-myc oncogene (for review see refs 5, 6). In UBLs carrying the t(8:14) translocation it has been shown that the human c-myc gene is located on the region of chromosome 8 (8q24) which is translocated to the immunoglobulin heavy-chain locus (IHC) on chromosome 14. Although it is known that the chromosomal breakpoints can be variably located within or outside the c-myc locus and within the IHC mu (refs 9, 11) or IHC gamma locus, the recombination sites have not been exactly identified and mapped in relation to the functional domains of these loci. We report here the identification and characterization of two reciprocal recombination sites between c-myc and IHC mu in a Burkitt lymphoma. Nucleotide sequencing of the cross-over point joining chromosomes 8 and 14 on chromosome 14q--shows that the onc gene is interrupted within its first intron and joined to the heavy-chain mu switch region. This recombination predicts that the translocated onc gene would code for a rearranged mRNA but a normal c-myc polypeptide.     

Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) chromosome translocation

The t(11;14) (q13;q32) chromosome translocation has been reported in diffuse small and large cell lymphomas and in chronic lymphocytic leukaemia (B-CLL) and multiple myeloma. Because chromosome band 14q32 is involved in this translocation, as well as in the t(8;14) (q24;q32) translocation of the Burkitt tumour, interruption of the immunoglobulin heavy-chain locus was postulated for this rearrangement. We have cloned the chromosomal joinings between chromosomes 11 and 14 and also between chromosomes 14 and 18, in B-cell tumours carrying translocations involving these chromosomes, and suggested the existence of two translocated loci, bcl-1 and bcl-2, normally located on chromosomes 11 (band q13) and 18 (band q21) respectively, involved in the pathogenesis of human B-cell neoplasms. The results indicate that in the leukaemic cells from two different cases of CLL, the breakpoints on chromosome 11 are within 8 nucleotides of each other and on chromosome 14 involve the J4-DNA segment. Because we detected a 7mer-9mer signal-like sequence with a 12-base-long spacer on the normal chromosome 11, close to the breakpoint, we speculate that the t(11;14) chromosome translocation in CLL may be sequence specific and may involve the recombination system for immunoglobulin gene segment (V-D-J) joining.     

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.     

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.     

Immunoglobulin-like nature of the alpha-chain of a human T-cell antigen/MHC receptor

Although the receptor with which T cells bind specific antigen can, like immunoglobulin, distinguish between antigens which differ only slightly in structure, it is unique in recognizing antigen only in conjunction with one of the self proteins of the major histocompatibility complex (MHC restriction). The receptor was identified and characterized in mouse and man by using monoclonal antibodies to receptor idiotypes, and consists of two disulphide-linked polypeptides, and acidic alpha-chain and a neutral to slightly basic beta-chain. Peptide maps have shown that, like immunoglobulin, both chains vary for receptors of different specificities. T-cell-derived cDNA clones have recently been identified in mouse and man encoding immunoglobulin-like molecules. These were identified as derived from beta-chain genes through a partial N-terminal protein sequence of the beta-chain isolated from a human T-cell tumour. We have now purified the alpha- and beta-chains of the receptor of the human T-cell leukaemia line HPB-MLT, and have determined the amino acid sequence of several tryptic peptides derived from each chain. Our results further confirm that the previously reported cDNA clones encode beta-chains. The sequence of the alpha-chain peptides identify this as another immunoglobulin-like polypeptide chain. Particularly striking was an alpha-chain peptide with high homology to the conserved portion of the immunoglobulin J segment and T-cell receptor beta-chains. Surprisingly, the alpha-chain peptides show little similarity to the sequence predicted by two overlapping putative murine alpha-chain cDNA clones.     

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.     

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.     

T-cell receptors of human suppressor cells

Cells which can suppress the immune response to an antigen (TS cells) appear to be essential for regulation of the immune system. But the characterization of the TS lineage has not been extensive and many are sceptical of studies using uncloned or hybrid T-cell lines. The nature of the antigen receptor on these cells is unclear. T cells of the helper or cytotoxic lineages appear to recognize their targets using the T-cell receptor (TCR) alpha beta-CD3 complex. TCR beta-gene rearrangements are also found in some murine and human suppressor cell lines but others have been shown not to rearrange or express the beta-chain or alpha-chain genes. We previously established TS clones derived from lepromatous leprosy patients which carry the CD8 antigen and recognize antigen in the context of the major histocompatibility complex (MHC) class II molecules in vitro. We here report the characterization of additional MHC-restricted TS clones which rearrange TCR beta genes, express messenger RNA for the alpha and beta chains of the TCR and express clonally unique CD3-associated TCR alpha beta structures on their cell surface but do not express the gamma chain of the gamma delta TCR on the cell surface. We conclude that antigen recognition by at least some human CD8+ suppressor cells is likely to be mediated by TCR alpha beta heterodimers.     

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.     

Correlation between immunoglobulin light chain expression and variant translocation in Burkitt's lymphoma

Burkitt's-type lymphomas-leukaemias (BL) are monoclonal proliferations of malignant B lymphocytes. Irrespective of whether they carry the Epstein-Barr virus (EBV) genome, these tumour cells have been shown consistently to have one of the specific reciprocal chromosome translocations, t(8; 14), t(2; 8) or t(8; 22), involving the long arm of chromosome 8 (on 8q24) and chromosome 14, 2 or 22 (on 14q32, 2p12 and 22q11, respectively). The latter chromosomes have been shown recently to carry genes for immunoglobulin (Ig) heavy chains, and kappa and lambda light chains, respectively. Furthermore, the localization of kappa light chains within 2pcen-2p13 encompasses the breakpoint observed in Burkitt's translocation (2p12). It was therefore considered of interest to determine whether the expression of immunoglobulin chains in BL cells is related to the type of chromosomal anomalies observed. We report here that there is a direct relationship between expression of immunoglobulin light chains and specific type of translocation: BL cells with t(8; 22) express lambda chains, whereas those with t(2; 8) express kappa chains.     

T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria

Endemic Burkitt's lymphoma, a tumour of children in which B lymphocytes are infected with Epstein-Barr virus (EBV), is common in areas of Africa where malaria is holoendemic. The tumour is characterized by chromosome translocations; usually the terminal portion of chromosome 8 containing the c-myc gene is translocated to chromosome 14, near the enhancer of the immunoglobulin heavy-chain locus. Less frequent are translocations of chromosome 8 to the kappa light-chain locus of chromosome 2 or to the lambda light-chain locus of chromosome 22. In vitro, EBV induces B cells to proliferate and secrete immunoglobulin and antibody. However, in vivo the infected B lymphocytes are under immunological control, so that abnormal proliferation is found only in immunosuppressed patients. Such patients are subsequently liable to develop lymphomas. Burkitt believed that the tumour he had described resulted from interaction between a virus(es) and a "reticuloendothelial system altered by chronic and heavy infection by malarial or other parasites". We report here that during an attack of Plasmodium falciparum malaria, T-cell subpopulations are radically altered so that, in vitro, B lymphocytes infected with EBV proliferate abnormally to secrete large amounts of immunoglobulin and antibody. This phenomenon offers some explanation for the increased incidence of Burkitt's tumour and the high levels of immunoglobulin found in people living in areas where P. falciparum malaria is common.     

CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist alpha-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR alpha-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR beta-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.     

Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor.

The T-cell receptor is necessary and sufficient for recognition of peptides presented by major histocompatibility complex molecules. Other adhesion molecules, like CD4 or CD8, play an auxiliary role in antigen recognition by T cells. Here we analyse T-cell receptor (TCR) binding using a soluble rather than a cell-bound receptor molecule. A TCR-immunoglobulin chimaera is constructed with the variable and the first constant regions of both the TCR alpha- and beta-chains linked to the immunoglobulin light-chain constant regions. This soluble TCR is expressed, assembled and secreted as an alpha beta heterodimer by a myeloma cell line transfected with the recombinant genes. Furthermore, the soluble TCR is biologically active: it specifically inhibits antigen-dependent activation of the relevant T-cell clones and thus discriminates between proper and irrelevant peptides presented by major histocompatibility complex molecules.     

Deletion of the human T-cell receptor delta-gene by a site-specific recombination

The newly described T-cell receptor (TCR) delta locus is located inside the TCR alpha locus, between variable region (V)alpha and joining region (J)alpha. Although the delta and alpha TCR genes are physically linked on the same chromosome, they are sequentially expressed during T-cell development. This implies the existence of a highly efficient regulatory mechanism by which these two genes are independently rearranged. We have recently described a genetic element 'T early alpha' (TEA) in humans transcribed in foetal thymocytes, spliced alternatively to constant region (C)alpha, and located between the TCR-delta locus (5') and the group of J alpha segments (3'). Importantly, TEA flanks a common site of rearrangement in the thymus, and distinguishes cells using TCR-gamma/delta (TEA in germline configuration) from cells using TCR-alpha/beta (TEA deleted on both chromosomes). In order to understand this TEA-associated recombination we analysed genomic clones representing these thymic rearrangements. We show that the TEA-associated recombination deletes the delta locus before productive (V delta D delta J delta) rearrangement. The diversity (D)delta and J delta regions, which provide the major source of delta gene diversity, are eliminated as a consequence of delta gene deletion and cannot then be used in conjunction with an alpha-TCR. We propose that the TEA-associated deletion of TCR-delta precedes the formation of an alpha-TCR and could down-regulate TCR-delta formation in maturing thymus.     

The murine T-cell receptor uses a limited repertoire of expressed V beta gene segments

Only 10 different V beta gene segments were found when the sequences of 15 variable (V beta) genes of the mouse T-cell receptor were examined. From this analysis we calculate that the total number of expressed V beta gene segments may be 21 or fewer, which makes the expressed germline V beta repertoire much smaller than that of immunoglobulin heavy-chain or light-chain genes. We suggest that beta-chain somatic diversification is concentrated at the V beta-D beta-J beta junctions.