Complexity of T cell receptor recognition sites for defined alloantigens

Three monoclonal antibodies react with the T cell receptor on the tumor line HPB-ALL and in addition with 3 to 13% of human peripheral blood T cells of normal donors. These antibodies are shown to react with an epitope encoded by the V beta 5 family of T cell receptor beta-chain variable region gene segments. Cells expressing V beta 5 gene segments can have cytotoxic or helper function, be of the T4+ or T8+ phenotype, and have specificity for either class I or class II major histocompatibility complex alloantigens. Seven T cell clones were generated, which express V beta 5 and are specific for the HLA-A2 molecule. With the use of these clones, we illustrate how isoelectric focusing can be used to analyze T cell receptor alpha- and beta-chain structure. The seven clones recognize five distinct conformational determinants on HLA-A2. They procure different binding sites by the use of different alpha-chains, J beta sequences, or both.     

T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining

We have identified circular DNA containing T cell receptor (TCR) beta gene sequences in mouse thymocytes, thereby providing direct evidence for the intramolecular DNA deletion model of V-D-J joining in TCR beta genes. Two types of excision products of V-D-J joining have been identified. Type I, a circular reciprocal recombinant of normal V-D or D-J joining, contains a 7mer-7mer head-to-head structure expected from an excised product of normal V-D or D-J joining. Type II contains a D beta 2-J beta 1 structure on the circular DNA; the recombination event producing this molecule occurs between an upstream J and a downstream D segment, probably leaving the reciprocal 7mer-7mer structure on the chromosome. Some type I molecules seem to represent excision products of secondary joining after formation of the first D-J or V-D-J structure. The recombination mechanism that generates the circular DNA is discussed.     

Molecular analysis of antigen recognition by insulin-specific T-cell hybridomas from B6 wild-type and bm12 mutant mice.

Molecular analysis of the heterodimeric T-cell antigen receptor of insulin-specific class II-restricted T-cell hybridomas (THys) derived from C57BL/6 (B6) wild-type and B6.C-H-2bm12 (bm12) mutant mice revealed that such T cells use a diverse V gene repertoire. Analysis of three THys that use related V genes, however, showed a number of novel features. Two THys that share major histocompatibility complex restriction use V alpha genes that are 98.6% homologous. Two THys sharing the same antigen fine specificity use a particular germ line V beta D beta J beta combination. A 21-base-pair deletion in the 5' segment of the J beta gene occurs in one THy, suggesting a novel mechanism for generating diversity in T-cell antigen receptor beta genes. The first amino acid encoded by N sequences at the V-D junction is conserved in a pair of T cells which recognize identical antigenic epitopes. The implications of these findings for the structural mechanisms underlying major histocompatibility complex-restricted antigen-specific T-cell recognition are discussed.     

Linkage disequilibrium among RFLPs at the insulin-receptor locus despite intervening Alu repeat sequences.

Multiple mutations of the insulin receptor (INSR) gene have been identified in individuals with extreme insulin resistance. These mutations have included recombination events between Alu repeat units in the tyrosine kinase-encoding beta-chain region of the gene. To evaluate the influence of Alu and dinucleotide repetitive sequences on recombination events within the insulin receptor gene, I examined the degree of linkage disequilibrium between RFLP pairs spanning the gene. I established 228 independent haplotypes for seven RFLPs (two each for PstI, RsaI, and SstI and one for MspI and 172 independent haplotypes which included an additional RFLP with BglII) from 19 pedigrees. These RFLPs span > 130 kb of this gene, and my colleagues and I previously demonstrated that multiple Alu sequences separate RFLP pairs. Observed haplotype frequencies deviated significantly from those predicted. Pairwise analysis of RFLP showed high levels of linkage disequilibrium among RFLP in the beta-chain region of the insulin receptor, but not between alpha-chain RFLPs and those of the beta-chain. Disequilibrium was present among beta-chain RFLPs, despite separation by one or more Alu repeat sequences. The very strong linkage disequilibrium which was present in sizable regions of the INSR gene despite the presence of both Alu and microsatellite repeats suggested that these regions do not have a major impact on recombinations at this locus.     

Thymocyte circular DNA excised from T cell receptor alpha-delta gene complex.

We have characterized thymocyte circular DNA excised from the T cell receptor alpha-delta gene complex. Some delta gene clones contained unusual recombinant structures derived from V-(D)-J joining: (i) a reciprocal joint of direct V to J delta joining, skipping the D delta segment; (ii) a V-D delta coding joint lacking an adjacent D delta-J delta coding joint; (iii) a V- D structure containing two D delta segments. Many of the alpha gen clones contained both coding and reciprocal joints of V alpha-to-J alpha joining on the same structure. Most of these coding joints were out of phase; however, in one clone there was an in-phase V-J alpha structure. Interestingly, some alpha gene clones contained the same V gene sequence as rearranged in the delta gene clone, indicating that the same V gene family, at least in part, could be utilized for both the alpha and delta gene systems.     

A complex of serine protease genes expressed preferentially in cytotoxic T-lymphocytes is closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14

A complex of genes encoding serine proteases that are preferentially expressed in cytotoxic T-cells was shown to be closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14. A striking difference in recombination frequencies among linkage crosses was reported. Two genes, Np-1 and Tcra, which fail to recombine in crosses involving conventional strains of mice, were shown to recombine readily in interspecific crosses involving Mus spretus. This difference in recombination frequency suggests chromosomal rearrangements that suppress recombination in conventional crosses, recombination hot spots in interspecific crosses, or selection against recombinant haplotypes during development of recombinant inbred strains. Finally, a mutation called disorganization, which is located near the serine protease complex, is of considerable interest because it causes an extraordinarily wide variety of congenital defects. Because of the involvement of serine protease loci in several homeotic mutations in Drosophila, disorganization must be considered a candidate for a mutation in a serine protease-encoding gene.     

Caspase-independent cell death by Fas ligation in human thymus-derived T cell line, HPB-ALL cells

In HPB-ALL cells, a human thymus-derived T-cell line, Fas (CD95)-mediated cell death was inhibited by about only 50% as a result of treatment with an amount of benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-CH(2)F (zVAD-fmk) sufficient to block the caspase activity. Fas-mediated caspase-independent cell death was not observed in other lymphoblast cell lines or mouse activated splenocytes, but this type of cell death was observed in mouse and rat thymocytes, the same as for HPB-ALL cells. This suggests that Fas-mediated caspase-independent cell death is a common feature in thymocytes. The signaling pathway of caspase-independent cell death has not yet been fully elucidated. In HPB-ALL cells, DNA fragmentation, one of the features of apoptotic cells, did not occur in the caspase-independent cell death after Fas ligation. On the other hand, this type of cell death and the surface exposure of phosphatidylserine were recovered by pretreatment with geldanamycin, which brought about a decrease in receptor interacting protein (RIP) kinase expression. These results suggested that HPB-ALL cells have a caspase-independent RIP kinasedependent pathway for Fas ligation.     

Analysis of the integrant in MyK-103 transgenic mice in which males fail to transmit the integrant.

Males in the transgenic mouse pedigree MyK-103, although fertile, do not transmit the integrant to offspring. The integrant is on chromosome 6 near the T-cell receptor beta-chain locus. It contains four fragments of the plasmid pMK (a metallothionein-thymidine kinase fusion gene) and a 532-base-pair fragment of displaced mouse DNA originating from a previously uncharacterized repetitive DNA family. The integration complex is flanked on either side by a 5-kilobase duplication of mouse DNA normally found in a single copy at this locus. Sequence analysis of the six novel junctions and their donor sequences shows that plasmid-plasmid junctions occurred at patches of limited homology, whereas chromosome-plasmid junctions were nonhomologous.     

A unique loop extension in the serine protease domain of haptoglobin is essential for CD163 recognition of the haptoglobin-hemoglobin complex

Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (alpha-chain) and a serine protease domain (beta-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the beta-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin beta-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin.     

Signal transduction through the T cell receptor-CD3 complex. Evidence for heterogeneity in receptor coupling

T cell receptor-CD3 complex (TCR-CD3)-mediated signal transduction was analyzed in HPB-ALL and Jurkat T cell lines. Both cell lines express high levels of TCR-CD3 complex on the cell surface, but provide different model systems for TCR-CD3 signaling in T cells. Jurkat responds with both inositol phosphate generation and intracellular Ca2+ mobilization after triggering of TCR-CD3, whereas TCR-CD3 triggering of HPB-ALL induces Ca2+ mobilization without detectable inositol phosphate generation. By employing a permeabilized cell system, we show that the HPB-ALL line expressed normal levels of Ca2(+)-induced phospholipase C activity. However, the TCR-CD3 on this cell line seems to be uncoupled from phospholipase C activation. In agreement with this result we also show, by analysis of protein kinase C-dependent phosphorylation of three distinct substrates, that TCR-CD3 in HPB-ALL is apparently uncoupled from protein kinase C activation. These findings may have implications for understanding signal-transducing pathways in T cells at various stages of differentiation.     

Heterogeneity of T-cell beta-chain gene rearrangements in human leukaemias and lymphomas.

The state of T-cell receptor beta-chain gene rearrangement in human T-cell leukaemias has been analysed. All forms of leukaemia tested (T-CLL, ALL, PLL, Sezary syndrome and ATL) exhibit rearrangements of C beta genes confirming the clonality of these neoplasias. However we find no evidence for common gene rearrangements nor for restricted rearrangement patterns within this type of neoplasia. We find evidence of T-cells with C beta 1 and C beta 2 rearrangements, sometimes associated with Igh JH rearrangements, but several cases of T-cell leukaemia with a marker inversion of chromosome 14 (q11;q32) do not have Igh JH rearrangements. The results suggest that TCR beta gene rearrangement occurs early in T-cell ontogeny but that this rearrangement is most often irrelevant to leukaemogenesis.     

Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes

The T3 complex has been defined by a group of monoclonal antibodies which react with all human peripheral blood T lymphocytes and a subpopulation of thymocytes. This membrane structure includes glycoproteins of 44 (alpha), 37 (beta), 25 (gamma), and 20 kDa (delta) as well as a nonglycosylated polypeptide of 20 kDa (epsilon). The characterization of the alpha and beta chains has been of particular interest because they may constitute the T cell receptor for antigen. Here we show that the T3 complex prepared by immunoprecipitation from T lymphocytes of a leukemic patient (Sezary syndrome) displays an unusually strong association of the alpha and beta chains with the 20/25-kDa T3 proteins. The alpha and beta chains (48 and 44 kDa) were co-precipitated by anti-20-kDa T3 monoclonal antibodies as a disulfide-linked 90-kDa heterodimer. A minor 220-kDa multimer composed of proteins similar to the alpha and beta chains was also present in these immunoprecipitates. This multimer could be independently precipitated with a new monoclonal antibody WT-31, which detects the larger polypeptide chains of the T3 complex on all human T lymphocytes. After removal of N-linked oligosaccharides, both the alpha and beta chain were found to have 33-kDa peptide backbones with distinct isoelectric points. Using a monoclonal reagent T40/25, a 90-kDa heterodimer, consisting of 40- and 49-kDa chains with peptide backbones of 34 kDa was found to be T3-associated on the T leukemic cell line HPB-ALL. When the alpha and beta chains from the Sezary patient were compared with the corresponding chains from HPB-ALL by peptide mapping, large differences were observed. Taken together, the data presented here provide strong evidence that the T cell receptor for antigen is part of the T3 complex on the surface of human T lymphocytes.     

Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx

Three monoclonal antibodies selected for their recognition of parts of the T3-T cell receptor complex on human T lymphocytes were found to induce an increase in cytoplasmic free Ca2+ (Ca2+i) in the T cell leukemia line HPB-ALL as measured by Quin2 fluorescence. These reagents are directed against T3 (OKT3), a nonvariable T3-associated structure (WT-31) and the variable region of the T3-associated antigen receptor (T40/25) of this cell line. The rise in Ca2+i was dependent on the presence of extracellular Ca2+, occurred within 30 sec of stimulation, and was sustained for at least 10 min. Fab fragments of OKT3 also caused a rapid increase in Ca2+i, indicating that cross-linking is not necessary to induce a Ca2+ response. Alterations in plasma membrane potential and La3+ blocked the Ca2+ influx induced by OKT3 and T40/25. These data suggest that the T3-T cell receptor complex of human T lymphocytes may be an antigen-regulated Ca2+ channel.     

Association between susceptibility to Theiler's virus-induced demyelination and T-cell receptor Jbeta1-Cbeta1 polymorphism rather than Vbeta deletion.

Theiler's murine encephalomyelitis virus (TMEV) induces demyelinating disease in susceptible mouse strains after intracerebral inoculation. The clinical symptoms and histopathology of the central nervous system appear to be similar to those of human multiple sclerosis (MS), and thus, this system provides an excellent infectious animal model for studying MS. The virus-induced demyelination is immune mediated, and the genes involved in the immune response such as those for the T-cell receptor beta-chain and major histocompatibility complex (MHC) haplotypes are known to influence disease susceptibility. To define whether the T-cell receptor Jbeta-Cbeta or Vbeta genes are associated with susceptibility, we have analyzed F2 mice from crosses of susceptible SJL/J (Vbeta(a)-JCbeta(b)) mice and resistant C57L (Vbeta(a)-JCbeta(a)) mice. Our results indicate that susceptibility to TMEV-induced demyelination is associated with restriction fragment length polymorphism reflecting the T-cell receptor Jbeta1-Cbeta1 region rather than the Vbeta polymorphism. This association becomes stronger when the MHC haplotype is considered in the linkage analysis. However, differences in the T-cell receptor alpha-chain haplotype have no significant influence on the pathogenesis of TMEV-induced demyelination.     

Pathogenesis of feline leukemia virus T17: contrasting fates of helper, v-myc, and v-tcr proviruses in secondary tumors.

A naturally occurring feline thymic lymphosarcoma (T17) provided the unique observation of a T-cell antigen receptor beta-chain gene (v-tcr) transduced by a retrovirus. The primary tumor contained three classes of feline leukemia virus (FeLV) provirus, which have now been characterized in more detail as (i) v-tcr-containing recombinant proviruses, (ii) v-myc-containing recombinant proviruses, and (iii) apparently full-length helper FeLV proviruses. The two transductions appear to have been independent events, with distinct recombinational junctions and no sequence overlap in the host-derived inserts. The T17 tumor cell line releases large numbers of FeLV particles of low infectivity; all three genomes are encapsidated, but passage of FeLV-T17 on feline fibroblast and lymphoma cells led to selective loss of the recombinant viruses. The oncogenic potential of the T17 virus complex was, therefore, tested by infection of neonatal cats with virus harvested directly from the primary T17 tumor cell line. A single inoculation of FeLV-T17 caused persistent low-grade infection culminating in thymic lymphosarcoma and acute thymic atrophy, which was accelerated by coinfection with the weakly pathogenic FeLV subgroup A (FeLV-A)/Glasgow-1 helper. Molecularly cloned FeLV-tcr virus (T-31) rescued for replication by a weakly pathogenic FeLV-A/Glasgow-1 helper virus was similarly tested in vivo and induced thymic atrophy and thymic lymphosarcomas. Most FeLV-T17-induced tumors manifested either v-myc or an activated c-myc allele and had undergone rearrangement of endogenous T-cell antigen receptor beta-chain genes, supporting the proposition that the oncogenic effects of c-myc linked to the FeLV long terminal repeat are targeted to a specific window in T-cell differentiation. However, neither the FeLV-T17-induced tumors nor the T-31 + FeLV-A-induced tumors contained clonally represented v-tcr sequences. Only one of the FeLV-T17-induced tumors contained detectable v-tcr proviruses, at a low copy number. While v-tcr does not have a readily transmissible oncogenic function, a more restricted role is not excluded, perhaps involving antigenic peptide-major histocompatibility complex recognition by the T-cell receptor complex. Such a function could be obscured by the genetic diversity of the outbred domestic cat host.