Molecular analysis of the human T-cell-antigen receptor.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
O Acuto
Publications and source records attributed to O Acuto.
Genes encoding the T-cell receptor alpha and beta subunits are transcribed in an ordered manner during intrathymic ontogeny.
To further characterize sequential events involved in activation of genes encoding the T-cell receptor (a complex of T3 molecules and a disulfide-linked heterodimer designated Ti, T3-Ti) for antigen and the major histocompatibility complex during intrathymic ontogeny, cDNA probes specific for Ti alpha and Ti beta subunits were used for transcriptional analysis. Ti beta transcript levels were minimal in stage I thymocytes, maximal in stage II thymocytes, and intermediate in state III thymocytes. In contrast, Ti alpha transcriptional activity was virtually undetectable in stage I, was low in stage II, and achieved high levels only in the stage III compartment. Analysis of tumor populations derived from individual stages of thymic differentiation confirmed these observations and demonstrated that clonal stage I-II cells often express Ti beta RNA in the absence of Ti alpha RNA. The latter was not a consequence of functional Ti alpha-subunit isotypy because each of 13 interleukin 2-dependent T-cell clones, including inducer, suppressor, and cytotoxic T cells, contain transcripts that hybridize with the Ti alpha probe. From these data it is concluded that Ti beta gene activation precedes Ti alpha gene activation. Moreover, the high level of Ti beta mRNA preceding Ti alpha mRNA expression implies that Ti beta mRNA and/or its protein products may regulate Ti alpha gene transcription.
Aging↗
Molecular aspects of human T lymphocyte antigen recognition.
Explore the source record for details and available documents.
Amino Acid Sequence↗
The human T lymphocyte receptor complex for antigen and MHC.
Recent studies using cloned antigen specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 KD Ti heterodimer and the in-variant 20 and 25 KD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors. The implications of these findings for understanding of human T cell growth and its regulation in disease states are discussed.
Amino Acid Sequence↗
Chromosomal location of human T-cell receptor gene Ti beta.
A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.
Animals↗
Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex.
Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) showed rearrangement of the TiC beta 1 gene on Southern blot analysis with or without deletion of the other TiC beta 1 allele. In contrast, TiC beta 2 always remained in germline configuration. Moreover, the finding that one additional suppressor clone deleted both TiC beta 1 alleles, maintained a germline TiC beta 2 configuration, and yet actively transcribed TiC beta 2 message suggested that TiC beta 2 is not a pseudogene. Rather, it appeared to be used less frequently than the TiC beta 1 gene and in the absence of detectable DNA rearrangements. Together, these results demonstrate that the functional repertoire (or isotype) of a given subclass of T cells is not encoded within the Ti beta genes.
Clone Cells↗
Natural killer-like function of activated T lymphocytes: differential blocking effects of monoclonal antibodies specific for a 90-kDa clonotypic structure.
A monoclonal antibody termed anti-NKTb has been generated following immunization of mice with cloned human cells (JT9) displaying natural killer (NK)-like activity. This antibody has the capacity to block cytotoxicity of the immunizing clone against several targets. In the present study, anti-NKTb was compared with a monoclonal antibody termed anti-NKTa that had previously been generated against JT9 cells and that had also been shown to block the NK-like function of these cells. The expression of a NKTb determinant, like that of NKTa, was found to be restricted to two NK active clones derived from the same individual, JT9 and JT10, both of which have the same mature T-cell phenotype (T3+, T8+, T11+). Comodulation, immunoprecipitation, and competitive binding experiments showed that both antibodies are directed to the same 90-kDa heterodimer associated with the T3 structure on the cell surface. However, cytotoxicity blocking studies suggested that NKTa and NKTb may represent functionally distinct epitopes of this 90-kDa molecule. Anti-NKTa uniformly blocked the cytotoxicity of both JT9 and JT10 cells when tested against 11 randomly selected target cell lines. In contrast, anti-NKTb totally blocked the cytotoxicity of these cloned cells against some targets (i.e., HPB-ALL, Nalm-1) but had very little effect when cytotoxicity was measured against other target cells (i.e., K562, U937, KG-1). This selective blocking effect, therefore, supports the notion that the heterodimer defined by the NKT antibodies is involved in the process of target cell recognition rather than in the cytolytic pathway of the cloned effector cells. Moreover, the unique functional effects of anti-NKTb suggest that additional levels of complexity exist in the specific recognition mechanisms of these clonal populations of NK active mature T lymphocytes.
Antibodies, Monoclonal↗
Genes encoding the Ti beta subunit of the antigen/MHC receptor undergo rearrangement during intrathymic ontogeny prior to surface T3-Ti expression.
To obtain further information about the ontogeny of the T-cell antigen/MHC receptor, a Ti beta subunit cDNA probe and heteroantisera specific for the Ti alpha and Ti beta subunits were utilized to characterize human T-lineage cells. Analysis of thymic tumors and normal thymocytes at both the DNA and protein levels demonstrates that Ti beta gene rearrangement is evident in stage II (T11+T6+T3-) and stage III (T11+T6-T3+), but not stage I (T11+T6-T3-) thymocytes. In contrast, surface expression of Ti alpha and Ti beta molecules is exclusively restricted to stage III thymocytes. Thus human T-lineage ontogeny is characterized by an orderly series of differentiation steps wherein Ti beta gene rearrangement precedes surface expression of the T3-Ti molecular complex.
Cloning, Molecular↗
An alternative pathway of T-cell activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein.
A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11(1), the SRBC binding site expressed on all T lymphocytes and thymocytes; T11(2), an epitope unrelated to the SRBC binding site but with a similar distribution; and T11(3), a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11(2) and T11(3) epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.
Animals↗
Identification and characterization of human T lymphocyte antigens.
Explore the source record for details and available documents.
Animals↗
Purification and NH2-terminal amino acid sequencing of the beta subunit of a human T-cell antigen receptor.
To obtain information about the structural basis for T-cell antigen recognition, a T3-associated Ti receptor molecule was isolated from crude membranes of the REX human thymic tumor line and purified by affinity chromatography with an anti- clonotypic monoclonal antibody in conjunction with preparative gel electrophoresis. NH2-terminal amino acid sequencing of the beta subunit unambiguously identified the amino acids in positions 2-12. Comparative protein sequence analysis by computer search demonstrated that this Ti beta sequence bore weak, but definite, homology to the first framework of the variable region of human lambda light chain. Anti-sera to a synthetic peptide corresponding to positions 2-11 precipitated the denatured Ti beta subunit from REX, thus confirming the above sequence. This information suggests that the Ti beta subunit is distantly related to human immunoglobulin lambda light chain and, moreover, should be of use in the molecular cloning of the Ti beta gene.
Amino Acid Sequence↗
Clonotypic surface structure on human T lymphocytes: functional and biochemical analysis of the antigen receptor complex.
Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90KD Ti heterodimer and the monomorphic 20/25KD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and fully expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate genes. The presence of unique peptides following proteolysis of different Ti molecules isolated by noncrossreactive anticlonotypic monoclonal antibodies supports the notion that variable regions exist within both the alpha and beta subunits. Moreover, N-terminal amino acid sequencing of the Ti beta subunit shows that it bears homology to the first V-region framework of immunoglobulin light chains and represents the product of a gene that rearranges specifically in T lymphocytes. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand (antigen/MHC), enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor crosslinking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.
Antibodies, Monoclonal↗
The human T-cell receptor.
Explore the source record for details and available documents.
Antibodies, Monoclonal↗
Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.
By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.
Animals↗
Identification of a clonally restricted 90 kD heterodimer on two human cloned natural killer cell lines. Its role in cytotoxic effector function.
The present studies were carried out to identify surface molecules involved in the cytotoxic effector function of a human natural killer (NK) clone termed JT9. This clone represents a mature T lymphocyte (T3+T8+T11+) mediating NK-like activity. Using JT9 for immunization, one monoclonal antibody termed anti-NKTa was selected that blocked the cytotoxicity of the clone towards K562 cells. Reactivity of anti-NKTa antibody was assessed using a large panel of lymphoid and nonlymphoid cells including a variety of cloned cell lines with either cytotoxic T lymphocyte (CTL) or NK-like activity. Among all cells tested, only two individual clones, JT9 and JT10, were found to express NKTa antigen. JT10 was derived independently from the same individual as JT9 and also represents a mature T cell (T3+T8+T11+) mediating NK-like activity. Like the Ti structure on CTL clones, the molecule defined by anti-NKTa was shown to be membrane associated with T3 in co-modulation experiments. Moreover, anti-NKTa precipitated a 90 kD heterodimeric structure in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 125I surface-labeled JT9 cells. The blocking capacity of anti-NKTa was evaluated in cytotoxicity assays using a panel of target cells. The influence of anti-T3 was tested in parallel and it was found that both anti-NKTa and anti-T3 blocked the cytotoxicity of the cloned cells against all targets. Given the potential role of 90 kD molecules as antigen-receptor structures, the specificity of the two NKTa+ NK clones was compared and found superimposable when assessed using 15 in vitro established cell lines. However, in contrast to conventional CTL clones, the expression of cytotoxicity by JT9 and JT10 was not dependent upon recognition of class I or class II major histocompatibility complex gene products on the target cells. In addition, the cytotoxicity of these T8+ NK active clones could not be blocked by anti-T8 antibodies. Taken together, the present data suggest that the specificity of one population of human NK active lymphocytes is determined by clonotypic structures. The NKTa determinant identified here appears to belong to the same family of molecules as Ti structures, previously identified on antigen-specific T lymphocytes.
Antibodies, Monoclonal↗
Peptide variability exists within alpha and beta subunits of the T cell receptor for antigen.
The T cell receptor for antigen (Ti) has recently been identified as a 90-kdalton T3-associated clonotypic structure composed of one 49-51-kdalton alpha and one 43-kdalton beta subunit, which are disulfide linked. Here, Ti molecules from two alloreactive CTL clones derived from the same donor but of differing specificities (CT8III and CT4II) are directly compared following isolation with anticlonotypic monoclonal antibodies. Isoelectric focusing shows that the alpha subunits (pI 4.4-4.7) are more acidic than the beta subunits (pI 6.0-6.2) but that each glycoprotein species is distinctive. More importantly, two-dimensional peptide maps of 125I-labeled surface receptors indicate that the beta chains of Ti1 and Ti2 appear unique and share only two peptides in common. In contrast, peptide maps of Ti1 and Ti2 alpha chains are more related although not identical. These results suggest that the human T cell receptor is composed of constant as well as variable regions and that at least one of the latter is located within the beta subunit.
Antibodies, Monoclonal↗
Evidence for the T3-associated 90K heterodimer as the T-cell antigen receptor.
Several surface molecules appear to be involved in antigen recognition by human T lymphocytes including the monomorphic 20/25K T3 structure present on all mature T lymphocytes and the subset-specific associative recognition elements, T4 and T8 (refs 1-8). More recently, Ti1, a clonally unique antigen recognition structure comprised of a 49,000 molecular weight (49K) alpha-chain and a 43K beta-chain, linked to T3 was identified on a major histocompatibility complex (MHC) class I specific T8+ T-cell clone, CT8III (ref. 9). To determine whether analogous receptor molecules could be found on other T-cell clones of differing specificity, we produced monoclonal antibodies against a clonal structure (Ti2) on an MHC class II specific T4+ lymphocyte, CT4II, derived from the same donor as CT8III. The Ti2 structure on CT4II is shown here to be a disulphide-linked heterodimer like Ti1 on CT8III and is composed of subunits of similar molecular weight. Monoclonal antibodies against Ti2 or Ti1 block antigen specific functions of the respective clone without showing any cross-reactivity. These findings suggest that each T lymphocyte, regardless of subset derivation or specificity, uses an analogous Ti heterodimer for antigen specific function. The latter is linked to T3 and expressed on the cell surface at an identical density (30,000-40,000 sites per cell).
Antibodies, Monoclonal↗
The human T cell receptor: appearance in ontogeny and biochemical relationship of alpha and beta subunits on IL-2 dependent clones and T cell tumors.
The human T cell receptor for antigen (Ti) has recently been identified on IL-2 dependent T cell clones as a 90 kd disulfide-linked heterodimer comprised of one 49-51 kd alpha (alpha) and one 43 kd beta (beta) chain. These subunits are noncovalently associated with a monomorphic 20-25 kd T3 molecule. Here, we produce monoclonal antibodies to a human tumor (REX) derived from an earlier stage of thymic differentiation in order to determine whether clonotypic structures are expressed and to define the ontogeny of Ti. The results of SDS-PAGE and peptide map analyses indicate that an homologous T3-associated heterodimer is synthesized and expressed by REX. This glycoprotein shares several peptides in common with clonotypic structures on an IL-2 dependent T cell clone. In addition, similar Ti related molecules appear during intrathymic ontogeny in parallel with surface T3 expression. The latter findings provide the structural basis for the immunological competence observed exclusively within the T3+ thymocyte compartment.
Antibodies, Monoclonal↗