The T cell receptor V beta 6 domain imparts reactivity to the Mls-1a antigen

A monoclonal antibody secreting hybridoma was established by fusing spleen cells from a rat immunized with a murine T cell clone, OI11, which has I-Ab restricted specificity for the male H-Y antigen and unrestricted specificity for the minor lymphocyte stimulating antigen, Mls-1a, to the mouse myeloma P3X63AG8.653 and screening for the capacity of the hybridoma supernatants to stimulate the OI11 T cell clone. An antibody (RR4-7) was found to be specific not only for the immunizing T cell clone but virtually for all T cells using the V beta 6 TCR gene product as part of their surface antigen receptor. When the expression of the V beta 6 gene in various strains of mice was analyzed, it was found that strains expressing the Mls-1a antigen contained few T cells expressing V beta 6-encoded TCRs. The majority of T cell hybridomas which expressed V beta 6-encoded TCRs were found to be reactive to the Mls-1a antigen. These data confirm the finding of H. R. MacDonald et al. (Nature (London) 332, 40, 1988) that most TCRs encoded by the V beta 6 gene have a biased specificity for the Mls-1a antigen.     

T cell receptor alpha-chain influences reactivity to Mls-1 in V beta 8.1 transgenic mice.

Most, but not all, V beta 8.1+ T cells respond to M1s-1 and are clonally deleted in the thymus of M1s-1-expressing animals. To formally examine the role of the TCR alpha-chain in reactivity and tolerance to M1s-1, we have analyzed M1s-1 reactivity in a large panel of CD4+ hybridomas generated from TCR V beta 8.1 transgenic mice, that express an identical, potentially M1s-1-reactive beta-chain. The data show that the alpha-chain strongly influences the M1s-1 reactivity of the hybridomas and that the differences in reactivity had relevance for tolerance. Thus, V alpha 11+ hybridiomas were biased toward M1s-1 reactivity and V alpha 11+ T cells were correspondingly absent from the peripheral repertoire of M1s-1-expressing transgenic mice. V alpha 2+ hybridomas, on the other hand, were biased against M1s-1 reactivity, and V alpha 2+ T cells were correspondingly amplified in the M1s-1-expressing transgenic mice. Structural analysis of the alpha-chains revealed that the M1s-1 reactivity of the V alpha 11+ hybridomas segregated precisely with family member, such that V alpha 11.1+ hybridomas were M1s-1-reactive and V alpha 11.3+ hybridomas were not M1s-1-reactive. On the other hand, there was not a clear correlation between family member and M1s-1 reactivity in the V alpha 2+ hybridomas. The hybridomas also showed striking variation in their reactivity to staphylococcal enterotoxin B (SEB), and the SEB reactivity of the V alpha 11+ hybridomas correlated precisely with family member and with M1s-1 reactivity. In contrast, there was not a clear correlation with V alpha 2+ alpha-chain structure and SEB reactivity. Also, there was no correlation between M1s-1 reactivity and SEB reactivity in individual V alpha 2+ hybridomas, suggesting that the recognition of the two superantigens by the same TCR is not equivalent. Taken together, these data define a role for the TCR alpha-chain in superantigen reactivity and T cell tolerance, and provide a structural explanation for the different fates of M1s-1-reactive T cells in normal and transgenic mice.     

Preferential use of a T cell receptor V beta gene by acetylcholine receptor reactive T cells from myasthenia gravis-susceptible mice.

Experimental autoimmune myasthenia gravis (EAMG) is an important model for testing current concepts in autoimmunity and novel immunotherapies for autoimmune diseases. The EAMG autoantigen, acethylcholine receptor (AChR), is structurally and immunologically complex, a potential obstacle to the application of therapeutic strategies aimed at oligoclonal T cell populations. Inasmuch as we had previously shown that the clonal heterogeneity of T cell epitope recognition in EAMG was unexpectedly limited, we examined TCR V beta expression. AChR primed lymph node T cells and established AChR reactive T cell clones from EAMG-susceptible C57BL/6 (B6; H-2b, Mls-1b) mice showed preferential utilization of the TCR V beta 6 segment of the TCR. After in vivo priming and in vitro restimulation for 7 days with AChR or a synthetic peptide bearing an immunodominant epitope, V beta 6 expressing lymph node cells (LNC) were expanded several-fold, accounting for up to 75% of recovered viable CD4+ cells. The LNC of B6.C-H-2bm12 (bm12; H-2bm12, Mls-1b) mice, which proliferated in response to AChR but not to the B6 immunodominant peptide, failed to expand V beta 6+ cells. Inasmuch as nonimmune bm12 and B6 animals had similar numbers of V beta 6+ LNC (4-5%), this suggested that structural requirements for TCR recognition of Ag/MHC complexes dictated V beta usage. Results concerning peptide reactivity and V beta 6 expression among T cells from (B6 x bm12)F1 animals also suggested that structure-function relationships, rather than negative selection or tolerance, accounted for the strain differences between B6 and bm12. To examine the potential effects of thymic negative selection of V beta 6+ cells on the T cell response to AChR, CB6F1 (H-2bxd, Mls-1b; V beta 6-expressing) and B6D2F1 (H-2bxd, Mls-1axb; V beta 6-deleting) strains were analyzed for AChR and peptide reactivity and V beta 6 expression. Both F1 strains responded well to AChR but the response of B6D2F1 mice to peptide was significantly reduced compared to CB6F1. Short and long term cultures of peptide-reactive B6D2F1 LNC showed no expansion of residual V beta 6+ cells, although similar cultures of CB6F1 LNC were composed of more than 60% V beta 6+ cells. The results from the F1 strains further indicated that the T cell repertoire for peptide was highly constrained and that non-V beta 6 expressing cells could only partially overcome Mls-mediated negative selection of V beta 6+ TCR capable of recognizing peptide.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

V beta gene polymorphism and a major polyclonal T cell receptor idiotype

Genetic polymorphism in the beta variable gene pool (V beta) is responsible for the strain-specific distribution of the KJ16 T cell receptor (TcR) marker on 20% of peripheral T cells. KJ16+ strains carry two homologous V beta genes that are absent in KJ16- strains. All functional KJ16+ T cell hybrids tested express a member of this V beta subset. mRNA hybridizing to this variable-region probe can be easily detected in total splenic T cells of a prototype KJ16+ strain. Thus, 20% of the TcR from the cytotoxic and helper T cell population, with various MHC restrictions and antigen reactivities, can be generated from two V beta genes. However, their deletion appears to have no effect on the functionality of the T cell repertoire.     

Characterization of a rat monoclonal antibody specific for a determinant encoded by the V beta 7 gene segment. Depletion of V beta 7+ T cells in mice with Mls-1a haplotype.

We have generated a rat mAb, TR310, which recognizes a determinant encoded by the murine V beta 7 gene segment of the TCR. TR310 immunoprecipitates TCR from cell lysates, co-modulates with CD3, and can be used for immunofluorescence staining of T cells. By using this antibody, we found that the average percentage of V beta 7+ peripheral T cells in Mls-1b mice was 3.8%, but only 0.8% in Mls-1a mice. A similar difference was also observed in the mature TCRhi thymocyte subsets, suggesting that V beta 7+ T cells are deleted during intrathymic maturation in Mls-1a mice. TR310 should prove to be a valuable reagent in further studies of the TCR repertoire and the analysis of factors which alter it.     

Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.     

Characterization of a new minor lymphocyte stimulatory system. I. Cluster of self antigens recognized by "I-E-reactive" V beta s, V beta 5, V beta 11, and V beta 12 T cell receptors for antigen.

In the mouse, two sets of V beta gene products have been shown to be associated with T cell recognition of endogenous self Ag. One of these is the set of V beta associated with T cell reactivities to stimulatory Mls gene products, Mlsa (V beta 6, V beta 8.1, V beta 9) or Mlsc (V beta 3); another is the set of V beta, such as V beta 5, V beta 11, V beta 12, or V beta 17a, which were originally found to be related to I-E recognition. Although the Mls system has been well characterized, little is known about the nature of the ligands for the second set of V beta. In this work, we describe the evidence that the natural ligand or ligands of V beta 5, V beta 11, and V beta 12 may be novel Mls determinants that are recognized by naive T cells at a high precursor frequency and function as the ligand for clonal deletion of self-reactive T cells by negative selection. However, surprisingly, unlike the conventional Mls system, in which all V beta associated with Mlsa recognition or Mlsc recognition are uniformly deleted in those animals expressing the relevant Mls type, expression of these three V beta segregates independently among strains. Based on these observations, the nature of T cell recognition for this new Mls gene product(s) is discussed.     

Growth inhibition of human T cells by antibodies recognizing the T cell antigen receptor complex

Monoclonal antibodies that bind to the T cell MHC-antigen recognition complex (anti-T3 or anti-Ti) are known to either mimic ligand binding and activate T cells or block ligand binding, leading to an inhibition of T cell activation. In the present experiments, we demonstrate a direct inhibitory effect on the growth of human T cells by anti-T3 or anti-Ti antibodies. The proliferation of human peripheral blood T cells preactivated by exposure to PHA was inhibited in a specific manner by anti-T3. Colony formation in soft agar by REX cells, a leukemic cell line of early T cell phenotype, was completely inhibited by anti-T3 or anti-Ti antibodies, whereas isotype-matched antibodies to a variety of other T cell markers had no effect. Growth of REX cells in suspension culture was not affected by anti-T3 or anti-Ti. A cell line, T3.N1, was established from an agar colony of anti-T3-resistant REX cells. T3.N1 was phenotypically identical to REX except for failure to express any detectable T3 or Ti surface antigen. T3.N1 colony formation in soft agar was not inhibited by anti-T3 or anti-Ti. There was no rise in [Ca2+]i of T3.N1 cells after anti-T3 or anti-Ti exposure. These results indicate that in addition to the well-known positive regulatory effects of ligand binding to the T3/Ti complex, T3/Ti binding can also result in a down-regulatory signal for human T cell growth.     

Expression of the Mls-1a superantigen results in an increased frequency of V beta 14+ T cells.

Minor lymphocyte-stimulating (Mls) Ag are alloantigens that stimulate T cells expressing specific V beta regions. Recent studies have established that Mls Ag are examples of endogenous superantigens encoded by the products of endogenous mouse mammary tumor virus (MLV) genomes. In a mouse strain that expresses a given mammary tumor virus (Mls) Ag, reactive T cells expressing the corresponding V beta region are profoundly deficient, due at least in part to clonal deletion of the cells during their development. Expression of Mls and other endogenous superantigens, therefore, results in profound alterations in the ultimate repertoire of T cells in an animal. A role for endogenous superantigens in positive selection of T cells has not been previously established. Here we present evidence that expression of Mls-1a leads to a specific increase in the abundance of V beta 14+ T cells. Genetic studies indicate linkage of the effect to the Mls-1a gene. Neonatal tolerance studies argue against the possibility that the increase is due solely to the deletion of Mls-reactive V beta 14- T cells. The results are consistent with the Mls-1a product playing a role in the positive selection of V beta 14+ T cells.     

Redirecting T lymphocyte specificity by T cell receptor gene transfer--a new era for immunotherapy

The therapeutic efficacy of adoptively transferred cytotoxic T lymphocytes (CTL) has been demonstrated in clinical trials for the treatment of chronic myelogenous leukemia, cytomegalovirus-mediated disease, and Epstein-Barr virus-positive B cell lymphomas. It is however limited by the difficulty of generating sufficient amounts of CTLs in vitro, especially for the treatment of solid tumors. Recent gene therapy approaches, including two clinical trials, successfully apply genetic engineering of T cell specificity by T cell receptor (TCR) gene transfer. In this review we want to elucidate several principles of the redirection of T cell specificity. We cover basic aspects of retroviral gene transfer, regarding transduction efficacy and transgene expression levels. It was demonstrated that the number of TCR molecules on a T cell is important for its function. Therefore, an efficient transfer system that yields high transduction efficiency and strong and stable transgene expression is a prerequisite to achieve effector function by redirected T cells. Furthermore, we consider more recent aspects of T cell specificity engineering. These include the possibility of co-transferring coreceptors to create for example functional T helper cells by engrafting CD4(+) T cells with a MHC class I restricted TCR and the CD8 coreceptor and vice versa. Also, risks related to the adoptive transfer of TCR gene-modified T cells and possible safety mechanisms are discussed. Finally, we summarize recent findings describing transferred TCRs capable of displacing endogenous TCRs from the cell surface.     

T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice.

To study T cell tolerance, transgenic mice were generated that expressed the Mlsa-reactive T cell receptor (TCR) beta chain V beta 8.1 (cDNA) under the control of the H-2Kb promoter/immunoglobulin heavy chain enhancer on approximately 90% of peripheral T cells. In transgenic mice bearing Mlsa, thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H-2. RNA analysis and immunofluorescence with TCR V beta-specific antibodies revealed that expression of endogenous TCR beta genes was suppressed. Both Mlsa and Mlsb TCR beta chain transgenic mice mounted a T-cell-dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus-specific cytotoxic T cells was impaired in TCR beta chain transgenic Mlsa, but not in transgenic Mlsb mice.     

Evidence for involvement of lymphocyte function-associated antigen 1 in T cell migration to epidermis.

Although it is well known that in various T cell-mediated skin diseases T cells migrate preferentially to epidermis, no direct evidence has been presented in which molecules on T cells are important in directing T cell traffic to epidermis. We have previously established CD4+ autoreactive cloned T cells with a special tropism for epidermis in vitro as well as in vivo. Antibody inhibition studies demonstrated that only anti-lymphocyte function associated Ag 1 (anti-LFA-1) mAb completely inhibited the in vitro migration of the T cells toward the epidermis, whereas mAb against other T cell surface molecules had little or no effect. Monovalent F(ab) fragment of the anti-LFA-1 mAb, although less efficient, also inhibited the T cell migration. The apparent dependency of the inhibition on the anti-alpha-chain mAb suggested a major role for the alpha-chain of LFA-1 in T cell migration to epidermis. The relevance of an LFA-1-dependent mechanism to the epidermotropic migration of T cells was further strengthened by the findings that the T cell migration to epidermis was inhibited by divalent cation depletion, cytochalasin B, and low temperature. These findings indicate that the LFA-1 molecule, which is thought to be primarily involved in cell-to-cell adhesions, also plays an important role in directing T cell migration to epidermis.     

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.     

Ia antigen: a murine B lymphocyte receptor for transformed cell induction of interferon-alpha/beta

A receptor on the surface of nonsensitized mouse spleen cells that recognizes a glycoprotein from transformed mouse L-929 cells is described. The interaction of the receptor and glycoprotein inducer results in the production of MoIFN alpha/beta. An assay was developed to assess certain biologic and physicochemical characteristics of the receptor. The receptor and glycoprotein inducer bound in a concentration-dependent manner, which tends to indicate a direct interaction between the two. The receptor was not ubiquitous; spleen cells but not normal mouse embryo cells appeared to be the source. It was specific for MoIFN alpha/beta inducers from transformed cells, but not from other MoIFN alpha/beta or gamma inducers such as NDV, LPS, PWM, or SEA. The receptor appeared to be a cell surface protein in that its activity was abolished by trypsinization of whole spleen cells. Previous studies indicated that the receptor was probably located on B cells. Gel filtration indicated that the receptor had a m.w. of 30,000 to 60,000. Because the receptor appeared to be: 1) B lymphocyte associated, 2) a surface protein, and 3) 30,000 to 60,000 daltons, a similarity to Ia antigen was suggested. This possibility was confirmed by showing binding of the receptor to an anti-IaK antibody-Sepharose affinity column. PAGE analysis of the affinity-purified receptor revealed a single protein band with a m.w. of approximately 60,000. ELISA of the above gel slices with anti-Ia antibody further confirmed the specificity of the column. A physical association of the receptor and inducer was demonstrated by showing binding of the glycoprotein inducer to a receptor (Ia antigen)-Sepharose affinity column. Furthermore, the receptor (Ia antigen) was highly purified by a glycoprotein inducer-Sepharose affinity column.     

Protection against experimental encephalomyelitis. Idiotypic autoregulation induced by a nonencephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene.

The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti-idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope.     

The human T cell antigen receptor is encoded by variable, diversity, and joining gene segments that rearrange to generate a complete V gene

A cDNA clone YT35 , synthesized from poly(A)+ RNA of the human T cell tumor Molt 3, exhibits homology to the variable (V), joining (J), and constant (C) regions of immunoglobulin genes. We have isolated and sequenced the germ-line V and J gene segment counterparts to YT35 from a human cosmid library, and these failed to encode 14 nucleotides of the cDNA clone between the V and J regions. We postulate that these 14 nucleotides are encoded by a third gene segment analogous to the diversity (D) gene segments of immunoglobulin heavy chain genes. This T cell antigen receptor V gene appears to be assembled from three gene segments, V, D, and J, and accordingly most closely resembles immunoglobulin heavy chain V genes.     

Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase C-gamma1

The small GTPase, Rap1, is a potent activator of leukocyte integrins and enhances the adhesive activity of lymphocyte function-associated antigen-1 (LFA-1) when stimulated by the T cell receptor (TCR) or chemokines. However, the mechanism by which Rap1 is activated remains unclear. Here, we demonstrate that phospholipase C (PLC)-gamma1 plays a critical role in the signaling pathway leading to Rap1 activation triggered by the TCR. In Jurkat T cells, TCR cross-linking triggered persistent Rap1 activation, and SDF-1 (CXCL12) activated Rap1 transiently. A phospholipase C inhibitor, U73122, abrogated Rap1 activation triggered by both the TCR and SDF-1 (CXCL12). PLC-gamma1-deficient Jurkat T cells showed a marked reduction of TCR-triggered Rap1 activation and adhesion to intercellular adhesion molecule-1 (ICAM-1) mediated by LFA-1. In contrast, SDF-1-triggered Rap1 activation and adhesion were not affected in these cells. Transfection of these cells with an expression plasmid encoding PLC-gamma1 restored Rap1 activation by the TCR and the ability to adhere to ICAM-1, accompanied by polarized LFA-1 surface clustering colocalized with regulator of adhesion and polarization enriched in lymphoid tissues (RAPL). Furthermore, when expressed in Jurkat cells, CalDAG-GEFI, a calcium and diacylglycerol-responsive Rap1 exchange factor, associated with Rap1, and resulted in enhanced Rap1 activation and adhesion triggered by the TCR. Our results demonstrate that TCR activation of Rap1 depends on PLC-gamma1. This activity is likely to be mediated by CalDAG-GEFI, which is required to activate LFA-1.