Direct binding of secreted T-cell receptor beta chain to superantigen associated with class II major histocompatibility complex protein.

The interaction of the T-cell receptor (TCR) with peptide antigen plus major histocompatibility complex (MHC) protein requires both alpha and beta chains of the TCR. The "superantigens" are a group of molecules that are recognized in association with MHC class II but that do not appear to conform to this pattern. Superantigens are defined as such because they cause the activation or thymic deletion of many or all T cells bearing specific TCR beta-chain variable region (V beta) elements. The strong association of particular V beta S with T-cell responses to superantigens suggests that their interaction with the TCR is fundamentally different from that of most antigens. We have directly investigated the involvement of the beta chain in recognition of a superantigen by using a secreted, truncated TCR beta chain and the bacterial superantigen staphylococcal enterotoxin A complexed to cell-surface MHC class II. We demonstrate that this interaction is specific for the enterotoxin and is dependent on MHC class II expression by the cell. The reaction can be inhibited by antibodies against the three components of the reaction: V beta, enterotoxin, and class II. This shows that the TCR beta chain is sufficient to mediate the interaction with a superantigen-class II complex. The TCR alpha chain and co-receptors such as CD4 are not required.     

Binding of soluble natural ligands to a soluble human T-cell receptor fragment produced in Escherichia coli.

An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.     

T-cell receptor alpha/beta chain-CD3 protein complex defect in systemic lupus erythematosus: T-cell function.

We describe the first case of systemic lupus erythematosus (SLE) in which peripheral blood T cells were deficient in cell surface expression of T-cell receptor alpha/beta chain (TcR alpha beta) and the CD3 protein. Because of the uncommon phenotype and because of the notion that coexpression of TcR alpha beta and CD3 is essential for antigen-specific T-cell function, in vitro functional assays were performed, showing a highly decreased proliferative response to anti-CD3 antibody and other T-cell mitogens, deficient interleukin-2 (IL-2) secretion, and impaired function to respond in autologous and allogeneic mixed lymphocyte reactions. However, the helper-inducer function of T cells was unaffected by deficient expression of the TcR alpha beta/CD3 protein complex. The relative increase of CD4+ CDw29+ helper-inducer subsets in T cells accounted for elevated secretion of two terminal B-cell stimulating factors, B-cell growth factor (BCGF) and B-cell differentiation factor (BCDF). Hence, our results suggest that the regulation of secretion of lymphokines, IL-2, and BCGF and BCDF is independently controlled in T cells, and this case illustrates the pathologic sequelae of a unique defect in T cells characteristic of SLE.     

Major histocompatibility complex determinants select T-cell receptor alpha chain variable region dominance in a peptide-specific response.

Dominant expression of T-cell receptor (TCR) alpha or beta chain variable region (V alpha or V beta) gene families has been observed in the T-cell response to some conventional peptide antigens. Current models for the interaction of TCR V region elements with different determinants of a major histocompatibility complex (MHC)-peptide complex, the normal TCR ligand, suggest that the TCR V-J junctional region (CDR3, where J is joining) is the primary contact with a peptide epitope and that other TCR V region segments may interact directly with neighboring MHC determinants. This suggests that V alpha or V beta dominance in a specific response can be MHC-selected. In this case, if related peptides bind to an MHC molecule in a similar orientation, they could select for identical V alpha or V beta dominance even if they are noncrossreactive at the level of T-cell activation. We have screened for this possibility by introducing minimal conservative substitutions in a synthetic peptide, YYEELLKYYEELLK, that is presented to T cells in association with an uncommon A beta E alpha d mixed Ia isotype. We report here that the peptide variant FFEELLKFFEELLK is noncrossreactive with YYEELLKYYEELLK but appears to preserve the same MHC binding motif since T-cell responses are restricted to the same mixed A beta E alpha isotype. Although the two peptides are noncrossreactive in either direction, the same members of the V alpha 4 gene family are dominantly expressed in T cells specific for either peptide. We conclude that the similar topography of the two MHC-peptide complexes gives functional significance to a unique A beta E alpha determinant that selects for V alpha 4 dominance.     

Engineered secreted T-cell receptor alpha beta heterodimers.

We have produced a soluble form of a mouse alpha beta T-cell antigen receptor (TCR) by shuffling its variable (V) and constant (C) domains to the C region of an immunoglobulin kappa light chain. These chimeric molecules composed of V alpha C alpha C kappa and V beta C beta C kappa chains were efficiently secreted (up to 1 micrograms/ml) by transfected myeloma cells as noncovalent heterodimers of about 95-kDa molecular mass. In the absence of direct binding measurement, we have refined the epitopic analysis of the soluble V alpha C alpha C kappa-V beta C beta C kappa dimers and shown that they react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TCR alpha and beta chains. Conversely, we have raised three distinct monoclonal antibodies against the soluble TCR heterodimers and shown that they recognize surface-expressed TCRs. Two of these antibodies were found to react specifically with the products of the V alpha 2 (V delta 8) and V beta 2 gene segments, respectively. When considered together, these data suggest that these soluble TCR molecules are folded in a conformation indistinguishable from that which they assume at the cell surface.     

Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.     

Genes for the tumor necrosis factors alpha and beta are linked to the human major histocompatibility complex.

The human major histocompatibility complex (MHC) includes the closely linked genes for the tumor necrosis factors alpha and beta. Their location is within the chromosomal segment between HLA-DR and HLA-A or centromeric of HLA-DP. This assignment is based on Southern blot analysis of a number of different MHC deletion mutants and is corroborated by chromosome in situ hybridization.     

An analysis of T-cell receptor variable region gene expression in major histocompatibility complex disparate mice.

To define the impact of major histocompatibility complex (MHC)-encoded glycoproteins on the selection of the T-cell receptor repertoire, we have determined the frequency with which T-cell receptor variable region (V alpha and V beta) genes are expressed in T cells from MHC disparate mice. Approximately 500 T-cell hybridomas were generated from each of three strains of MHC congenic mice [B10 (H-2b), B10.BR (H-2k), and B10.Q (H-2q)] by fusing mitogen-stimulated lymph node T cells with the thymoma BW5147. RNA was prepared from 1629 individual hybridomas and analyzed for the expression of 10 V alpha and 16 V beta gene families. These experiments reveal significant differences in the relative contributions of 1 V alpha gene family (V alpha 3) and several V beta gene segments (V beta 5.1, -5.2, -11, and -12) to the T-cell receptor repertoire of MHC disparate mice.     

CDR1 T-cell receptor beta-chain peptide induces major histocompatibility complex class II-restricted T-T cell interactions.

T-T cell interactions have been proposed in postulated network theories of immunoregulation and autoimmunity. Despite previous reports of protection induced by T-cell receptor (TcR)-derived peptides in experimental autoimmunity, no evidence for T-T cell interactions by direct recognition of processed TcRs on native T cells was obtained. Here we report that immunization of rats with overlapping sets of peptides of the TcR alpha or beta chain allowed us to detect immunogenic TcR peptides. Remarkably enough, these TcR peptides appeared to cluster within the hypervariable complementarity-determining regions of the TcR. Immunization of rats with these TcR peptides induced CD4+ TcR peptide-specific T cells, which recognized both rDNA TcR proteins and the original, arthritogenic T cell in a major histocompatibility complex class II-restricted way. These findings indicate that activated T cells can process and present their own TcR in the context of major histocompatibility complex class II molecules and, furthermore, that such peptides can be recognized by TcR variable gene-specific T cells.     

Highly lytic CD8+, alpha beta T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in beta 2-microglobulin, MHC class I-deficient mice.

Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibility complex (MHC) class I deficiency and virtual disappearance of functional CD8+ cytotoxic T lymphocytes (CTLs) in beta 2m-deficient (beta 2m-/-) mice. We asked whether the beta 2m-/- mice are able to reject tumor cells injected i.p. and what is the cellular composition of peritoneal exudate leukocytes (PELs) from such mice. We found that beta 2m-/- mice do reject MHC class I-bearing tumor cells injected i.p. Surprisingly, analysis of PEL CTLs obtained from i.p. tumor-injected beta 2m -/- mice revealed the presence of a large proportion of functional, tumor-destroying CD8+, CD4-, alpha beta T-cell receptor-positive, CD3+, Thy-1+, MHC class I-negative CTLs with strong MHC class I-directed cytotoxic activity. These results call for careful studies of local accumulation of CD8+ CTLs in beta 2m -/- mouse models and suggest that the dramatic decrease in MHC class I expression caused by beta 2m gene disruption does not prevent CD8+/CD4- cell selection and expansion.     

Specific binding of antigenic peptides to separate alpha and beta chains of class II molecules of the major histocompatibility complex.

Class II molecules of the major histocompatibility complex bind antigenic peptides and present them to T-helper cells. Class II molecules are heterodimers consisting of one alpha and one beta chain. Here we report that each isolated alpha and beta chain binds antigenic peptides and that this binding is specific. The specificity of peptide binding was investigated by employing the murine major histocompatibility complex haplotypes I-Ad and I-Ek and fluorescence-labeled peptides of chicken ovalbumin and pigeon cytochrome c, respectively, which are known to be specific for these haplotypes. The major histocompatibility complex molecules were incubated with these peptides and subjected to SDS/PAGE under nondenaturing conditions. The gels were then scanned for the fluorescent peptides and, after silver staining, for proteins. We found that the fluorescence-labeled peptide fragment of ovalbumin bound preferentially to the isolated alpha and beta chains of I-Ad, whereas the fluorescence-labeled peptide fragment of pigeon cytochrome c bound preferentially to the isolated alpha and beta chains of I-Ek. The alpha and beta chains of each haplotype bound their specific peptides about equally well, suggesting comparable affinities. Our results indicate that in vivo the kinetic pathway for the formation of antigenic peptide complexes with the alpha/beta heterodimers may involve the initial formation of complexes of the alpha and/or beta chains with the specific antigenic peptides.     

Role of interferon alpha/beta receptor chain 1 in the structure and transmembrane signaling of the interferon alpha/beta receptor complex.

A previously cloned cDNA encodes one subunit of the human interferon alpha/beta receptor (IFN alpha R), denoted IFN alpha R1. To study the expression and signaling of IFN alpha R1, we used monoclonal antibodies (mAbs) generated against the baculovirus-expressed ectodomain of IFN alpha R1. Immunoprecipitation and immunoblotting of lysates from a variety of human cell lines showed that IFN alpha R1 has an apparent molecular mass of 135 kDa. Binding analysis with 125I-labeled mAb demonstrated high levels of cell surface expression of IFN alpha R1 in human cells and in mouse cells transfected with IFN alpha R1 cDNA, whereas no cross-reactivity was observed in control mouse L929 cells expressing only the endogenous mouse receptor. The subunit was rapidly down-regulated by IFN alpha (80% decrease within 2 hr) and degraded upon internalization. The IFN alpha R1 chain appeared to be constitutively associated with the 115-kDa subunit of the IFN alpha/beta receptor, since the mAbs coprecipitated this protein. IFN alpha/beta treatment induced tyrosine phosphorylation of IFN alpha R1 within 1 min, with kinetics paralleling that of the IFN-activated protein-tyrosine kinases Jak1 and Tyk2. Ligand-induced tyrosine phosphorylation of IFN alpha R1 was blocked by the kinase inhibitors genistein or staurosporine. Although IFN alpha R1 cDNA-transfected mouse cells expressed high levels of this subunit when compared with empty vector-transfected cells the number of binding sites for human IFN alpha (50-75 sites per cell) was not increased. Human IFN alpha induced the expression of a mouse IFN alpha/beta-responsive gene (the 204 gene) in mouse L929 cells transfected with the IFN alpha R1 cDNA, but not in mock-transfected cells. These results suggest that the IFN alpha R1 subunit acts as a species-specific signal transduction component of the IFN alpha/beta receptor complex.     

Recognition of a self major histocompatibility complex TL region product by gamma delta T-cell receptors.

Ligand specificity of a murine gammadelta T-cell receptor-expressing hybridoma (KN6) derived from adult thymocytes has been analyzed in detail. The molecule recognized by the KN6 gammadelta T-cell receptor is expressed on syngeneic cells of various sources (peritoneal macrophages, thymocytes, spleen cells, and Abelson murine leukemia virus-transformed cell lines) and on transformed cells arrested at an early stage of development (e.g., PCC3 embryonal carcinoma cells). Linkage of the gene coding for the KN6 ligand to the major histocompatibility complex genes could be demonstrated by testing KN6 hybridoma reactivity to cells from congenic strains that differ only at H-2. In addition, analysis of recombinant strains indicates that the gene controlling the KN6 ligand is located in or distal to the TL region. Involvement of the KN6 gammadelta T-cell receptor in this recognition process could be directly demonstrated by transferring the KN6 TL specificity after introduction of the productively rearranged KN6 gamma and delta genes into an alphabeta T-cell clone or into the germ line in transgenic mice. These observations raise the possibility that at least some gammadelta cells regulate hemopoietic cell maturation and activation.     

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.     

Predominant role of amino-terminal sequences in dictating efficiency of class II major histocompatibility complex alpha beta dimer expression

Cell surface expression of class II major histocompatibility complex-encoded (Ia) molecules depends on association of the component alpha and beta chains into a stable heterodimer. In the mouse, two isotypes of class II molecules have been identified, A beta A alpha and E beta E alpha. However, experiments from this laboratory have shown that, following DNA-mediated gene transfer into murine L cells, an A beta E alpha-mixed-isotype molecule can be assembled and expressed at the cell surface. In the present study, we have investigated the structural features of the beta chain that control the extent of association and level of membrane expression of A beta E alpha interisotypic pairs. The use of intact allelic A beta genes demonstrated that only A beta d chains, but not A beta b or A beta k chains, can be coexpressed on the surface membrane with E alpha chains. Transfection of recombinant A beta genes that encode all or half of the beta 1 domain from one allele and the rest of the chain from another allele revealed that the 5-7 polymorphic residues in the amino-terminal 50 residues of the A beta chain completely controlled this variation in expression with E alpha. Isotypically mixed beta genes encoding the A beta 1 domain of either A beta d or A beta k chains and the beta 2, transmembrane, and intracytoplasmic portions of E beta chains were used to assess the role of isotypically conserved structures in alpha beta pairing and expression. In marked contrast to the major alterations in expression accompanying changes in the amino-terminal polymorphic residues, exchange of these carboxyl-terminal isotypic segments had no detectable influence on the efficiency of expression with either A alpha or E alpha chains. These results argue strongly that variations in the efficiency with which distinct Ia alpha beta dimers assemble and are transported to the membrane is determined almost exclusively by a critical chain interaction involving the amino-terminal domains of the molecules.     

Identification of a shark sequence resembling the major histocompatibility complex class I alpha 3 domain.

Elasmobranchs (sharks and rays) branched off from the vertebrate line leading to the mammals more than 400 million years ago. They do not exhibit acute allograft rejection, and so far no report has pointed to the presence of major histocompatibility complex (MHC) antigen-like molecules in elasmobranchs. Here we demonstrate the existence of a gene in the shark (Triakis scyllia) genome with a sequence that resembles the MHC class I alpha 3 domains of other vertebrates. Several amino acids considered to be important for the interaction of the T-cell coreceptor CD8 with the MHC class I alpha 3 domain are conserved in the shark sequence.     

Binding of an invariant-chain peptide, CLIP, to I-A major histocompatibility complex class II molecules.

Invariant chain (Ii) associates with major histocompatibility complex (MHC) class II molecules and is crucial for antigen presentation by class II molecules. The exact nature of Ii interaction with MHC class II molecules remains undefined. A nested set of Ii peptides, CLIPs (class II-associated Ii peptides), have been eluted from various MHC class II molecules, suggesting that CLIPs correspond, at least in part, to the Ii motif which blocks the conventional peptide binding site in MHC class II molecules. Here we report how CLIPs interact with class II MHC molecules, I-A. We have identified regions critical for binding of CLIPs and I-A class II molecules. In most cases, the binding of CLIPs to a number of I-A molecules is modulated by the steric bulk of methionine residues at positions 93 and 99. In addition, the binding of CLIPs to an I-A molecule, I-Au, is sensitive to substitutions at aspartic acid-59 in the alpha chain and threonine-86 in the beta chain, whereas the binding of an antigen-derived peptide is not. Taken together, these results provide an insight as to how CLIPs bind to MHC class II heterodimers.     

Thymic depletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice.

Injection of mice transgenic for a class I major histocompatibility complex-restricted T-cell receptor with a soluble peptide antigen from influenza virus nucleoprotein results in clonal depletion of double-positive immature thymocytes in the thymus and activation of mature T cells in the periphery, accompanied by a transient up-regulation of the T-cell receptor and CD3 and CD8 coreceptor molecules.     

Antigen/major histocompatibility complex-specific activation of murine T cells transfected with functionally rearranged T-cell receptor genes.

The genes encoding the alpha and beta chains of the T-cell antigen receptor isolated from a cytochrome c-specific, major histocompatibility complex (MHC)-restricted murine T-cell hybridoma were introduced into a mouse T-cell line of helper lineage by electroporation. In order to examine the contributions of those gene products to antigen and/or MHC specificity, the resultant transfectants were tested for functional antigen and/or MHC recognition. Only those transfectants that express both the introduced genes (alpha and beta) contributed by the normal T cell can respond specifically to the appropriate antigen/MHC pair. None of the transfectants that express only one of the introduced genes (alpha or beta) of the normal T cell, or paired hybrid genes (i.e., one gene from the normal T cell and the other from the fusion partner), can respond to the same combination of antigen and MHC product recognized by the donor T cell. However, one clone expressing the transfected genes that encode the alpha and beta chains of the fusion partner shows reactivity to the antigen-presenting cells even in the absence of the antigens. These data suggest that the alpha beta heterodimer of the T-cell receptor is required to define the fine specificity of a T cell.     

Restricted and conserved T-cell repertoires involved in allorecognition of class II major histocompatibility complex.

The nature of the alloreactive T-cell response is not yet clearly understood. These strong cellular responses are thought to be the basis of allograft rejection and graft-vs.-host disease. The question of the extent of responding T-cell repertoires has so far been addressed by cellular cloning, often combined with molecular T-cell receptor (TCR) analysis. Here we present a broad repertoire analysis of primed responder cells from mixed lymphocyte cultures in which two different DR1/3 responders were stimulated with DR3/4 cells. Repertoire analysis was performed by TCR spectratyping, a method by which T cells are analyzed on the basis of the complementarity-determining region 3 length of different variable region (V) families. Strikingly, both responders showed very similar repertoires when the TCR V beta was used as a lineage marker. This was not seen when TCR V alpha was analyzed. A different pattern of TCR V beta was observed if the stimulating alloantigen was changed. This finding indicates that alloreactive T cells form a specific repertoire for each alloantigen. Since conservation appears to be linked to TCR V beta, the question of different roles of alpha and beta chains in allorecognition is raised.