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N R Gascoigne

Publications and source records attributed to N R Gascoigne.

15 recordsLinked to original sources
PubMed1992

Enterotoxin residues determining T-cell receptor V beta binding specificity.

Superantigens such as the staphylococcal enterotoxins bind to major histocompatibility complex (MHC) class II molecules and activate T cells through a specific interaction between the V beta region of the T-cell antigen receptor (TCR) and the toxin. The TCR beta-chain alone is sufficient to produce the interaction with the enterotoxin-class II complex. Identification of the regions of enterotoxins that interact with TCR has so far proved equivocal because of difficulties in distinguishing between direct effects on T-cell recognition and indirect effects resulting from alteration of binding to class II. For example, amino-terminal truncations of SEB abrogated T-cell stimulation whereas carboxy-terminal truncation of SEA stopped its mitogenic activity. The most comprehensive study to date, accounting for both enterotoxin binding to class II and enterotoxin interactions with the TCR, identified two functionally important regions for SEB binding to TCR. Although the amino-acid sequences of staphylococcal enterotoxins A and E are 82% identical, they activate T cells bearing different V beta elements. We have assayed the binding of cells coated with these enterotoxins to soluble secreted TCR beta-chain protein and find that V beta 3 binds enterotoxin A but not E, whereas V beta 11 binds enterotoxin but not A. To map the amino-acid residues responsible for these different binding specificities, we prepared a series of hybrids between the two staphylococcal enterotoxins. We report that just two amino-acid residues near the carboxy terminus of the enterotoxins are responsible for the discrimination between these molecules by V beta 3 and V beta 11.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

T-cell antigen receptor binding sites for the microbial superantigen staphylococcal enterotoxin A.

We have examined the interaction of the microbial superantigen staphylococcal enterotoxin A (SEA) with peptides corresponding to overlapping regions of the T-cell antigen receptor beta chain variable region V beta 3. SEA is known to stimulate murine T cells bearing certain V beta elements, among them V beta 3. Five peptides were synthesized representing amino acids 1-24, 20-44, 39-60, 57-77, and 74-95 of V beta 3. We demonstrate here that soluble V beta 3-bearing beta chains can bind to a complex of SEA and major histocompatibility complex class II and that the synthetic peptide V beta 3-(57-77) blocked this interaction. The peptide V beta 3-(57-77) also inhibited SEA-induced interferon-gamma production and SEA-induced proliferation of B10.BR spleen cells. Conversely, the peptide corresponding to amino acids 57-77 of V beta 8.2, a V beta element that is not recognized by SEA, decreased staphylococcal enterotoxin C-2-induced proliferation but did not affect SEA-induced proliferation. The peptide inhibition of SEA-induced function was due at least in part to inhibition of V beta 3-bearing T-cell activity, since the percentage of T cells reactive with an anti-V beta 3 monoclonal antibody was significantly reduced by V beta 3-(57-77). These data suggest that the region of V beta 3 encompassing amino acids 57-77 is an area that displays the appropriate sequence and conformation for binding of the SEA molecule and blocking of the resultant interaction with the T-cell antigen receptor.

Amino Acid Sequence↗
PubMed1992

Chromosome 14 in B10.A(18R) mice is recombinant and includes Tcra-Va alleles.

Analysis of mouse Tcr genes has previously defined at least five different Tcra-V haplotypes among inbred strains of mice. For mice of the Tcra-Vb haplotype, including C57BL/10 (B10), T-cell expression of the Tcra-V11 gene subfamily can be detected with a monoclonal antibody, 1.F2. In the course of further characterizing the specificity of 1.F2, we found that it fails to recognize Tcra-V11-expressing T-cell hybrids derived from the B10 congenic strain, B10.A(18R)/SgIcr. Moreover, staining analysis indicated that the Va11 epitope recognized by 1.F2 is not expressed by peripheral T cells from several different B10.A(18R) colonies with the exception of that at the Research Institute of Scripps Clinic. Nucleotide sequences were determined for cDNA representing rearranged Tcra-V11 genes from two independent, B10.A(18R)/SgIcr derived T-cell hybrids. The two Tcra-V11 gene segments were identical and the predicted amino acid sequence differed by at least five residues from Tcra-V11 sequences previously obtained from B10.A mice. Southern blot analysis of restriction fragment length polymorphisms (RFLP) associated with Tcra-V11, as well as Tcra-V1, subfamily genes revealed that the B10.A(18R) mouse has inherited Tcra-Va alleles rather than the expected Tcra-Vb alleles from the B10 strain. RFLP analysis of the Rib-1 locus, located in close proximity to the Tcra locus on chromosome 14, showed that B10.A(18R) carries the Rib-1b allele from B10. These results indicate that the B10.A(18R) mouse has inherited a recombinant chromosome 14 with a recombination event having occurred between the Rib-1 locus and the Tcra-V gene subfamilies examined. Inheritance of Tcra-Va alleles in B10.A(18R) probably originated from strain 129/J which breeding records show was used in the first cross with B10.A in the production of B10.A(18R) and which we found exhibits Tcra-V11a RFLPs.

Alleles↗
PubMed1991

Profound alteration in an alpha beta T-cell antigen receptor repertoire due to polymorphism in the first complementarity-determining region of the beta chain.

Amino acid residues that are critical in maintaining the framework structure of immunoglobulin heavy- and light-chain variable (V) regions are strongly conserved in the V alpha and V beta proteins of the alpha beta T-cell antigen receptor (TCR alpha beta). Consequently, it has been proposed that TCR alpha beta has a conformation similar to that of an immunoglobulin Fab fragment and that the regions of the TCR homologous to the three immunoglobulin complementarity-determining regions (CDRs 1, 2, and 3) bind to the peptide antigen-major histocompatibility complex (MHC) molecule ligand. A single amino acid substitution in the predicted CDR1 of the V beta 3 protein of certain mouse strains dramatically altered TCR alpha beta usage in an antigen-specific MHC-restricted immune response but did not abrogate V beta 3 specificity for the superantigens minor lymphocyte stimulatory locus (Mls)c and staphylococcal enterotoxin A (SEA). The results confirm the importance of the V beta CDR1 in antigen-MHC molecule recognition, supporting the Fab-like structural model of TCR alpha beta, and provide further evidence that conventional antigen-MHC recognition and superantigen recognition are mediated by distinct regions of the TCR beta chain. They also suggest that allelic polymorphism may be a significant source of diversity in the TCR repertoire.

Animals↗
PubMed1991

The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele-specific antibodies.

Allelic polymorphism in TCR loci may play an important role in shaping the T cell repertoire and in disease susceptibility. We have used a combination of antibody and sequence analysis to investigate polymorphism in the murine V alpha 11 family. Two different antibodies have been analyzed that recognize particular V alpha 11 family members of the V alpha b and V alpha d haplotypes. One antibody shows J alpha dependency, suggesting a conformational element to the epitope. Investigation of the anti-V alpha 11 staining pattern on different mouse strains indicates that there is a marked influence of MHC haplotype on V alpha 11 selection and that V alpha 11 is preferentially expressed on CD4+ cells. Sequence analysis of V alpha 11 genes from the V alpha a, V alpha b, and V alpha d haplotypes shows two potential regions for the haplotype-specific epitopes. The relatedness of the different V alpha 11 family members from different haplotypes suggests that the V alpha 11.1/11.2 gene duplication is relatively recent, but that V alpha 11.3 separated much earlier. Differences between V alpha 11.3 and V alpha 11.1/11.2 are concentrated in the putative complementarity determining regions (CDR), whereas differences between alleles are not clearly clustered. However, the V alpha 11.1a and V alpha 11.1d alleles differ from V alpha 11.1b and V alpha 11.2b in CDR1. A V alpha 11.2-expressing anti-cytochrome c T cell has the same V-J junction as a V alpha 11.1-bearing cell with a similar fine specificity, indicating that V alpha 11.1b and V alpha 11.2b do not contribute different Ag specificities.

Alleles↗
PubMed1991

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.

Antibodies, Monoclonal↗
PubMed1990

A T cell receptor V alpha region selectively expressed in CD4+ cells.

The peripheral TCR V beta repertoire is strongly influenced by the processes of negative selection (deletion) and positive selection in the thymus. In order to investigate whether such selection events influence the V alpha repertoire, we have produced an anti-V alpha 11 mAb. This antibody was made by immunization with a chimeric TCR:Ig protein containing V alpha 11 in place of the VH of an IgG2a, lambda Ig. This scheme optimizes the specificity of immunization and facilitates the screening procedure. The antibody recognizes a panel of V alpha 11-expressing T cell clones. Analysis of mouse strains indicates that the antibody recognizes V alpha 11 only in mice of the C57 background. The expression of the epitope on peripheral T cells is strongly biased to the CD4+ subset, suggesting positive selection of V alpha 11 on class II MHC molecules. In some strain comparisons, the percentage of V alpha 11-expressing T cells in the CD4+ subset was elevated in I-E+ relative to I-E- strains. These data suggest that V alpha 11 can differentially influence the selection of T cells into the CD4+/CD8+ subsets.

Animals↗
PubMed1990

Transport and secretion of truncated T cell receptor beta-chain occurs in the absence of association with CD3.

The T cell receptor (TCR) beta-chain is produced in the endoplasmic reticulum where it associates with the TCR alpha-chain and the members of the CD3 complex to form the complete receptor. When the other chains of the complex are not available, the beta-chain is rapidly degraded within the endoplasmic reticulum. When incomplete TCR.CD3 complexes are formed, they are transported through the Golgi apparatus and degraded in lysosomes. In this study, a truncated form of the TCR beta-chain has been made by removal of the transmembrane and cytoplasmic segments. Unlike the normal beta-chain, the truncated molecule is stable and is transported through the Golgi apparatus and secreted. This process occurs at a similar rate in both T and B cells, indicating that it is not affected by the presence or absence of CD3 components. These data suggest that an element in the transmembrane or cytoplasmic region of the beta-chain confers sensitivity to the degradative control mechanisms that regulate TCR expression.

Acetylglucosaminidase↗
PubMed1989

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor.

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.

Animals↗
PubMed1987

Secretion of a chimeric T-cell receptor-immunoglobulin protein.

To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable.

Amino Acid Sequence↗
PubMed1986

Expression of T cell receptor genes in an antigen-specific hybridoma and radiation-induced variants.

We have analyzed a series of mutants derived from a KLH-specific, I-E-restricted T hybridoma (FN1-18) which have lost antigen-reactivity while retaining both T cell receptor idiotypic determinants and the ability to respond to Con A. The variants have not gained any detectable alloreactivity, nor is there an obvious lesion in the mutants' beta chain DNA containing the utilized beta chain genes. This loss of antigen reactivity is due to a failure of stable production of the specific V beta-containing mRNA. Our results indicate that in FN1-18, the T cell receptor antigenic determinants are most likely carried by the alpha chain alone or by a complementation product of the V alpha FN1-18 with the V beta of BW5147. V beta FN1-18 represents a previously undescribed T cell receptor V region.

Animals↗
PubMed1986

T-cell receptor gene structure and function.

Recent progress in the serology, biochemistry, and now the molecular genetics of T-cell receptor molecules has brought within reach the prospect of solving some of the most basic questions about the nature of T-cell recognition. These include the exact nature of the receptor-major histocompatibility complex (MHC)-antigen recognition event and the sequential expression of T-cell receptor molecules in the thymus.

Animals↗
PubMedDate not supplied

Variability and repertoire size of T-cell receptor V alpha gene segments.

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

Animals↗