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J D Fraser

Publications and source records attributed to J D Fraser.

At least 37 records · Page 2Linked to original sources
PubMed1997

Crystal structure of the streptococcal superantigen SPE-C: dimerization and zinc binding suggest a novel mode of interaction with MHC class II molecules.

Bacterial superantigens are small proteins that have a very potent stimulatory effect on T lymphocytes through their ability to bind to both MHC class II molecules and T-cell receptors. We have determined the three-dimensional structure of a Streptococcal superantigen, SPE-C, at 2.4 A resolution. The structure shows that SPE-C has the usual superantigen fold, but that the surface that forms a generic, low-affinity MHC-binding site in other superantigens is here used to create a SPE-C dimer. Instead, MHC class II binding occurs through a zinc binding site that is analogous to a similar site in staphylococcal enterotoxin A. Consideration of the SPE-C dimer suggests a novel mechanism for promotion of MHC aggregation and T-cell activation.

Amino Acid Sequence↗
PubMed1996

Cross-linking of MHC class II molecules by staphylococcal enterotoxin A is essential for antigen-presenting cell and T cell activation.

Two binding sites for MHC class II have previously been identified on opposite sides of the superantigen, staphylococcal enterotoxin A (SEA). The sites mediate separate binding reactions with nonoverlapping regions of class II, and in solution cause SEA to complex with purified HLA-DR1 to form DR1.SEA2 trimers. Here, a set of complementary SEA class II-binding mutants was used to study the interaction of SEA with cell surface MHC class II. The results indicate that both class II binding sites are required on the same toxin molecule for maximal activity, demonstrating that simultaneous ligation of two MHC class II molecules on APCs by a single SEA is essential for effective superantigen function. Coalescence of MHC class II by SEA results in protein tyrosine kinase activation and contributes to the induction of cell:cell adhesion, pro-inflammatory cytokine gene transcription, and T cell proliferation.

Antigen Presentation↗
PubMed1996

TCR binding differs for a bacterial superantigen (SEE) and a viral superantigen (Mtv-9).

Both superantigens (SAG) and many anti-TCR monoclonal antibodies (mAb) have specificity for the V beta region of the TCR encoded by TCRBV genes. For instance the bacterial SAG staphylococcal enterotoxin E (SEE), the retroviral SAG MTV-9 and the mAb OT145 each react with human T cells expressing BV6S7. This BV gene encodes two common alleles. We found that SEE and the mAb preferentially activate T cells expressing BV6S7*1 as opposed to BV6S7*2, but Mtv-9 activates T cells expressing either allele. Thus binding to the TCR differs between the two SAGs. A mutation in the TCR HVR-4 region of BV6S7*1 (G72E), where the two BV6S7 alleles differ, indicated that HVR-4 is a component of the binding site for SEE and for the mAb OT145. BV6S7*2 has a charged E72 which may result in electrostatic repulsion of SEE, as SEE contains a similarly acidic aspartic acid residue at a TCR interaction site (204D).

Alleles↗
PubMed1996

The influence of a recombinant-HBsAg vaccine on the repertoire of transcribed T-cell receptor beta chain variable region genes in peripheral blood in humans.

The purpose of this study was to determine whether in vivo changes in the repertoire of Tcr beta chain variable region (V beta) genes could be detected in peripheral blood mononuclear cells after immunization of humans with recombinant hepatitis B virus envelope protein (rHBsAg). We measured the percentage of Tcr RNA transcripts carrying each of 20 V beta genes in human PBMC before and after immunization with rHBsAg in Polynesians (8 non-immunized controls, 26 immunized subjects) and Europeans (9 non-immunized controls, 11 immunized subjects). The per cent of RNA transcripts containing V beta 7.4 family genes was increased in immunized vs control Polynesian (+ 1.6 +/- 0.5%) vs -1.1 +/- 0.3%, P = 0.0002) and European (+1.6 +/- 0.6% vs -0.1 +/- 0.5%, P = 0.05) subjects at 48 h and 28 h post-immunization, respectively. No changes in V beta repertoire were found after 48 h in either race. Thus, there is a transient increase in frequency of T cells with Tcr containing V beta 7.4 family genes within 48 h of an immunization containing rHBsAg in humans. There are a number of explanations for this finding, including the possibility that V beta 7.4 gene family products may be preferentially involved in the primary immune response to HBsAg.

Adolescent↗
PubMed1996

Repertoire of transcribed peripheral blood T-cell receptor beta chain variable-region genes in acute rheumatic fever.

Patients with severe group A streptococcal infections have abnormalities in the Vbeta repertoire of peripheral blood T cells that are consistent with superantigen stimulation by cytoplasmic membrane proteins. The purpose of this study was to determine whether similar changes in Vbeta repertoire could be found for patients with acute rheumatic fever (ARF). The mean Vbeta repertoire of peripheral blood T cells in nine hospitalized ARF patients was similar to that of 34 controls and did not change during 6 months of follow-up in 6 of the ARF subjects. We were unable to detect changes in the Vbeta repertoire of peripheral blood T cells from patients with ARF that could be attributed to the influence of a superantigen.

Acute Disease↗
PubMed1995

Isolation of HLA-DR1.(staphylococcal enterotoxin A)2 trimers in solution.

Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.

B-Lymphocytes↗
PubMed1995

Staphylococcal enterotoxin A has two cooperative binding sites on major histocompatibility complex class II.

The superantigen staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules at two sites on either side of the peptide groove. Two separate but cooperative interactions to the human class II molecule HLA-DR1 were detected. The first high affinity interaction to the DR1 beta chain is mediated by a zinc atom coordinated by H187, H225, and D227 in SEA and H81 in the polymorphic DR1 beta chain. The second low affinity site is to the DR1 alpha chain analogous to SEB binding and is mediated by residue F47 in SEA. Binding of one SEA to the DR1 beta chain enhances the binding of a second SEA molecule to the DR1 alpha chain. The zinc site is on the opposite side of the SEA molecule from residue F47 so that one SEA molecule can readily bind two class II molecules. Both binding sites on SEA are required for maximal activity. Thus, unlike, SEB, SEA requires two separate binding sites for optimal activity, which may allow it to stabilize SEA interaction with T cell receptors, as well as to activate the antigen-presenting cell by cross-linking MHC class II.

Amino Acid Sequence↗
PubMed1995

The influence of a maternal chronic hepatitis B virus infection on the repertoire of transcribed T-cell receptor beta chain variable region genes in human cord blood.

We used an anchor polymerase chain reaction method to compare the repertoires of transcribed T-cell receptor beta chain variable region (V beta) genes in cord blood T cells from neonates of hepatitis B surface antigen (HBsAg) positive (n = 40) and HBsAg negative (n = 40) women. Fifteen of the HBsAg positive women were hepatitis B e antigen (HBeAg) positive, and 25 were HBeAg negative. The percentage of V beta 7.4 transcripts was lower in cord blood T cells from neonates of HBsAg-positive relative to HBsAg-negative women (9.7% +/- 0.5% vs. 12.7% +/- 0.6%, P = .002). The percent of V beta 5.1 transcripts was higher in cord blood T cells from neonates of HBeAg-positive relative to HBeAg-negative women (9.3% +/- 0.7% vs. 7.0% +/- 0.3%, P < .001). There were no correlations between neonatal maturity at birth and V beta repertoire. In summary, a maternal chronic hepatitis B virus (HBV) infection is associated with changes in the repertoire of transcribed T-cell receptor genes in neonatal cord blood T cells. It is possible that the T-cell response to the HBV is associated with a limited repertoire of V beta genes. The mechanism of vertical chronic HBV infection in human neonates may involve changes in the T-cell response to the virus that are induced in utero.

Adult↗
PubMed1995

Peripheral blood T lymphocytes in systemic vasculitis: increased T cell receptor V beta 2 gene usage in microscopic polyarteritis.

Antigen recognition by T lymphocytes is mediated by cell surface receptors T cell specificity depends on the variable, diversity and junctional (VDJ) regions of the alpha and beta polypeptide chains of the T cell receptor (TCR). The expression of the variable region genes of the beta chain (V beta) has been analysed to study the involvement of peripheral blood T cells in systemic vasculitis. RNA was extracted from peripheral blood lymphocytes of 12 patients with microscopic polyarteritis, 10 with Wegener's granulomatosis, six with unclassified vasculitis, and 28 healthy age- and sex-matched individuals. Complementary DNA was made from RNA and amplified by the anchored polymerase chain reaction (PCR) using redundant oligonucleotide primers for the TCR V beta genes. To determine if the dominant usage of a V beta gene family reflected the presence of particular T cell clones, cDNA was amplified with primers for the specific V beta gene family. The product was screened for sequence homogeneity by single-stranded conformational polymorphism (SSCP) and cloned to sequence the adjoining TCR (D beta) J beta region. A significant increase in the mean percentage expression of the V beta 2.1 gene was seen in vasculitis patients (11.4 + 1.0% (mean + s.e.m.)) compared with controls (6.6 + 0.6%; P < 0.003). The most marked increase was seen in microscopic polyarteritis (13.9 + 1.7%; P < 0.0001). There were also increases in the expression of V beta 3, 13 and 14 in peripheral blood of vasculitis patients compared with controls. SSCP analysis of V beta 2.1 amplified products indicated the presence of oligoclonal bands in a smaller proportion of patients (8/27) than controls (12/28). There was no strong evidence for the conservation of the TCR V beta 2.1 junctional region sequence data from a sample group of three patients with oligoclonal bands. Thus, a subset of patients with systemic vasculitis, particularly those with microscopic polyarteritis, have increased TCR V beta 2.1 gene expression in their peripheral blood T cell repertoire. As superantigens binding V beta 2.1 are postulated to activate T cells with diverse CDR3 sequences, it is proposed that a superantigen is involved in the immunopathogenesis of vasculitis.

Adult↗
PubMed1994

The cytoplasmic domain of CD28 is both necessary and sufficient for costimulation of interleukin-2 secretion and association with phosphatidylinositol 3'-kinase.

T-cell activation requires two signaling events. One is provided by the engagement of the T-cell antigen receptor, and the second represents a costimulatory signal provided by antigen-presenting cells. CD28 mediates a costimulatory signal by binding its ligands, B7-1 and B7-2, on antigen-presenting cells, but the signaling pathway activated by CD28 has not been identified. A homologous molecule, CTLA-4, expressed on activated T cells, also binds to B7-1 and B7-2, but whether it has a signaling function is not known. We performed a structure-function analysis of CD28 to identify the functional domain which activates signal transduction. Truncation of the 40-amino-acid CD28 cytoplasmic tail abrogated costimulatory signaling. Chimeric constructs containing the extracellular and transmembrane regions of CD8 linked to the cytoplasmic region of CD28 had a costimulatory signaling function. Similar chimeras containing the cytoplasmic tail of CTLA-4 did not signal. Thus, the cytoplasmic region of CD28, but not CTLA-4, is sufficient to mediate costimulatory signaling. In addition, after CD28 stimulation, the p85 subunit of phosphatidylinositol 3'-kinase and phosphatidylinositol 3'-kinase activity were found in CD28 immunoprecipitates. The CD8-CD28 chimera, which has a costimulatory signaling function, associates with p85, while the nonfunctioning CD8-CTLA-4 chimera and a CD8-zeta chimera do not associate with p85. These results suggest that phosphatidylinositol 3'-kinase is specifically activated by CD28 and may mediate proximal events in the costimulatory signaling pathway regulated by CD28.

Abatacept↗
PubMed1993

Two adjacent residues in staphylococcal enterotoxins A and E determine T cell receptor V beta specificity.

The T cell receptor (TCR) V beta-determining region of two bacterial superantigens, staphylococcal enterotoxin A (SEA) and SEE, has been mapped to the COOH-terminal region of SEA and SEE using a panel of recombinant SEA/SEE hybrids. Total TCR V beta mRNA enrichment in human peripheral blood T cell cultures was determined by a novel single-tube amplification technique using a redundant V beta-specific primer. SEA routinely enriched mRNA coding for hV beta 1.1, 5.3, 6.3, 6.4, 6.9, 7.3, 7.4, and 9.1, while SEE, which is 83% homologous to SEA, enriched hV beta 5.1, 6.3, 6.4, 6.9, and 8.1 mRNA. Exchanging residues 206 and 207 was sufficient to convert in toto the TCR V beta response of human peripheral T lymphocytes. In addition, an SEA-reactive murine T cell line, SO3 (mV beta 17), unresponsive to wild-type SEE responded to SEE-S206N207, while an SEE-specific human T cell line, Jurkat (hV beta 8.1), unresponsive to SEA was stimulated strongly by SEA-P206D207. Exchanging all other regions of SEA and SEE except residues 206 and 207 did little to change the V beta response. Thus, the V beta binding region appears to be a stable, discrete domain localized within the COOH-terminal region that is largely unaffected by the considerable amino acid variability between SEA and SEE. This region may interact directly with TCR V beta.

Base Sequence↗
PubMed1993

Signal transduction events leading to T-cell lymphokine gene expression.

The expression of T-cell derived lymphokines is regulated by signal transduction events initiated by the T-cell antigen receptor and other T-cell surface molecules. Substantial progress has been made in characterizing the signal transduction events initiated at the plasma membrane of the T cell and their targets which control lymphokine gene expression in the nucleus. This review will summarize recent progress in this area of investigation.

Animals↗
PubMed1993

Population study of T cell receptor V beta gene usage in peripheral blood lymphocytes: differences in ethnic groups.

The T cell receptor (TCR) V beta repertoire in peripheral blood lymphocytes (PBL) of a large number of healthy individuals was analysed by quantifying V beta-specific mRNA using the method of anchored multiprimer DNA amplification and a reverse dot blot assay. Among 16 V beta gene families examined, particular V beta genes were noted to be unequally expressed in the PBL of 70 healthy donors. The frequently used genes belong to the V beta 4, 5, 6, 8 and 13 (12) families, while V beta 1, 9 and 15 were the least frequently used gene families. This bias in gene usage was observed in all individuals. Marked deviation from the mean percentage usage was noted for some V beta genes in individuals when their PBL were examined serially, but the common pattern of biased usage was not grossly distorted. When the TCR repertoire of different ethnic groups was examined, a lower mean frequency of V beta 3.2 was seen in the repertoire of 19 Caucasians compared with 25 age-matched Samoans (P < 0.003). Conversely, the expression of V beta 5.1 and V beta 5.3 was higher in Caucasians than in 51 age-matched Polynesians (Maoris and Samoans, P < 0.003). Considering the 20% co-efficient of variation in the estimate of V beta gene usage, our data from 70 unrelated individuals suggest that in PBL, individual variations in the TCR repertoire were superimposed upon a common biased usage of V beta genes in the general population.

Base Sequence↗
PubMed1993

T-cell receptor beta-chain binding to enterotoxin superantigens.

The last few years have seen an enormous jump in our knowledge and understanding of T-cell activation by superantigens. Clearly, a great number of infectious and parasitic organisms utilize superantigens as part of a strategy to evade the immune response of their host. The ability to modulate superantigen effects will give us new means to fight infections, and the knowledge of T-cell activation that we have gained from study of superantigens will, in turn, allow us to modulate the immune system in new ways.

Animals↗
PubMed1992

Superantigen data.

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Antigens, Viral↗
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↗