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R D Salter

Publications and source records attributed to R D Salter.

At least 37 records · Page 2Linked to original sources
PubMed1992

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.

Animals↗
PubMed1992

Mutant HLA-A201 heavy chains with lowered affinity for beta 2m are transported after growth at reduced temperatures.

Forty-five site-directed mutants bearing single amino acid substitutions in the alpha 3 domain of the class I molecule HLA-A201 were previously transfected into CIR cells and screened for surface expression by antibody binding. Eight mutants are expressed at significantly reduced levels relative to HLA-A201, including two with substitutions at residues contacting beta 2m. One of the latter mutations, position 242 gln > lys (242K), is now shown to block assembly with beta 2m and prevent intracellular transport at 37 degrees C. At temperatures ranging from 21 degrees C to 30 degrees C, 242K heavy chains and beta 2m form dimers that are exported to the cell surface. Surface expression at 26 degrees C is not blocked by cycloheximide pretreatment, but is completely inhibited by Brefeldin A, suggesting that at 37 degrees C preformed heavy chains accumulate in the ER. Glycans on the retained heavy chains are sensitive to digestion by Endo H, but become Endo H resistant after cells are grown at 26 degrees C. Preincubation of 242K cells with synthetic peptides shown previously to bind HLA-A201 does not increase reactivity with anti-HLA-A2 antibodies, suggesting that the defective phenotype is not due to instability of cell surface mutant class I dimers, but derives instead from impaired assembly of 242K heavy chains with beta 2m inside the cell. This contrasts with mutant cells such as .174, T2 and RMA-S, which exhibit defects in internal peptide transporters, but assemble and export "empty" dimers to their surfaces that can be stabilized subsequently by exogenous peptides. 242K mutants may therefore be suited uniquely for studying assembly and peptide binding to class I molecules in the ER.

Amino Acids↗
PubMed1992

Role of endogenous peptide in human alloreactive cytotoxic T cell responses.

The T x B hybrid 174 x CEM.T2 (T2) has been shown to be defective in the processing of proteins for presentation by MHC class I molecules. It continues, however, to express significant quantities of HLA-A2.1, suggesting that this class I molecule is expressed either in a largely peptide-free form or in association with a small subset of peptides. In this paper T2 was used in conjunction with limiting dilution analysis to provide a direct estimate of the fraction of alloreactive cytotoxic T lymphocytes (CTLs) that were dependent upon the presence of peptides for their recognition of HLA-A2.1. Alloreactive cytotoxic T cell lines generated by stimulation with HLA-A2.1 expressing peripheral blood lymphocytes recognized T2 poorly. Split-well analysis of 240 clonal limiting dilution cultures demonstrated that this reflected the existence of two subpopulations. An average 85% of HLA-A2.1 specific CTLs recognized HLA-A2.1 on normal cells but not on T2. The remainder recognized HLA-A2.1 on both T2 and normal targets. CTL lines with the latter specificity could be generated by using T2 as a stimulator cell. Using target cells that either expressed a lower density of HLA-A2.1 or that expressed HLA-A2 molecules that had been mutated to affect CD8 binding, no significant differences in avidity between T2-reactive and T2-unreactive CTLs were seen. Thus the failure of the majority of alloreactive CTLs to recognize T2 is not a consequence of the lower level of HLA-A2.1 surface expression on this cell, but is instead due to the absence of appropriate epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗
PubMed1991

Class I-induced resistance to natural killing: identification of nonpermissive residues in HLA-A2.

Structural characteristics of major histocompatibility complex class I antigens associated with natural killer (NK)-resistance phenomena were examined. Previous research has shown that transfection of class I genomic DNA clones into class I-deficient, NK-sensitive target cell lines results in transfectants exhibiting class I+, NK-resistant phenotypes. In contrast to the HLA-A3, -B7, -B27, and -Bw58 class I molecules, the HLA-A2 class I molecules were shown not to protect target cells from NK activity. Here we show that this nonprotective phenotype maps to the alpha 1 domain of the HLA-A2 molecule by examining the NK-protective capacity of the natural interdomain recombinant HLA-Aw69 molecule. HLA-Aw69, which consists of an alpha 1 domain exhibiting homology with HLA-Aw68, and alpha 2/alpha 3/transmembrane-cytoplasmic domains, exhibiting homologies with HLA-A2, mimics HLA-Aw68 and provides HLA-A,B null target cell (C1R) transfectants with increased resistance to NK. Further, the inability of transfected HLA-A2 to confer protection against NK activity can be completely attributed to the expression of a "nonpermissive" residue at position 74 in the alpha 1 domain. Site-directed mutation of the His-74 residue in HLA-A2 to the Asp-74 (HLA-A3, -Aw68, -Aw69, -B7) residue generates a mutant that provides C1R cell line transfectants an NK-resistant phenotype. As His-74 blocks access to a side pocket in the HLA-A2 antigen-binding cleft, these results support the critical involvement of residues within the peptide-binding groove of class I molecules in determining the NK susceptibility phenotype of class I+ target cells.

Alleles↗
PubMed1991

CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA-Aw68.1.

The crystal structure of the HLA-Aw68.1 antigen binding site revealed a negatively charged pocket centred on aspartic acid 74 (Garrett et al. 1989). Access to this '74 pocket' is blocked in HLA-Aw68.2 and HLA-Aw69 by two substitutions at positions 97 and 116. This key feature suggests that the Aw68.1-peptide-specific interactions may involve salt bridges between oppositely charged residues. In this paper, the influenza epitope recognized by virus-specific HLA-Aw68.1-restricted cytotoxic T lymphocytes (CTL) has been defined in vitro with a synthetic peptide corresponding to residues 89-101 of the nucleoprotein (NP). Amino acid substitutions of the peptide NP 89-101 showed that the arginine at position 99 is an anchor point of the peptide within the Aw68.1 antigen binding site. Consistent with this we find that neither HLA-Aw68.2 nor HLA-Aw69 positive cells can present peptide NP 89-101 to Aw68.1-restricted CTL. Our results therefore suggest a model in which presentation of NP 89-101 by HLA-Aw68.1 is dependent upon interaction of the positively charged arginine residue at position 99 of the peptide, with the negatively charged aspartic acid in the '74 pocket' of HLA-Aw68.1. We also show that influenza-virus-specific HLA-Aw68.1-restricted CTL are CD8 independent. This result is consistent with the low affinity of HLA-Aw68.1 for CD8 (Salter et al. 1989) and reveals a unique example of CD8-independent priming of CTL by natural infection with a common pathogen in humans.

Amino Acid Sequence↗
PubMed1990

A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2.

Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.

Antigens, CD↗
PubMed1990

Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification.

A method for cloning full-length HLA-A,B cDNA (1.1 kilobases) by using the polymerase chain reaction (PCR) is described. Six HLA-A,B alleles (HLA-A2, -A25, -B7, -B37, -B51, and -B57) were cloned, and their structures were determined. Multiple PCR clones for each allele were sequenced to obtain both an accurate consensus sequence and an "authentic" clone having that sequence. Sequences from 50 clones encoding five different alleles permit assessment of the frequency and nature of PCR-produced errors. These include recombinations, deletions, and insertions in addition to point substitutions. Authentic clones were obtained at a frequency of between 30% and 70%, and analysis of three or four clones generally should be sufficient for characterization of an allele.

Alleles↗
PubMed1989

Polymorphism in the alpha 3 domain of HLA-A molecules affects binding to CD8.

Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

HLA-B51 and HLA-Bw52 differ by only two amino acids which are in the helical region of the alpha 1 domain.

Genes encoding the serologically cross-reactive HLA-B51 and HLA-Bw52 molecules were isolated and the exons sequenced. HLA-B51 genes obtained from Caucasian and Oriental individuals were identical. HLA-Bw52 differs from HLA-B51 by four nucleotide substitutions in exon 2 encoding the alpha 1 domain. These comprise one isolated silent substitution in codon 23 and a cluster of three coding substitutions in codons 63 and 67. Amino acid substitutions of N----E at position 63 and F----S at position 67 are the only differences between HLA-B51 and HLA-Bw52 and these residues are postulated to form HLA-B51 specific epitopes. HLA-B51 could have been formed from HLA-Bw52 by the combination of a genetic exchange with HLA-B8 and a point mutation. Similarity of HLA-B51 and HLA-Bw52 with HLA-Bw58 suggest they also share a common ancestor.

Amino Acid Sequence↗
PubMed1988

Cell-cell adhesion mediated by CD8 and MHC class I molecules.

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Nature of polymorphism in HLA-A, -B, and -C molecules.

Diversity in 39 HLA-A, -B, and -C molecules is derived from 20 amino acid positions of high variability and 71 positions of low variability. Variation in the structurally homologous alpha 1 and alpha 2 domains is distinct and may correlate with partial segregation of peptide and T-cell receptor binding functions. Comparison of 15 HLA-A with 20 HLA-B molecules reveals considerable locus-specific character, due primarily to differences at polymorphic residues. The results indicate that genetic exchange between alleles of the same locus has been a more important mechanism in the generation of HLA-A, -B, and -C diversity than genetic exchange events between alleles of different loci.

Amino Acid Sequence↗
PubMed1987

In vitro mutagenesis at a single residue introduces B and T cell epitopes into a class I HLA molecule.

We have studied the interaction of HLA class I antigens with alloreactive cytotoxic T lymphocytes and monoclonal antibodies using site-directed mutagenesis and expression of an HLA-Aw68.1 gene. Two mutants containing distinct substitutions at polymorphic residues near the NH2-terminal end of the alpha 2 domain were made. One mutant with substitutions at positions 95 and 97 corresponding to residues found in HLA-A2.1 showed no alterations in binding of HLA-Aw68- or HLA-A2-specific monoclonal antibodies, but was reactive with some HLA-A2-specific CTL clones. A second mutant, in which glycine at position 107 was replaced with tryptophan found at that position in HLA-A2.1, was recognized by HLA-A2-specific CTL clones and HLA-A2, Aw69-specific monoclonal antibodies. Thus, substitution of a single amino acid residue at position 107 of the HLA-Aw68.1 molecule generates an allospecific determinant shared with HLA-A2.1 and recognized by both B and T lymphocytes.

Amino Acid Sequence↗
PubMed1986

Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid.

Biosynthesis of HLA class I antigens has been studied in a variant B-LCLxT-LCL hybrid, 174XCEM.T2. This cell line encodes HLA-A2 and -B5, but expresses only small amounts of A2 antigen and undetectable B5 antigen at the cell surface due to a mutation inactivating a trans-acting regulatory gene encoded within the class II region of the human major histocompatibility complex. Northern blot analysis with HLA-A- and HLA-B-specific probes shows that 174XCEM.T2 synthesizes quantities of A and B locus mRNA comparable with its class I antigen-positive parent cell line. Immune precipitation studies indicate that 174XCEM.T2 synthesizes normal HLA heavy chains and beta 2-microglobulin which fail to form dimers. The heavy chains are N-glycosylated normally, but processing of the glycan to the complex form does not occur. In addition, free heavy chains in this cell line are not phosphorylated. Thus, the majority of class I heavy chains in 174XCEM.T2 do not combine with beta 2-microglobulin, and are not processed or transported to the cell surface. As both subunits are synthesized in normal amounts, we propose that an additional molecule absent from 174XCEM.T2 and encoded by an HLA-linked gene is necessary for efficient assembly of class I antigen subunits.

Animals↗
PubMed1986

Expression of differentiation antigens by hybrids of human lymphoblastoid cells.

In previous communications, we have described the expression of class I and class II histocompatibility antigens by hybrids of human B and T lymphoblastoid cell lines (B- and T-LCL). In all cases, such hybrids were found to resemble their B-LCL parents, expressing high levels of class I and class II antigens encoded by both parent cell lines. In the current study, we have conducted a more extensive analysis of B-LCLxT-LCL hybrids with a panel of monoclonal antibodies recognizing a variety of B and T lymphocyte differentiation markers. Rather than exhibiting a B-LCL-dominant phenotype, most hybrids were found to express a majority of both T and B lymphocyte antigens expressed by their parent cell lines. Several hybrids of pairs of dissimilar T-LCL were also produced and analyzed. Again, a majority of parental antigens was expressed on the hybrids. However, eight of eight hybrids of the T-LCL CEM and HSB failed to express HNK-1, an antigen strongly expressed by HSB; and two hybrids of the T-LCL CEM and SKW3 expressed CD3, an antigen expressed by neither parent cell line.

Antibodies, Monoclonal↗
PubMed1985

Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids.

Regulation of HLA class I and class II antigen expression was studied in hybrids of human T and B lymphoblastoid cell lines (LCL). The T-LCL CEMR.3 expresses no HLA class II antigens. It expresses little total HLA class I antigen and no HLA-B antigens. The B-LCL 721.174 is a radiation-induced variant immunoselected for loss of class II antigen expression. In addition to showing a deletion of all HLA-DR and DQ structural genes, 721.174 expresses no HLA-B antigens and a decreased level of HLA-A antigen compared with the parental cell line. A hybrid of 721.174 and CEMR.3 expresses class II antigens encoded by CEMR.3. Increased expression of HLA class I antigens encoded by both 721.174 and CEMR.3 was also observed. Specifically, the previously undetectable HLA-B5 and HLA-Bw6 antigens encoded by 721.174 and CEMR.3, respectively, were present on the hybrid. Increased expression of the HLA-A2 antigen encoded by 721.174 was also observed. An immunoselected variant of the hybrid lacking both CEMR.3-derived copies of chromosome 6 lost expression of the HLA-B5 antigen encoded by 721.174 and expressed a decreased amount of HLA-A2. From these data, we infer that two complementary trans-acting factors mediate enhanced expression of HLA class I antigens in the hybrid. One of these factors is provided by a gene located on chromosome 6 derived from CEMR.3. The second factor, introduced by 721.174, is the gene previously postulated to induce expression of CEMR.3-encoded class I antigens in hybrids of CEMR.3 with B-LCL.

B-Lymphocytes↗
PubMed1985

Evidence for methylation as a regulatory mechanism in HLA-DR alpha gene expression.

We examined the possibility that one mechanism for controlling HLA-DR alpha gene expression occurs through DNA hypomethylation. We employed the restriction enzyme Hpa II, which recognizes the sequence 5'CCGG3' but not 5'CmCGG3', to study DNA methylation. We first compared a DR-positive B lymphoblastoid cell line (LCL) with an isogenic DR-negative T-LCL. Using a genomic probe for the DR alpha gene, we showed that an Hpa II digestion of DNA from the B-LCL resulted in bands of lower molecular weight than that of the T-LCL. This indicates that the B-LCL DR gene is hypomethylated relative to the T-LCL gene. Demethylation of the gene from the B-LCL is incomplete, suggesting that complete demethylation is not required for its expression. We also examined somatic cell hybrids of T-LCL and B-LCL since the DR alpha gene, which is inactive in the T-LCL, is expressed in the hybrids, providing a system to study DR alpha gene induction. We examined the hybrid line 174 X CEM.T1, which contains and expresses solely the DR alpha gene from the T-LCL parent since both copies of the DR alpha gene from the B-LCL parent, 174, are deleted. The expressed DR alpha gene from the hybrid was compared with the unexpressed gene from the T-LCL parental line, and again an association between DR alpha gene expression and DNA hypomethylation was observed. In contrast to the DR alpha gene from B-LCL, which is not completely demethylated, the DR alpha gene in this hybrid line is not methylated at either of the Msp I sites covered by our probe. This suggests that different regulatory mechanisms operating through DNA methylation may be involved in the expression of DR alpha genes from T-LCL and B-LCL. Examination of another hybrid line which has DR alpha genes from both parental lines supports this contention. The implications of these findings are discussed.

B-Lymphocytes↗