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R DeMars

Publications and source records attributed to R DeMars.

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

New genes in the MHC that encode proteins for antigen processing.

Most cells process proteins into short peptides that are displayed on the cell surface bound to class I or class II proteins encoded by the major histocompatibility complex (MHC). These protein-peptide complexes can then be recognized by the circulating lymphocytes of the immune system. Several genes found recently in the MHC encode proteins with possible roles in the supply of peptides to class I molecules. The results imply that the peptides are produced in the cytoplasm by proteasomes and are translocated into the endoplasmic reticulum by 'peptide transporters' related to the multidrug resistance proteins. While there is little biochemical evidence to validate these ideas, Robert DeMars and Thomas Spies discuss here the arguments supporting this view. New data indicate that there may also be factors for class II peptide-processing hidden in the MHC.

Journal Article↗
PubMed1992

No evidence for germline mutations in exons 5-9 of the p53 gene in 25 breast cancer families.

Recent studies have demonstrated that families with the Li-Fraumeni syndrome carry inherited point mutations of the p53 gene. In the present study 25 families with strong histories of breast cancer were screened for the presence of such mutations. Polymerase chain reaction products of exons 5-9 of the p53 gene were examined by single-stranded conformational polymorphism analysis and, in addition, exon 7 was further screened by direct sequencing. No mutations were detected in constitutive DNA by either method. These results indicate that familial breast cancer does not usually result from germline point mutations in the p53 gene.

Breast Neoplasms↗
PubMed1991

Restored expression of major histocompatibility class I molecules by gene transfer of a putative peptide transporter.

Cytotoxic T lymphocytes recognize antigen-derived peptides bound to major histocompatibility complex (MHC) class I molecules with which they assemble in the endoplasmic reticulum or in an undefined subcompartment. There is genetic evidence that the peptides that are products of cytosolic protein degradation are transported into this compartment by a peptide supply factor (PSF), encoded in the MHC class II region. Like the corresponding genes RING4, HAM1 and mtp1, PSF is related to the multidrug-resistance family of transporters and may be a peptide pump, as translocation of peptides across membranes must occur independently of the secretory pathway. There is, however, no functional evidence for this role so far. Here we report gene transfer experiments showing that expression of PSF complementary DNA in the human lymphoblastoid cell line mutant 721.134 restores normal levels of surface HLA-A2 and -B5. No similar effect was observed in 721.174 mutant cells, in which a homozygous deletion includes PSF among several other closely linked genes. At least one of these genes may therefore also be required for PSF function.

Animals↗
PubMed1990

Recognition by human V gamma 9/V delta 2 T cells of a GroEL homolog on Daudi Burkitt's lymphoma cells.

All human gamma delta T cells coexpressing the products of the variable (V) region T cell receptor (TCR) gene segments V gamma 9 and V delta 2 recognize antigens from mycobacterial extracts and Daudi cells. Exogenous and endogenous ligands on the cell surface, homologous to the groEL heat shock family, induced reactivities that resembled superantigen responses in this major subset of human peripheral blood gamma delta T cells. Stimulation of human V gamma 9/V delta 2 T cells is not restricted by human leukocyte antigens (HLA), including nonpolymorphic beta 2-microglobulin (beta 2M)-associated class Ib molecules. These data may be important for understanding the role of gamma delta T cells in autoimmunity and in responses to microorganisms and tumors.

Antigen-Presenting Cells↗
PubMed1990

Human T cell receptor-gamma delta-expressing T-cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA-A, -B, -C, -DR, -DQ, or -DP.

HLA-loss variants of an EBV-transformed B lymphoblastoid cell line (EBV-LCL) 721 were used to investigate whether human MHC molecules other than known class I or class II were involved in autologous T cell responses. Bulk lymphocyte cultures of purified T cells primed to an autologous variant EBV-LCL that fails to express HLA-class II and has reduced cell surface HLA-class I expression, and oligoclonal TCR-gamma delta-bearing lines derived from them, could lyse both this EBV-LCL and an independently derived, class II expressing autologous variant EBV-LCL that bears no HLA-A, -B, or -C, suggesting the presence of additional HLA-like restriction elements. Cold target inhibition of cytolysis mediated by these lines indicated that a shared or cross-reactive MHC controlled restriction element other than the known MHC determinants was retained by the EBV-LCL variants. Single-cell derived clones from these T cell lines which expressed only the TCR-gamma delta showed this same target cell specificity pattern, proving recognition of MHC-controlled determinants by autologous gamma delta T cells. Anti-gamma delta antibody could inhibit cytolysis by the gamma delta-expressing lines, suggesting that the TCR-gamma delta was involved in recognition of the EBV-LCL targets. Flow cytometric analysis with separate HLA-reactive antibodies indicated that the restriction element for these cytolytic responses is a molecule serologically cross-reactive with HLA-B and -C Ag, yet is a determinant that cannot be HLA-A, -B, -C, -DR, -DQ or -DP.

Antigens, CD↗
PubMed1990

A class I antigen, HLA-G, expressed in human trophoblasts.

The alpha chain of the human histocompatibility antigen HLA-G was identified as an array of five 37- to 39-kilodalton isoforms by the use of two-dimensional gel electrophoresis. Both cell-associated and secreted HLA-G antigens are prominent in first trimester villous cytotrophoblasts and are greatly reduced in third trimester cytotrophoblasts. Allelic variation was not detected, an indication that HLA-G is not obviously polymorphic in cytotrophoblasts. Among the following choriocarcinoma cell lines studied, HLA-G is expressed in JEG but not in Jar or BeWo. Expression of endogenous HLA-G genes has not been found in normal lymphoid cells. Thus, HLA-G is subject to both cell type-specific and developmental regulation and is expressed in early gestation human cytotrophoblasts.

Antibodies, Monoclonal↗
PubMed1989

Sequence and gene transfer analyses of HLA-CwBL18 (HLA-C blank) and HLA-Cw5 genes. Implications for the control of expression and immunogenicity of HLA-C antigens.

Our previous studies suggested that a serologically undetectable HLA-C blank allele (HLA-CwBL18) is either a variant Cw5 allele or a novel HLA-C Ag. To examine these possibilities, the CwBL18 and Cw5 genes from the TCC (HLA-A1, -A2, -B52, -B18, -Cw-, -Cw-) and QBL (HLA-A26, -B18, -Cw5) EBV-transformed B lymphoblastoid cell lines (LCL) were cloned, sequenced, and transferred into HLA-A, -B, -C null LCL mutant .221 cells. The CwBL18 Ag was detected on the cell surface of CwBL18 transferents by flow cytometry with the anti-class I mAb W6/32 but not by complement-mediated cytotoxicity with currently available HLA-C specific antisera. Sequence analysis of the Cw-BL18 gene indicated that the CwBL18 Ag is "C"-like because it contains all C-locus-specific residues and amino acid replacements commonly found in HLA-C alleles. However, the amino acid sequence of the CwBL18 Ag is unusual; CwBL18 lacks unique allele-specific residues when compared with the sequences of other HLA-C alleles. Moreover, apart from the C-locus-specific differences, the sequence of CwBL18 is identical to the HLA class I consensus sequence. This striking homology of CwBL18 to other HLA class I alleles suggests that CwBL18 may be a weak Ag. Taken together, these data demonstrate that CwBL18 is not a variant Cw5 Ag but is a newly described HLA-C Ag. In contrast to CwBL18, the Cw5 Ag is serologically detectable on the cell surface of Cw5 transferents with HLA-specific allo-antisera. Rather unexpectedly, Cw5 was usually expressed at a lower level than CwBL18 on the surface of .221 transferents as evaluated by W6/32 mAb binding analyses. The sequence of Cw5 revealed several unique amino acid replacements. Two of these substitutions, at residue 35 of the alpha 1 domain and residue 275 of the transmembrane domain, may be responsible for the reduced cell surface expression of Cw5. Additional unique replacements at residues 138 and 177 of the alpha 2 domain suggest that these amino acids may be important in the formation of an epitope recognized by a Cw5-specific antibody.

Amino Acid Sequence↗
PubMed1989

Production of human cells expressing individual transferred HLA-A,-B,-C genes using an HLA-A,-B,-C null human cell line.

We detail in this report the characterization of a human B-lymphoblastoid cell line, .221, that does not express endogenous HLA-A, HLA-B, or HLA-C class I Ag due to gamma-ray-induced mutations in the HLA complex. Mutant .221 is characterized by: 1) complete absence of HLA-A,-B,-C mRNA transcripts and alpha-chains, and 2) intracellular expression of two non-A,-B,-C class I alpha-chains with an abundance less than or equal to 1% of normal HLA-A,-B,-C expression on similar cells. However, transferred HLA-A, HLA-B, and HLA-C genomic genes are expressed as cell surface Ag in amounts similar to expression of the same endogenous genes in human B-lymphoblastoid cells. The amount of class I transcript produced from transferred class I genes is roughly proportional to the number of gene copies but, in every case studied, post-transcriptional processes limited cell surface Ag expressions to amounts approximately normal for the cell type. The ability of mutant .221 to express quantitatively normal amounts of transferred class I genes suggests that: 1) it can serve as a recipient for, and then express, any cloned HLA-A,-B, or -C gene that would normally be expressible in human B-lymphoblastoid cells; 2) the absence of a background of HLA-A,-B,-C Ag permits its use for studying the expression of normal non-A,-B,-C class I genes and of class I genes that have mutations; 3) mutant .221 can be used to create human cells that express on their surfaces just one defined class I Ag encoded by a transferred class I gene.

B-Lymphocytes↗
PubMed1989

Chromosomal organization of the human major histocompatibility complex class I gene family.

17 HLA class I genes have been isolated from the genome of B-lymphoblastoid cell line 721. Sequence analysis and transfection studies indicate that three genes, in addition to those encoding the HLA-A, -B, and -C antigens can direct the synthesis of a class I alpha protein (4, 5, 21). Using gene-specific DNA probes to analyze the presence of restriction fragment-length polymorphisms within a large pedigree and in panel of HLA deletion mutant cell lines, we show here that two of these genes, designated HLA-G and HLA-F, are located on the short arm of chromosome 6 telomeric to the HLA-A locus. The third expressed non-A, -B, and -C class I gene, HLA-E, is located between HLA-A and HLA-C (4). In addition, the remaining 11 class I pseudogenes and gene fragments are localized relative to established markers on chromosome 6p.

B-Lymphocytes↗
PubMed1989

Demonstration by class I gene transfer that reduced susceptibility of human cells to natural killer cell-mediated lysis is inversely correlated with HLA class I antigen expression.

HLA antigen-loss mutants and class I gene transferents were used to analyze the influence of class I expression on natural killer (NK) cell-mediated lysis. Only HLA antigen-loss mutants that had lost expression of either HLA-A and HLA-B antigens (mutant .184) or of HLA-A, B and C antigens (mutant .221) were distinctly susceptible to NK-mediated lysis. Mutants with reduced expression of class II antigens but unaltered expression of class I antigens remained resistant to NK lysis. A direct demonstration of the effect of class I antigen expression on human cells was made by analyzing a variety of gene transferents of the HLA-A, B, C null mutant .221 expressing only one transferred HLA-A, B or C gene. These results specifically show that expression of class I antigens, with a possible preferential effect of HLA-B expression, reduces the susceptibility of mutant .221 to NK-mediated lysis.

Cytotoxicity, Immunologic↗
PubMed1989

Mapping of prolactin and tumor necrosis factor-beta genes on human chromosome 6p using lymphoblastoid cell deletion mutants.

A collection of human B lymphoblastoid cell lines (LCLs) was used to map two genetic sequences for which polymorphism had not been identified: human prolactin (PRL) and tumor necrosis factor-beta (TNFB). The LCLs have overlapping deletions on chromosome 6p produced by gamma-irradiation of LCL 721. After using two chromosome 6p sequences for which LCL 721 is heterozygous to validate our scanning densitometry (SD) method for inferring gene copy number, SD was used to map TNFB and PRL. TNFB maps to the interval between the C4 complement and HLA-B loci within the MHC on chromosome 6p. PRL lies within the 6p21.3-6p22.2 interval distal to HLA-C. We found that LCL 721 is heterozygous for PRL DNA fragment lengths generated by HpaII but not MspI digestion, indicating that the two copies of PRL in LCL 721 are differentially methylated. This novel methylation RFLP was used to corroborate the region PRL assignment.

Blotting, Southern↗
PubMed1989

Restriction of Epstein-Barr virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules.

HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytolytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells.

Antibodies, Monoclonal↗
PubMed1989

Secretion and cell surface expression of IgG1 are impaired in human B lymphoblasts that lack HLA-A, -B, and -C antigens.

B-lymphoblastoid cell line (LCL) 721.221 lacks HLA-A, -B, and -C class I antigens and transcripts as a result of gamma-ray-induced mutations. LCL 721, from which mutant .221 was derived, produces membrane and secreted forms of IgG1(kappa). In contrast, IgG expression in .221 had these characteristics: (i) gamma 1 heavy chains were diminished by 98% but were detectable with chain-specific antibodies in cell lysates; (ii) kappa light chains were present at normal levels in cell lysates and free kappa chains were secreted; (iii) cell-surface-associated IgG and secreted IgG were absent. Mutants that had partially reduced amounts of class I antigens continued to secrete IgG; however, both the absolute amount of IgG secreted and the relative amount of kappa vs. intact IgG secreted were abnormal in such partially class I-deficient cells. The failure to export IgG and the deficiency of HLA-A, -B, and -C were not merely coincidental in mutant .221, since production of IgG was restored by transferring a functional HLA-A, -B, or -C gene into .221. Cell surface antigen expression of cloned HLA-A, -B, and -C transgenes introduced into .221 was comparable to that of the same genes in their normal chromosomal locations. These observations reveal a relation between production of HLA class I gene products and production of IgG.

Antibodies, Monoclonal↗
PubMed1988

HLA-E. A novel HLA class I gene expressed in resting T lymphocytes.

A MHC class I gene, HLA-6.2, has been identified. Examination of its primary structure indicates that HLA-6.2 is the most divergent member of the class I gene family characterized to date. Tissue distribution of HLA-6.2 RNA differs from other class I genes in that the highest level of RNA is detected in resting T lymphocytes. After transfer of the HLA-6.2 gene into a class I null human lymphoblastoid cell line, a protein associated with beta 2-microglobulin is detected. Analysis of HLA deletion mutant cell lines places the HLA-6.2 gene between the HLA-C and HLA-A loci on the short arm of chromosome 6. These data support the fact that the HLA-6.2 gene is a distinct expressible HLA class I gene, which we propose be designated HLA-E.

Amino Acid Sequence↗
PubMed1988

Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin.

We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We determined the kinetic and equilibrium binding parameters for the fluorescent b2m derivatives associating with the class I H chains of mouse and human cells. Peaks B and C exhibited biphasic binding to the mouse lymphoma cells EL-4(G-CSA-) (Kd1 = 1 x 10(-9) M; K2 = 1.5 to 3.0 x 10(-8) M whereas peak A bound to a small number of low affinity binding sites. In contrast to the biphasic binding observed with EL-4(G-CSA-), only monophasic binding was observed for peak C binding to RDM4 cells. Biphasic binding was also observed with the human B cell line LCL 721. Analysis of a series of LCL 721 class I MHC loss mutants and gene transferents revealed that the heterogeneity in binding is due to differences in the affinity of different class I encoded H chains for b2m.

Animals↗
PubMed1988

A-11: cell type-specific and single-active-X transcription controls of newly found gene in cultured human cells.

We describe the isolation and characterization of a human X-chromosomal gene that is subject to both single-active-X control and tissue-specific control. The A-11 gene was identified by a cDNA that hydridizes to a 3.2-kb EcoR1 fragment of genomic DNA on the long arm of the human X chromosome. A-11 transcripts are normally present in fibroblasts but not in B- or T-lymphoblasts. However, A-11 transcription was activated in four of 11 independent, gamma ray-induced B-lymphoblastoid HLA antigen-loss mutants. Cell hybrids with a human fibroblast-derived active X contained A-11 transcripts but hybrids carrying the human inactive X did not. Azacytidine, a potent inhibitor of DNA methylation, readily reactivated the A-11 locus on the inactive X in hybrid cells, indicating that differential methylation is likely to be involved in the single-active-X control of A-11 transcription in fibroblasts. Failure of cells to remethylate DNA synthesized to repair gamma ray-induced damage may also have resulted in the activation of A-11 transcription among the lymphoblastoid mutants. The A-11 locus provides an opportunity to study the relationship between two types of transcriptional regulation of a gene.

Azacitidine↗