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D Pious

Publications and source records attributed to D Pious.

At least 37 records · Page 2Linked to original sources
PubMed1984

Mutant HLA-A2 antigens as restricting elements for virus-specific cytotoxic T cells.

Mutants of the EB virus-transformed cell line T5-1 (HLA-A1, 2; B8, 27), bearing well-characterized alterations in HLA-A2 antigen expression and unable to bind the HLA-A2-specific monoclonal antibody BB7.2, have been tested for their susceptibility to EB virus-specific cytolysis using effector T-cell preparations functionally restricted through relevant HLA antigens. Initial experiments first confirmed that the parent line T5-1 was susceptible to cytolysis by both "common" A2-restricted and B27-restricted effector cells. While those T5-1 mutants with little or no surface A2 expression were not lysed by A2-restricted effectors, those targets with quantitatively normal expression of mutant A2 molecules were as susceptible to A2-restricted lysis as the parent line itself. In contrast, all the T5-1 mutant lines were susceptible to B27-restricted cytolysis. The results demonstrate that experimentally induced mutations of HLA-A2 antigen structure, affecting a serologically defined site on the molecule, can occur without altering that same molecule's expression of the T cell-restricting determinant(s). Such experimentally induced mutations are quite different from the naturally occurring "variant" A2 antigens which are present within the serologically defined A2 antigen group and which show changes at the T cell-restricting site.

Antibodies, Monoclonal↗
PubMed1984

Revertants from the HLA class II regulatory mutant 6.1.6: implications for the regulation of Ia gene expression.

Class II (Ia) genes of the human MHC code for developmentally regulated, highly polymorphic cell surface glycoproteins. The control of expression of this highly complex genetic region has not been extensively investigated. We used 6.1.6, a variant of a normal B lymphoblastoid cell that has lost expression of all class II molecules, to study this question. In previous studies, we have shown that the structural genes for class II molecules are intact but are not expressed in 6.1.6 (6), and that the defect is at the transcriptional level (7). In this report, we describe the isolation and characterization of revertants of 6.1.6 that reexpress class II molecules. These revertants have four unusual properties: 1) they are found at a higher frequency (0.7%) than would be expected for a mutational event; 2) they are unstable; 3) they reexpress only a subset of class II molecules; and 4) they have only been found in tetraploid cells. On the basis of previous work with 6.1.6 and the properties of reversion in 6.1.6, we propose a hypothesis for the nature of the defect in 6.1.6 and a model for the regulation of class II molecules.

Antibodies, Monoclonal↗
PubMed1984

Characterization of a B lymphoblastoid cell line mutant that secretes HLA-A2.

HLA-A2 antigen mutants were obtained previously from the B lymphoblastoid cell line T5-1 by mutagenesis followed by immunoselection. Here we present biochemical studies of one particular mutant, clone 8.14.1. These cells synthesize two forms of HLA-A2: a minor form, which remains cell-associated at all times, and an abundant form, which is secreted. The former appears by SDS-PAGE to be slightly larger than T5-1 HLA-A2, whereas the latter appears to be 4000 to 5000 daltons smaller. In vitro translation and in vivo pulse-chase studies suggest that these species are not related to each other by post-translational processing. Proteolytic digestion studies localize the resulting structural alteration in the mobility difference between wild-type and secreted HLA-A2 to a region near the carboxy terminus of the HLA-A2 heavy chain; however, their extreme carboxy termini appear similar, if not identical. We suggest that the secreted form may result from a pattern of RNA splicing in which the exon encoding the hydrophobic, membrane-spanning region is frequently deleted.

Antibodies, Monoclonal↗
PubMed1983

HLA-A2 mutants immunoselected in vitro. Definition of residues contributing to an HLA-A2-specific serological determinant.

The HLA-A2-specific mouse monoclonal antibody BB7.2 plus complement has been used to immunoselect variant clones of the lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, and -B27). Members of one class of variant clones appear to express cell surface HLA-A2 molecules that display reduced reactivity with the selecting antibody, but normal or near normal reactivities with some other HLA-A2-specific monoclonal antibodies and human alloantisera. The HLA-A2 heavy chains derived from two of these variant clones were characterized by comparative double-label tryptic peptide mapping in conjunction with microsequence analysis. These heavy chains were found to carry distinct mutations in the same peptide in the molecule. We conclude that residues within this short segment of the polypeptide contribute to an HLA-A2-specific serological determinant.

Amino Acid Sequence↗
PubMed1983

Mapping of the genes encoding the HLA-DR alpha chain and the HLA-related antigens to a chromosome 6 deletion by using genomic blotting.

We have used genomic blotting with DNA from a human cell line that has a small deletion on chromosome 6 (6.3.6) and from its parent cell line (T5-1) to map DNA fragments complementary to cloned DNA sequences encoding the HLA-B7 antigen (class I) and the alpha chain of the HLA-DR antigen (class II). The 6.3.6 variant fails to express the HLA-A, -B, -C, and -DR and MB specificities associated with one of the parental T5-1 haplotypes and has a visible deletion in the short arm of one chromosome 6 (1). The gene locus assignment was based on the expectation that, if the chromosomal location of the DNA sequences used as a hybridization probe were within the deletion, then the relative amount or size (or both) of genomic restriction fragments that hybridize to the probe in T5-1 and in 6.3.6 DNAs should differ predictably. By comparing the genomic blot patterns from T5-1 and 6.3.6 DNAs, we have shown directly that the loss of haplotype expression was due to deletion of the structural genes and have mapped the structural gene for the HLA-DR alpha chain to the chromosomal location (6p2105-6p23) defined by the 6.3.6 deletion. A cDNA clone encoding the alpha chain of the HLA-DR antigen hybridized to two genomic fragments, 4.2 and 3.8 kilobases long, generated by Bgl II digestion of T5-1 DNA. The 4.2-kilobase fragment was absent from DNA derived from the 6.3.6 deletion variant. Thus, this fragment could be assigned to the parental chromosome 6 with the A1, B8, DR3 haplotype, and the 3.8-kilobase fragment, to the chromosome 6 with the A2, B27, DR1 haplotype. In addition, comparison of the T5-1 and 6.3.6 genomic blot patterns obtained with the HLA-B7 probe revealed dosage differences for all of the class I genomic fragments generated by BamHI digestion, suggesting that all of the class I loci map to the region 6p2105-6p23.

Chromosome Deletion↗
PubMed1983

Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line.

HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule.

Animals↗
PubMed1983

Structural analysis of HLA-A2 antigen from immunoselected mutant 8.6.1: further definition of an HLA-A2-specific serological determinant.

The HLA-A2 mutant cell line 8.6.1 was isolated previously from the lymphoblastoid B cell line T5-1 (HLA-A1, -A2, -B8, and -B27) by immunoselection with the mouse HLA-A2-specific monoclonal antibody BB7.2 and complement. The HLA-A2 molecules synthesized by 8.6.1 do not react with either the selecting antibody or with a second HLA-A2-specific monoclonal antibody, PA2.1. In this study, HLA-A2 heavy chains derived from 8.6.1 and those from the parent T5-1 cells have been analyzed by double-labeled tryptic peptide mapping by using reverse-phase HPLC, cation exchange chromatography, and microsequence analysis. We detect only a single difference between these molecules: 8.6.1 HLA-A2 differs from T5-1 HLA-A2 by the substitution of lysine for glutamic acid at position 161. This result is consistent with data derived from other immunoselected mutants, which implicate the second heavy chain domain (alpha 2) in the expression of the PA2.1 and BB7.2 epitopes, and suggests a crucial role for glutamic acid at position 161 in the formation of an HLA-A2-specific determinant.

Amino Acid Sequence↗
PubMed1982

Human histocompatibility antigen mutants immunoselected in vitro. Biochemical analysis of a mutant which synthesizes an altered HLA-A2 heavy chain.

Immunoselection with HLA-A2 or HLA-A1 specific alloantisera has been utilized to isolate spontaneously arising and mutagen-induced variants from the B lymphoblastoid cell line T5-1 (HLA haplotypes DR3, B8, A1 and DR1, B27, Cw1, A2). Such variants are characterized by reduced reactivity with alloantisera of the selecting specificity, but normal reactivity with alloantisera directed to the other HLA specificities of T5-1. Biochemical analysis reveals two classes of variants. In all HLA-A1 and some HLA-A2 variants, the heavy chain in question cannot be detected; however, in other HLA-A2 variants, a structurally altered HLA-A2 heavy chain is found. In the HLA-A2 variant 6.6.5, this heavy chain is glycosylated and thus has presumably been inserted into the rough endoplasmic reticulum membrane in vivo. However, unlike all other HLA heavy chains, the 6.6.5 HLA-A2 heavy chain does not associate with beta 2-microglobulin, does not undergo processing of its high mannose oligosaccharide, and does not migrate to the cell surface, although it is relatively stably expressed within the cell. We suggest that the primary defect in these cells is the failure of the 6.6.5 HLA-A2 heavy chain to associate stably with beta 2-microglobulin. It is likely that the observed structural alteration in this heavy chain reflects a change in amino acid sequence, and thus, a mutation in the structural gene encoding HLA-A2.

Cell Line↗
PubMed1982

Monoclonal antibodies as a tool for phylogenetic studies of major histocompatibility antigens and beta 2-microglobulin.

The cross-reactivity of several monoclonal antibodies recognizing monomorphic determinants of human HLA-A, B, C, and DR antigens and human beta 2-microglobulin (beta 2m) has been studied on peripheral blood leukocytes in 24 different species. An monoclonal HLA-A-, B-, and C-specific antibody and four monoclonal HLA-DR-specific antibodies cross-reacted with cells from all the primate species tested. Furthermore, antibodies HLA-DR-specific were positive with peripheral blood leukocytes (PBL) from cows, goats, sheep, horses, and dogs. Two monoclonal beta 2m-specific antibodies, which were positive with PBL from certain primates, also reacted with cells from cows, goats, sheep, horses, and dogs. Two other beta 2-m-specific antibodies reacted only with PBL from chimpanzees. No reaction could be detected with all our reagents in other classes tested (birds, reptiles, amphibians, and Teleostei).

Animals↗
PubMed1982

Control of HLA-DR antigen gene expression at the pretranslational level: comparison of an HLA-DR-positive B lymphoblastoid cell line and its HLA-DR-negative variant.

An HLA-DR-positive human B lymphoblastoid cell line, T5-1, and its HLA-DR negative variant, 6.1.6, were studied to elucidate mechanisms resulting in the nonexpression of HLA-DR genes in 6.1.6. The cell lines were labeled with 35S-methionine in vivo, their proteins immunoprecipitated with a monoclonal HLA-DR-specific antibody, and their two-dimensional gel electrophoresis patterns compared. The T5-1 map showed DR-antigen heavy and light chains, while the 6.1.6 map showed neither chain. When the cells were labeled in the presence of tunicamycin, the two-dimensional map of T5-1 showed nonglycosylated heavy and light chains of DR antigen while that of 6.1.6 did not. RNA was extracted from T5-1 and 6.1.6 cells and translated in rabbit reticulocyte lysates. Two-dimensional gel analysis of the immunoprecipitated proteins from T5-1 revealed spots which were identified as HLA-DR light chain and I invariant on the basis of their precipitation by monoclonal and specific allo- and heteroantibodies, and their molecular weight and pI values. These spots were absent in the 6.1.6 maps, indicating that 6.1.6 has no detectable translatable messenger RNA for HLA-DR light chains. The addition of dog pancreas microsomes to the T5-1 cell-free translation mixture resulted in an increase in the molecular weight of the precursor HLA-DR proteins consistent with glycosylation. Together with earlier cell fusion studies showing that DR structural genes were intact in 6.1.6, these data suggested that the lesion in 6.1.6 is an alteration in a regulatory element required for transcription of DR genes or mRNA processing.

B-Lymphocytes↗
PubMed1982

HLA antigen structural gene mutants selected with an allospecific monoclonal antibody.

The HLA-A2 antigen-specific monoclonal antibody BB7.2 and complement were used to immunoselect mutants from an ethyl methanesulfonate-mutagenized human B lymphoid cell line, T5-1. Surviving colonies were screened by radioimmune binding with BB7.2 and with a monospecific HLA-A2 alloantiserum, Stewart, and HLA antigens of selected clones were immunoprecipitated and studied by isoelectric focusing. Several classes of mutants could be distinguished: mutants that expressed no HLA-A2 heavy chain; mutants that expressed an HLA-A2 heavy chain that was unable to associate with beta 2-microglobulin (beta 2m) and was not expressed at the cell surface; mutants with reduced HLA-A2 heavy chain-beta 2m association and cell surface expression of HLA-A2 dimer with or without heavy chain charge alterations; mutants with normal HLA-A2 heavy chain-beta 2m association and normal quantitative cell surface HLA-A2 expression but with HLA-A2 heavy chain charge alterations; and mutants with as yet incompletely defined lesions. Mutants with altered cell surface HLA antigens were not found in previous selections with alloantisera and should be useful for epitope mapping and structure-function studies of HLA molecules.

Antibodies, Monoclonal↗
PubMed1982

Gene dosage and gene expression in the HLA region: evidence from deletion variants.

Among variants selected in a human cell line for nonexpression of a single gene product in the HLA complex, most are single-gene variants, but several have been isolated that are cis-acting and abolish expression of a series of closely linked genes. The two most plausible mechanisms by which such variants could arise are mitotic crossing-over and chromosome deletion. In two HLA variants the presence of a visible chromosome deletion, a 50% reduction in activity of glyoxalase I (a closely linked marker), or both provided evidence for deletional origins. In a third variant these changes were not demonstrable. All three variants showed reductions in amount of cell surface HLA antigens: 40% for the Ia antigens (HLA-DR) and 20-25% for HLA-ABC antigens. The reductions in cell surface antigen in deletion variants have an important implication: in the case of the HLA-A, -B, and -C heterodimer, which consists of a subunit coded for within the major histocompatibility complex and another subunit (beta(2)-microglobulin) coded for on a different chromosome, it is the gene of the major histocompatibility complex that is limiting. The nonmutant haplotype includes A2; binding of an A2 monoclonal antibody in two of the mutants was found to be approximately equal to that in the wild-type cells. Thus, loss of one copy of HLA-ABC genes does not lead to gene dosage compensation-i.e., increased activity by the remaining ABC alleles. The results with the two types of antibodies support a deletional mechanism and are inconsistent with mitotic crossing-over. Of interest with respect to the potential use of deletion variants for purposes of mapping is the fact that each of these variants has distinctive breakpoints. The absence of mitotic crossing-over in 1.2 x 10(7) cells selected suggests that the event is rare in this autosomal region, if it occurs at all.

Cell Line↗
PubMed1980

Identification of a trans-acting function regulation HLA-DR expression in a DR-negative B cell variant.

Somatic cell hybridizations were performed between an HLA-DR negative variant of a human B lymphoid cell line (B-LCL) and normal unrelated B-LCLs. The HLA-DR codes for polymorphic determinants on a heterodimeric cell surface lymphocyte differentiation glycoprotein. A variant subline which was selected in a single step from a diploid heterozygous DR-1 DR-3 B-LCL had lost expression of both DR-1 and DR-3 and the heretodimer; it has been described earlier. In a fusion with a DR-2 B-LCL, the hybrids expressed DR-2 and reexpressed the DR-1 and DR-3 alleles. Similar results were seen in a fusion with a different normal B-LCL. Hybrid clones from both fusions were tested with a large number of alloantisera and essentially all informative sera showed reexpression. The results show that (1) the variant did not arise by mutations in the structural genes for DR-1 and DR-3; (2) the normal cells are supplying a missing gene product needed for expression of DR; (3) this gene product is capable of acting in trans. Chromosome counts showed that the apparent recessiveness of the variant in the hybrids was not due to chromosomal segregation.

B-Lymphocytes↗
PubMed1980

Linked marker analysis of spontaneous HLA variants of somatic cells.

Fourteen independent, spontaneously arising HLA-A1 and -B27 variant clones were isolated from the pseudodiploid B lymphoid line T5-1 by selection using A1 and B27 alloantiserum, respectively, and complement. T5-1 is heterozygous for the tightly linked loci HLA-DR, -B, and -A and probably for -C as well. Following recloning, each of the variants was tested for the HLA specificities of T5-1. None of these spontaneous variants had a genetic lesion which extended to the nearest flanking HLA marker, less than 1 cM distant. On the other hand, a variant isolated from mutagenized cells had a lesion which extended completely through one HLA region in cis, suggesting that haploid expression in the HLA region is compatible with viability, that there are no recessive lethals in the opposite HLA region, and that spontaneously arising variants with extended lesions could have been recovered had they occurred. From these results, we conclude that extended genetic lesions of 0.8 cM or longer contribute less than 10% to the overall rate of spontaneous variation for HLA alleles, which we previously estimated at about 5 x 10(-7) per cell per generation.

B-Lymphocytes↗
PubMed1979

Absence of allogeneic restriction in human T-cell-mediated cytotoxicity to Epstein-Barr virus-infected target cells. Demonstration of an HLA-linked control at the effector level.

Peripheral T lymphocytes from patients with infectious mononucleosis (IM) are sensitized in vivo against the Epstein-Barr virus (EBV). The expression of HLA-A, B, or C molecules at the target cell surface is necessary for the cytotoxic reaction because (a) EBV-positive Daudi cells lacking HLA-A, B, and C determinants are resistant to anti-EBV T-cell lysis, (b) cytolysis of EBV-positive target cells can be consistently inhibited by anti-HLA-A, B, and C and anti-beta 2 microglobulin antibodies. However, no evidence for allogeneic restriction in this system was apparent as (a) cytotoxic T lymphocytes (CTL) from one given individual could exert a cytotoxicity of a similar magnitude on different EBV-positive target cells, regardless of the number of HLA-A or B specificities shared by the effectors and targets; (b) CTL from IM patients were able to kill target cells without any HLA-A or B antigen in common; and (c) T5-1 variants lacking one or two HLA antigens at the A, B, or D locus are killed to the same extent as the parental cells. 7 of the 9 IM patients with detectable circulating anti-EBV CTL carried the HLA-A1 antigen, whereas none of the 16 IM patients lacking detectable peripheral CTL were HLA-A1 positive (mean specific lysis of T5-1 target cells by T cells from HLA-A1 positive patients: 29.3 vs. 0.6% in HLA-A1-negative patients) (P less than 10(-9)). These data suggest an HLA-A1-linked gene control of the magnitude of the anti-EBV CTL response. Thus, the HLA region appears to act at two different level sin the T-cell-mediated lysis of EBV-infected cells by controlling first, the development of anti-EBV and second, the expression of HLA-A, B, and C molecules involved as recognition structures at the target cell surface.

Antigen-Antibody Reactions↗