Biomedical subjects

D Pious

Publications and source records attributed to D Pious.

At least 37 records · Page 2Linked to original sources

PubMed1986

Genes for the tumor necrosis factors alpha and beta are linked to the human major histocompatibility complex.

The human major histocompatibility complex (MHC) includes the closely linked genes for the tumor necrosis factors alpha and beta. Their location is within the chromosomal segment between HLA-DR and HLA-A or centromeric of HLA-DP. This assignment is based on Southern blot analysis of a number of different MHC deletion mutants and is corroborated by chromosome in situ hybridization.

Chromosome Deletion↗

PubMed1985

HLA class II regulation and structure. Analysis with HLA-DR3 and HLA-DP point mutants.

Point mutations that affect HLA-DR structure or expression have not previously been described. In the present study, we isolated such mutants by immunoselection of an ethyl methanesulfonate-mutagenized HLA-DR3 cell line with an anti-HLA-DR3 monoclonal antibody, 16.23. To facilitate analysis, we used a parent cell line with a preexisting deletion of one haplotype encompassing DR and DQ alpha and beta. The selection yielded two sets of mutants, one with defects in DR3 structure, the other with defects in different steps leading to DR expression. Of the expression-defective mutants, one had undergone a second deletion removing the remaining DR alpha gene but no other class II genes. It had a normal abundance of DR beta mRNA but had lost binding of DR monomorphic antibodies, indicating that DR beta chains do not form noncognate dimers. A second mutant had an abnormally large DR alpha mRNA, probably resulting from a splice site mutation. Several mutants had marked reductions in DR beta mRNA levels; in two of these, the lesion appeared to be transcriptional because the reduction in DR beta mRNA was paralleled by an altered methylation pattern of one of the DR beta genes. Other expression-defective mutants had different posttranscriptional defects. Some of the mutations were similar to those that have been found in mouse strains defective in I-E expression, whereas others have no known natural counterpart. The matrix of reactivities of anti-HLA class II monomorphic antibodies with these and similar mutants allowed us to define the gene products recognized by these antibodies. A set of seven mutants were "epitope defective," that is, they expressed normal or near normal levels of HLA-DR3 but no longer bound 16.23. Unexpectedly, each of the epitope mutants had decreased DR dimer stability. These mutants should be useful in localizing the DR3 alloepitope and in elucidating its contribution as a restriction element in the presentation of soluble antigen to immune T cells.

Antibodies, Monoclonal↗

PubMed1985

Different roles for cytosine methylation in HLA class II gene expression.

We studied the role of cytosine methylation in the control of HLA class II gene expression in isogenic sets of cells whose members differ in their expression of HLA class II genes. These included: T5-1, 6.1.6, and P30, which are a class II expressing B-cell line, a class II nonexpressing mutant derived from T5-1, and an HLA-DR expressing partial revertant derived from 6.1.6, respectively; the class II expressing B-cell line, SB, and the class II non-expressing T-cell line, HSB, from the same individual. The use of sets of cells that differ in the way their class II genes are regulated allows us to study how that difference is reflected in the methylation state of their class II genes. At least five out of six class II genes in nonexpressing cells have a CpG site that is demethylated, when compared with the same class II gene in the respective expressing cells. The results presented in this paper indicate that most methylation changes in and around class II genes have a correlation with their state of expression. Some of these changes reflect rather than determine the state of expression. Other methylation changes appear to directly affect expression, whereas some methylation differences neither correlate with nor influence gene expression. Although 5-azacytidine does not affect class II expression in T5-1 or 6.1.6, it does induce expression in HSB. This indicates that the basis for nonexpression of class II genes is different in 6.1.6 and HSB.

B-Lymphocytes↗

PubMed1985

Deletion mapping of HLA and chromosome 6p genes.

We have analyzed a set of heterozygous mutants with deletions that encompass parts of HLA and surrounding regions of chromosome 6p. By a combination of Southern blotting, serologic, enzymatic, and cytogenetic analyses, we have ordered eight independent deletion break points into a sequence that divides chromosome 6p into six regions. The deletion mutants have been used in conjunction with the Southern blot technique to map HLA and other 6p gene probes into those regions. On the basis of these and other data we propose a genetic and physical map of HLA and surrounding regions of chromosome 6p. We find that for HLA probes, most of which hybridize with more than one gene, the multiple copies recognized by single probes map to single regions. Any chromosome 6p gene can now be regionally mapped by using these mutants.

Chromosome Deletion↗

PubMed1985

Clonal T lymphocyte recognition of the fine structure of the HLA-A2 molecule.

A human alloimmune cytotoxic T lymphocyte (CTL) clone (4E4) was generated against the HLA-A2 molecule. Lysis of 51Cr-labeled HLA-A2 target cells was blocked by monoclonal antibodies (mAb), including mAb PA2.1 (anti-HLA-A2), mAb BB7.2 (anti-HLA-A2), mAb 4B (anti-HLA-A2-plus-A28), mAb MA2.1 (anti-HLA-A2-plus-B17), and mAb W6/32 (anti-HLA-A,B,C), which are directed against different serologic epitopes on the HLA-A2 molecule. However, HLA-A2 mutant lines lacking the serologic epitope recognized by mAb BB7.2 (anti-HLA-A2) were efficiently lysed by CTL 4E4. Thus, although mAb may block cytolysis, the HLA-A2 epitope recognized the 4E4 CTL clone is distinct from the HLA-A2-specific epitope recognized by serologic reagents. Moreover, analysis of HLA-A2 population variants revealed that only the predominant HLA-A2.1 subtype molecule was recognized by CTL 4E4. No cross-reactivity on other, biochemically related HLA-A2 population subtypes was observed, including HLA-A2.2 cells (Hill, CVE, ZYL, M7), HLA-A2.3 cells (TENJ, DK1), or HLA-A2.4 cells (CLA, KNE). This CTL clone appears to recognize a single epitope and, like monoclonal antibody counterparts, can be used to discriminate among immunogenic cellular and serologic epitopes on closely related HLA-A2 molecules. On the basis of the known sequence changes in mutant and subtype HLA-A2 molecules, it appears that the sequence spanning residues 147 to 157 may be critical for cellular recognition of this Class I MHC molecule.

Amino Acid Sequence↗

PubMed1985

Evidence for two trans-acting genes regulating HLA class II antigen expression.

Previous studies have shown that a trans-acting regulatory factor is required for expression of HLA class II molecules in B-LCL, and that a trans-active factor derived from B-LCL induces de novo synthesis of T-LCL-encoded class II antigens in hybrids of T-LCL and B-LCL. To further examine the genetics of class II antigen regulation, we have now fused two different T-LCL with the class II antigen negative B-LCL 6.1.6, a regulatory mutant that does not synthesize a trans-acting factor necessary for transcription of class II genes. Northern blot analysis and cell surface immunofluorescence studies indicate that the hybrids express class II antigens encoded by both 6.1.6 and T-LCL parent cell lines. Thus, expression of class II antigens in these hybrids is controlled by a minimum of two trans-acting regulatory genes. One of these genes is inactivated in the 6.1.6 mutant and is provided by the T-LCL fusion partner. The second regulatory gene may still be active in 6.1.6, but it alone is insufficient for maintenance of class II antigen expression.

Antigens, Surface↗

PubMed1985

Transcriptional regulation of HLA class II and invariant chain genes.

Class II (Ia) antigens are coded for by a family of genes located in the human MHC (HLA). These genes are regulated in a complex manner, being constitutively expressed, inducibly expressed, or not expressed, depending on the cell type examined. 6.1.6 is a variant of a normal B lymphoblastoid line that has lost expression of all class II molecules and has previously been shown to have a defect in the regulation of class II genes. In this report, we have examined those genes by Southern and Northern blotting and have found that 6.1.6 is severely deficient in mRNA for all class II genes examined, although the genes are structurally intact. P30, a partial revertant of 6.1.6, re-expresses mRNA for a subset of class II genes. mRNA for the class II-associated invariant chain is substantially reduced but not absent in 6.1.6.

Genes↗

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↗