Biomedical subjects

D Pious

Publications and source records attributed to D Pious.

At least 19 recordsLinked to original sources

PubMed1998

Discoordinate surface expression of IFN-gamma-induced HLA class II proteins in nonprofessional antigen-presenting cells with absence of DM and class II colocalization.

We compared HLA class II expression in a human melanoma line (a nonprofessional APC), induced by IFN-gamma or by stable transfection with CIITA, with constitutive class II expression in an EBV-transformed B lymphoblastoid cell line (a professional APC) from the same donor. IFN-gamma-induced and CIITA-transfected melanoma cells expressed DR, DP, and DQ at levels similar to those expressed by the professional APC; however, DP and DQ proteins and DM-dependent DR epitopes were delayed in appearing on the cell surface when induced by IFN-gamma. The delay in cell surface expression of some IFN-gamma-induced class II epitopes was observed even though Northern blots demonstrated class II and DM genes to be coordinately transcribed and their mRNA levels to be equivalent to that in B lymphoblastoid cells. Confocal microscopy suggests that discoordinate cell surface expression of class II results from different intracellular trafficking for IFN-gamma-induced class II proteins in the melanoma line compared with that in professional APCs. Specifically, although DR and DM proteins were present 2 days after IFN-gamma induction, colocalization of DR and DM proteins intracellularly was not apparent in cells at any time after induction. Failure of DR and DM proteins to colocalize suggests that IFN-gamma-induced cells lack an intracellular MIIC-like compartment. The absence of a compartment containing DR and DM to facilitate interaction between the two proteins may account for the delayed surface expression of class II epitopes whose formation requires both class II and DM.

Antigen Presentation↗

PubMed1997

Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p.

We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM null mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

Antibodies, Monoclonal↗

PubMed1997

Exogenously provided peptides fail to complex with intracellular class II molecules for presentation by antigen-presenting cells.

Exogenously supplied antigenic peptides can bind to and be presented by cell surface class II molecules of APCs without prior processing. However, it has been unclear whether peptide Ags exogenously supplied to APCs can also form complexes with nascent intracellular class II molecules that contribute to Ag presentation. We found that exogenously provided peptide Ags, unlike whole protein Ags, are presented as efficiently by fixed as by unfixed B lymphoblastoid APCs, suggesting that intracellular processes do not contribute to the presentation of exogenously supplied peptides by unfixed APCs. Consistent with this finding, exogenously provided peptides do not bind detectably to nascent intracellular class II molecules. We studied the basis for this failure. First, as compared with whole proteins, exogenously supplied peptides accumulate very poorly intracellularly. Second, peptides are more rapidly exocytosed. The limited ability of APCs to accumulate exogenously supplied peptides intracellularly provides a likely explanation for the failure of these peptides to associate with nascent intracellular class II molecules. Exogenously supplied peptides probably never reach the intracellular vesicles in which peptide loading of class II molecules occurs. These findings have implications for the use of peptides therapeutically to block presentation of autoantigens in autoimmune disease.

Antigen Presentation↗

PubMed1995

HLA-DR beta chains enter into an aggregated complex containing GRP-78/BiP prior to their degradation by the pre-Golgi degradative pathway.

HLA class II molecules are membrane proteins which are assembled in the endoplasmic reticulum shortly after synthesis of the alpha and beta and invariant chain (Ii) monomers. DR beta chains, in the absence of DR alpha, are rapidly and completely degraded by the pre-Golgi degradative pathway. Here we have examined those factors which target DR beta chains for degradation in a DR alpha deficient cell line, 9.22.3. The DR beta monomers in 9.22.3 were initially incorporated into a proteinaceous complex containing BiP. With time, the DR beta complexes were further aggregated. In wild type cells, which can assemble DR alpha-beta dimers, the secondary phase of aggregation of DR beta was not seen. Additional evidence that aggregation of DR beta in 9.22.3 cells was progressive was that a more mature form of DR beta was found exclusively in the largest DR beta complexes. Furthermore, the most highly aggregated DR beta chains were degraded more rapidly than bulk DR beta chains. These data suggest that DR beta aggregates are intermediates in the pre-Golgi pathway of DR beta degradation. They further suggest that formation of large DR beta aggregates is a proximal event to DR beta degradation. We conclude that DR beta chains are targeted for degradation as a consequence of a change of state, coincident with their aggregation into slow forming, high molecular weight complexes.

Biological Transport↗

PubMed1994

Invariant-cognate peptide exchange restores class II dimer stability in HLA-DM mutants.

Class II presentation mutants have mutations in the HLA-DMA or B genes and are defective in the presentation of whole exogenous Ags restricted by HLA-DR, -DQ, and -DP. The functional defect in Ag presentation is accompanied by an altered conformation of cell surface class II molecules and instability of extracted class II dimers in SDS-PAGE; the latter can be corrected by incubation of mutant cells in an acidic pH in the presence of cognate peptide. Here we investigated the basis for correction of class II dimer instability by acid/cognate peptide treatment and the extent to which this treatment corrects the class II conformational defect in DMB mutants. We found that an acidic pH generates peptide binding sites in class II molecules of DMB mutants by eluting invariant chain (li)-derived peptides from them. Cognate peptides can then bind to the empty binding sites of class II molecules in a pH-independent manner, which results in stabilization of class II dimers. Acid/peptide treatment also restores the DR polymorphic epitope recognized by mAb 7.3.19.1 but not the DR polymorphic epitope recognized by mAb 16.23; low pH gradually destroys the 16.23 epitope in nonmutant cells. Mutant 10.24.6, which has a mutation in the DRA coding region creating an extra glycosylation site, also has unstable DR dimers whose stability is restored by acid/peptide treatment. These results suggest that the primary phenotypic defect in both the DMB and 10.24.6 mutants is the abundance of li peptides and lack of cognate peptides bound to class II molecules.

Amino Acid Sequence↗

PubMed1994

HLA-DMA and -DMB genes are both required for MHC class II/peptide complex formation in antigen-presenting cells.

Major histocompatibility complex (MHC) class II molecules are highly polymorphic cell-surface glycoproteins that present antigenic peptides to CD4+ T lymphocytes. The normal assembly of class II molecules with cognate peptides for antigen presentation requires an accessory function provided by a gene mapping to the class II region of the HLA complex. The isolation of somatic cell mutants of antigen-presenting cells (APC) has shown that at least one gene which maps between HLA-DP and HLA-DQ, provisionally designated c2p-1 (ref. 3), mediates this process. Here we describe a unique new mutant 2.2.93, which manifests defective formation of class II/peptide complexes like that described in c2p-1 mutants. We show that (1) mutant 2.2.93 contains a mutation in HLA-DMA, and a representative c2p-1 mutant, 9.5.3, contains a mutation in HLA-DMB; and (2) transfection and expression of DMA complementary DNA in 2.2.93, and DMB cDNA in 9.5.3, reverses their mutant phenotypes. These results show that HLA-DMA and -DMB, genes of previously unknown function mapping between HLA-DP and HLA-DQ, are required for the normal assembly of peptides with MHC class II molecules. They suggest that HLA-DMA and -DMB encode subunits of a functional heterodimer which is critical in the pathway of class II antigen presentation.

Antigen-Presenting Cells↗

PubMed1994

A mutant human histocompatibility leukocyte antigen DR molecule associated with invariant chain peptides.

From a human histocompatibility leukocyte antigen (HLA)-DR/DQ hemizygous, B lymphoblastoid progenitor, we isolated a cell line, 10.24.6, with a DR alpha missense mutation (96P-->96S), which results in an N-linked carbohydrate addition at position 94 in the DR alpha 2 domain. Several features of 10.24.6 cells suggest that the mutation disrupts normal intracellular formation of peptide/DR complexes. The mutant HLA-DR dimers, though expressed at the cell surface, lack the conformation of the mature, peptide-loaded class II molecules of the progenitor cell, as assessed by their loss of binding of certain antibodies and by the lack of stability in detergent (sodium dodecyl sulfate) solution. In addition, presentation of endocytosed antigen to HLA-DR-restricted T cells is defective in the mutant, but can be restored by transfection of a wild type DRA gene. Assays with synthetic peptides indicate that the 10.24.6 phenotype is not due to an intrinsic inability of the mutant DR molecules to bind peptides. Therefore, to directly evaluate peptide occupancy of the mutant molecules, we analyzed acid-eluted, HLA-DR-associated peptides. The predominant species from the 10.24.6 mutant is a nested set of invariant chain (Ii)-derived peptides that are undetectable in the DR eluate from progenitor cells. The region of DR alpha altered in the mutant molecules is thus implicated in normal formation of peptide/DR complexes. Further, the same set of Ii peptides associated with the DR molecules is present in the eluate from an antigen presentation mutant with a defect in an major histocompatibility complex (MHC)-linked gene. These results suggest that DR molecules in 10.24.6 and in certain presentation mutants are affected at the same or related steps in class II molecule biosynthesis, raising the possibility that class II molecules interact with an MHC-encoded accessory molecule during antigen presentation.

Amino Acid Sequence↗

PubMed1991

A gene required for class II-restricted antigen presentation maps to the major histocompatibility complex.

We have previously described a set of mutants (16.23-selected mutants) of a B lymphoblastoid cell line that are defective in the presentation of intact proteins to class II-restricted T cells, but effectively present immunogenic peptides. The mutations in these mutants are recessive in somatic cell hybrids and are not in Class II structural genes. Here, we report on a unique mutant, 5.2.4, in which a similar defect in class II-restricted antigen presentation has occurred in association with a one-megabase homozygous deletion in the class II region of the major histocompatibility complex (MHC). The defects in class II presentation among three of the 16.23-selected mutants, and between these mutants and 5.2.4, are noncomplementary in somatic cell hybrids. This suggests that the class II presentation-defective phenotype in all four mutants results from lesions in a single MHC-linked gene, a conclusion strengthened by the finding that in a hybrid made with a second, unrelated MHC deletion mutant, T2, the class II presentation defect in a 16.23-selected mutant is also not complemented. Mutant 5.2.4, in addition to its class II presentation defect, is also defective in surface expression of MHC class I molecules, most likely because its deletion encompasses the peptide supply factor 1 gene, whose function is known to be required for normal abundance of cell surface class I molecules. However, the surface abundance of class I molecules is normal in the 16.23-selected mutants, suggesting that the lesions affecting class I surface abundance and class II presentation result from mutations in different genes.

Antigen-Presenting Cells↗

PubMed1991

Defective HLA DRA X box binding in the class II transactive transcription factor mutant 6.1.6 and in cell lines from class II immunodeficient patients.

6.1.6 is one of several immunoselected mutants from EBV-transformed human B cell lines that have undergone coordinate loss of expression of all their HLA class II genes. Similar defects have been found in cells from some patients with class II immunodeficiencies. Previous studies have suggested that the defects in 6.1.6 and in the other class II regulatory mutants are in transactive factors required for class II transcription. The defective factors, however, have not been identified. Here we present two lines of evidence that serve to localize the site of action of the factor that is defective in 6.1.6. First, transfected indicator genes linked to HLA DRA promoter fragments that include the conserved X box region are transiently expressed at greatly reduced levels in 6.1.6, compared with the progenitor cell line T5-1. Second, a DNA-protein complex, termed X-A, formed by nuclear extracts from T5-1 with DRA sequences containing the X box and a few bases 5' and 3' to it, is missing with extracts from 6.1.6. Extracts from some but not all patients with class II-negative immunodeficiency also fail to form X-A, whereas extracts from class II-negative mutants derived from the Burkitt's line Raji do form an apparently normal X-A complex. The X-A complex contains proteins of approximately 22, 32, 82, and 92 kDa that can be cross-linked to a 5-bromodeoxyuridine-substituted X box probe by UV light. A defect in an X box-binding protein, or in a factor required for its binding, is a likely cause for the loss of transcription of the class II genes in 6.1.6.

Cell Line↗

PubMed1991

Mutations affecting antigen processing impair class II-restricted allorecognition.

Both exogenously derived and endogenously derived Ag generally require processing for their optimal binding and presentation by class I and class II major histocompatibility proteins. It is not known whether steps involved in Ag processing also affect the recognition of alloreactive T cells. We have recently described B cell mutants which have general defects in the processing and presentation of a variety of exogenous Ag to class II restricted T cells. In this report we have studied the ability of these processing mutants to stimulate a set of anti-DR3-specific alloreactive T cells clones. These processing/presentation mutants express normal MHC class II molecules, both in terms of primary sequence and cell surface abundance, but they appear unable to generate effective peptide-MHC complexes. When tested for their ability to stimulate MHC class II alloreactive T cell clones, only one of four T cell clones was stimulated by these mutants; the other three alloreactive T cell clones were not stimulated by either of two different mutants. Both of these mutants express normal levels of the accessory molecules, LFA-3 and ICAM-1. The inability of these mutants to stimulate three of four alloreactive clones indicates that the capacity to be recognized by many alloreactive T cells is linked to the Ag processing capacity of a stimulator cell.

Antigen-Presenting Cells↗

PubMed1990

Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes.

Presentation of an exogenous protein antigen to helper (CD4+)T-lymphocytes by antigen presenting cells (APC) generally requires that the APCs degrade the native protein antigen into an immunogenic peptide, a process termed 'antigen processing', and that this peptide bind to a major histocompatibility complex (MHC) class II molecule. The complex of peptide and MHC molecule on the APC surface provides the stimulatory ligand for the alpha beta T cell receptor. The intracellular pathways and molecular mechanisms involved in the generation of the peptide-MHC complex are not well understood. Here, we describe several mutant APCs which are altered in their ability to present native exogenous protein antigens but effectively present immunogenic peptides derived from these proteins. The lesions in these mutants are not in the class II structural genes, but they affect the conformation of mature class II dimers.

Antigen-Presenting Cells↗

PubMed1990

Point mutations define positions in HLA-DR3 molecules that affect antigen presentation.

Allelic differences in major histocompatibility complex (MHC)-encoded class II molecules affect both the binding of immunogenic peptides to class II molecules and the recognition of MHC molecule-peptide complexes by T cells. As yet, there has been no extensive mapping of these functions to the fine structure of human class II molecules. To determine sites on the HLA-DR3 molecule involved in antigen presentation to T cells, we used monoclonal antibodies specific for HLA-DR3 to immunoselect mutants of a B-lymphoblastoid line. We located the sites of single amino acid substitutions in the HLA-DR3 molecule and correlated these structural changes with patterns of recognition by HLA-DR3-restricted, antigen-specific T cells, allospecific T cells, and allospecific anti-DR3 monoclonal antibodies. We analyzed seven mutations. One mutation, at position 74 in domain 1 of the DR beta chain, affected recognition by all T cells tested, whereas others, at positions 9, 45, 73, 151, and 204 of the DR beta chain and position 115 of the DR alpha chain, altered recognition by some T cells, but not others. Each of the substitutions resulted in a unique pattern of T-cell stimulation. In addition, each T-cell clone recognized a different subset of the mutants. These results indicate that different residues of the DR3 molecule are involved in presentation of antigen to different DR3-restricted T cells. These studies further show that substitutions which most likely affect peptide binding alter recognition of DR3 molecules by an alloreactive T-cell clone and some allospecific antibodies.

Alleles↗

PubMed1989

mRNA abundance, rather than differences in subunit assembly, determine differential expression of HLA-DR beta 1 and -DR beta 3 molecules.

The class II major histocompatibility molecules HLA-DR are formed by the association of a single DR alpha chain with two nonallelic DR beta chains. In DR3 cells one DR beta chain is severalfold more abundant than the other. We have studied the mechanism that controls the differential expression of these DR beta genes. We determined the amino-terminal sequences of the two expressed DR beta chains. Comparison of these sequences with the nucleotide sequences of the DR3B1 and DRB3a genes indicates that the abundant chain is the B1 gene product. Supporting this conclusion, an informative mutant, 9.4.3, was found to have lost the abundant beta chain and beta 1 mRNA. This mutant expresses normal cell surface levels of the DR beta 3 chain and exhibits no significant dosage compensation of its beta 3 chain. The unchanged level of DR beta 3 dimer on the cell surface suggests that free DR alpha chains are not the limiting factor in the surface expression of the beta 3 chain and, further, that the differential regulation of the surface expression of the two DR beta chains occurs at a step prior to DR assembly. Quantitation of DR beta mRNAs by locus-specific oligonucleotide probes showed that beta 1 mRNA is 4.5-fold more abundant than beta 3 mRNA, strongly indicating that the greater surface expression of DR beta 1 is a direct consequence of greater beta 1 mRNA abundance.

Cell Line↗

PubMed1988

A single amino acid substitution in the human histocompatibility leukocyte antigen DR3 beta chain selectively alters antigen presentation.

Activation of T lymphocytes by immunogenic peptides bound to HLA molecules is a central event in the generation of an immune response. To determine the sites on HLA molecules involved in this process, we isolated mutant EBV-transformed B cell clones that express altered HLA-DR3 molecules. One mutant has lost the ability to stimulate a T cell clone specific for a mycobacterial protein, but retains the ability to stimulate other antigen-specific T cells. The DNA sequence of the complete DR alpha and beta coding regions revealed a single nucleotide change resulting in a glutamic acid to lysine substitution at amino acid 9 in the first hypervariable region of the DR beta chain. These results are discussed in relation to a recently proposed model of class II molecule structure.

Amino Acid Sequence↗

PubMed1988

Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines.

Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF.

B-Lymphocytes↗

PubMed1988

Transcription of HLA class II genes in the absence of B-cell-specific octamer-binding factor.

HLA-DR and other human class II histocompatibility genes are expressed by Epstein-Barr virus-transformed B-lymphocyte cell lines but not by most T-cell leukemia lines. We determined by transcriptional run-on experiments that regulation of class II expression in these cells is at the level of gene transcription; nuclei isolated from B-cell lines actively transcribe class II mRNA, whereas nuclei from non-class II-expressing T-cell lines and from the class II transactive factor-deficient B-cell mutant 6.1.6 do not. In searching for DNA-binding proteins which might regulate transcription, we found both a ubiquitous (B1) and a B-cell-specific (B2) factor which bind to the octamer sequence ATTTGCAT 52 base pairs 5' of the cap site in the DR alpha gene. We examined the relationship of these factors to DR alpha transcription. HUT-78, a T-cell line which expresses class II mRNA constitutively, contains only the ubiquitous B1 octamer-binding factor also found in non-class II-expressing T-cell leukemias. Human fibroblast, HeLa, and melanoma cell lines similarly contain only the ubiquitous factor, even when these cells are induced to express class II mRNA by treatment with gamma interferon. Both B1 and B2 binding factors are present in the B-cell mutant 6.1.6, which nevertheless fails to transcribe class II mRNA. Although we have not ruled out the requirement of B-cell-specific octamer-binding factor B2 for class II expression in B cells, it is clear that in other cells substantial DR alpha transcription occurs in the absence of this factor.

B-Lymphocytes↗

PubMed1987

Importance of HLA-DQ and -DP restriction elements in T-cell responses to soluble antigens: mutational analysis.

We describe a new approach to delineating the restriction elements used by antigen-specific human T-cell lines. EBV-transformed B cell lines and congenic HLA class II antigen-loss mutants are used to present soluble antigen to immune T cells. In this way it is possible to assess the independent contribution of individual class II loci to the restriction repertoire of the T cells. In contrast to results obtained with other methods of restriction element analysis, we find that approximately 40% of the T-cell response to several antigens is restricted by non-DR class II molecules. Both mutational analysis and blocking by class II specific monoclonal antibodies demonstrate that the non-DR restricted responses derive from DQ and DP-encoded determinants. We also find specific DR/DQ haplotype preferences for the presentation of some but not all antigens. Using a mutant that expresses only the DQ1 molecule, and is derived from a DR1, DQ1 parent line, we demonstrate a functional split of serologically defined DQ1 molecules consistent with the electrophoretic variation reported between DQ1 molecules linked to DR1 and those linked to DR2. Pairs of mutants that differ by expression of a single class II protein reveal a much broader use of available class II restriction elements than previously recognized.

Antibodies, Monoclonal↗

PubMed1987

Expression of HLA-DR antigen in human class II mutant B-cell lines by double infection with retrovirus vectors.

A new retrovirus vector containing the gene for hygromycin B resistance (hyg) as a selectable marker under the control of an internal simian virus 40 promoter was constructed. It was used, together with an analogous previously described vector, DO1, which contains the gene for G418 resistance, to introduce and express the genes for the two chains of a human class II major histocompatibility complex antigen in NIH 3T3 cells. In addition, these vectors were used to express DR antigens in two human mutant B-lymphoblastoid cell lines, one of which was deleted for both alleles of the DR alpha gene and the other of which expressed no class II antigens because of a genetic defect in a putative trans-acting regulatory factor.

Animals↗