MHC: orchestrating the immune response.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
R S Accolla
Publications and source records attributed to R S Accolla.
Evidence for a specific post-transcriptional mechanism controlling the expression of HLA-DQ, but not -DR and -DP, molecules.
It is generally believed that the various MHC class II molecules are expressed coordinately in B cells. To investigate this aspect in more detail, interspecies somatic cell hybrids were constructed between Raji or RJ 2.2.5 (a class II-negative derivative of Raji) human B cells and M12.4.1 mouse B cells. In both types of hybrids, HLA-DR and -DP, but not -DQ, molecules were expressed at the cell surface. The specific lack of expression of DQ Ags correlated with undetectability of newly synthesized DQ alpha beta heterodimers, as assessed by biosynthetic labeling and immunoprecipitation with a variety of DQ-specific mAbs. Studies at the mRNA level showed that apparently normal DQ alpha and DQ beta transcripts were present in the hybrids at levels comparable, if not higher, with the levels of DR- and DP-specific transcripts. From these results, we conclude that lack of appreciable amount of DQ molecules in the hybrids is caused by a post-transcriptional block. To date, these findings represent a rather unique example of noncoordinate expression of MHC class II Ags caused by distinct post-transcriptional mechanisms. These data may be relevant to a more correct interpretation of the functional role of the various MHC class II molecules, particularly with regard to the well-known association of HLA-DQ with many autoimmune diseases. Possible mechanisms at the basis of the distinct control of expression within the MHC class II molecular pool are discussed.
Animals↗
ras-family gene mutations in neoplasia of the ampulla of Vater.
Mutations in the first and second exons of Ha-, Ki- and N-ras oncogenes were investigated in 17 epithelial tumors of the ampulla of Vater by single-strand conformation polymorphism analysis and direct sequencing of DNA fragments amplified by polymerase chain reaction. The panel included 12 intestinal-type adenocarcinomas, 3 villous adenomas, 1 papillary carcinoma and 1 neuroendocrine carcinoma. Six cases (35%) contained ras mutations, affecting codon 12 of Ki-ras in 2 adenomas and 3 carcinomas, and of N-ras in 1 adenoma. All mutations were found in adenomas and among cancers with adenomatous areas, whereas none of the cases lacking adenomatous areas contained mutations. This suggested that ampullary cancers represent heterogeneous diseases with respect to the presence or absence of adenomatous areas and, among those with adenomatous areas, with respect to the presence of activated ras genes. Ki-ras mutated cases included 3 of 4 tumors which mainly involved the intraduodenal bile duct, thus suggesting that a proportion of Ki-ras-mutated ampullary cancers might correspond to those originating from the epithelium of the bile duct component of the ampulla.
Adenocarcinoma↗
In vivo modification of major histocompatibility complex class II DRA promoter occupancy mediated by the AIR-1 trans-activator.
RJ 2.2.5 is a human B cell mutant derived from the Burkitt lymphoma Raji cell which is defective in the AIR-1 locus function. This locus encodes a transcriptional trans-activator required for the constitutive expression of major histocompatibility complex (MHC) class II genes. Here we show, by in vivo DNase I footprinting, that the AIR-1 locus defect correlates with changes in the DRA promoter occupancy. Interestingly, reexpression of human MHC class II genes in RJ 2.2.5 x mouse spleen cell hybrids is associated with partial reversion of DRA promoter occupancy to the Raji pattern. DRA promoter occupancy in other class II-negative B cell lines, derived from patients with bare lymphocyte syndrome, is drastically different from the one observed in RJ 2.2.5 and Raji cells. Moreover, the use of the DNase I as an in vivo footprinting agent reveals that the patients' cell lines do not display a completely "bare promoter" as previously reported using dimethyl sulfate as the footprinting agent. Thus, the use of DNase I allowed us, for the first time, to correlate the AIR-1 locus defect with class II promoter occupancy alterations and distinguish these alterations from the ones observed in phenotypically similar but genetically distinct MHC class II-negative cells.
Base Sequence↗
Evidence for a trans-acting activator function regulating the expression of the human CD5 antigen.
Interspecies somatic cell hybrids were generated by fusing the mouse T-lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T-cell clone were used as human partners, respectively, in three independent fusions. Irrespective of the human cell partner used for fusion, a certain number of hybrids lost CD5 surface expression over a period of time in culture. Analysis at the phenotype and genetic level showed that lack of CD5 expression was due neither to segregation of human autosome 11, on which the CD5 gene has been mapped, nor to deletion of the CD5 structural gene. Furthermore, loss of CD5 surface expression correlated with the absence of specific mRNA. Since these hybrids preferentially segregate human chromosomes, these results indicate the existence of a non-syntenic trans-active locus, or loci, positively controlling the expression of the human CD5 gene.
Animals↗
Physiologic target of the Air-1 trans-activator revealed by stable transfection assay.
RJ 2.2.5 is a human B cell mutant, derived from Raji cells, which has lost expression of major histocompatibility complex (MHC) class II genes because of a defect in the AIR1 locus function. The MHC class II-positive phenotype can be restored by introducing an active AIR1 locus or its mouse equivalent, Air-1. An example of the latter is the H4 cell hybrid, derived by somatic cell fusion between RJ 2.2.5 and mouse class II-positive spleen cells. H4 contains a single mouse chromosome, autosome 16, in which the Air-1 locus maps, and an entire RJ 2.2.5-derived genome. In the present study we show that the physiologic target of the Air-1 locus product is contained within a limited HLA-DRA promoter sequence of 300 base pairs, encompassing the crucial Y, X, and W cis-acting elements. A plasmid construct, pDRA300neo, containing the HLA-DRA promoter sequence which drives the expression of the neomycin resistance gene, has been stably integrated in the genome of the H4 hybrid. Transfectants selected in the presence of G418 retain mouse chromosome 16 and express the DR genes. On the other hand, transfectants grown in a non-selective medium segregate mouse chromosome 16; this is accompanied by a loss of DRA gene expression and G418 resistance, although pDRA300neo is still integrated in the genome. These results offer scope for using this experimental model to clone the Air-1 gene in a straightforward way.
Blotting, Northern↗
Interspecies somatic T cell hybrids as biological tools for studying gene expression during T cell development.
Interspecies somatic cell hybrids were generated by fusing the mouse thymic lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T cell clone were used as human partners, respectively, in three independent fusions. Phenotype and genetic analysis demonstrated that these hybrids preferentially segregated human chromosomes while retaining a complete mouse genetic complement, irrespective of the human partner used for fusion. A large number of T cell differentiation antigens constitutively expressed throughout the T lymphocyte development remained constitutively expressed in the hybrids, irrespective of the maturation stage of human partner used for fusion. In contrast, the expression of other antigens related to a specific stage of T cell development (CD2, CD8), or to an activated state of T lymphocytes (HLA-DR, CD25), was to observed in the hybrids, with no apparent correlation with the segregation of human chromosomes other than, of course, the encoding chromosome. From these results we suggest that the developmental stage of the fusion partners strongly influences the pattern of expression by activating or silencing genes programmed to be expressed in distinct phases of T cell ontogeny.
Animals↗
The complex interplay of the DQB1 and DQA1 loci in the generation of the susceptible and protective phenotype for insulin-dependent diabetes mellitus.
IDDM patients of North East Italian region were molecularly typed for their HLA-DQB1 and DQA1 loci by using allele specific oligonucleotide probes and PCR amplified genomic DNA. IDDM status strongly correlated with DQB1 alleles carrying a non-aspartic acid residue in position 57 of DQ beta chain and DQA1 alleles with an arginine residue in position 52 of DQ alpha chain. Genotype analysis revealed that individuals with two DQB1 alleles having a non-aspartic residue in position 57 and two DQA1 alleles with an arginine residue in position 52 had the highest relative risk of disease: they constituted 41% of IDDM patients as compared to 0% of controls. Heterozygosity either at residue 57 of DQB1 or residue 52 of DQA1 was sufficient to abrogate statistical significance for disease association, although 43.6% of IDDM patients were included in these two groups as compared to 21.6% of normal controls. On the other hand the presence of two DQB1 alleles with aspartic acid in position 57 was sufficient to confer resistance to disease irrespective of the DQA1 genotype. Based on the number of possible susceptible heterodimers an individual can form, it was found that 85% of IDDM cases could form two or more heterodimers (two in cis and two in trans), but no IDDM case was found to form one susceptible heterodimer in cis. These results demonstrate that the complete HLA-DQ genotype, more than single DQB1 or DQA1 alleles or DQB1-DQA1 haplotypes, is associated with the highest risk of disease. Screening of the population for preventive purposes and/or early signs of IDDM should then take advantage of this result and "susceptible homozygous" individuals should be followed very closely and considered the first group of choice for possible new therapeutical trials.
Alleles↗
Assessment of the DQB1-DQA1 complete genotype allows best prediction for IDDM.
OBJECTIVE: To analyze the HLA-DQ (human leukocyte antigen) genetic association with insulin-dependent diabetes mellitus (IDDM) patients of the Northeast Italian population. RESEARCH DESIGN AND METHODS: Fifty-one IDDM patients and 52 healthy control subjects were molecularly typed for DQB1 and DQA1 loci by using allele-specific oligonucleotide probes and polymerase chain reaction amplified genomic DNA. DNA enzyme immunoassay was used to assess allele specificities. RESULTS: IDDM status strongly correlated with DQB1 alleles carrying a non-aspartic acid (non-Asp) residue in position 57 of DQ beta-chain and DQA1 alleles with an arginine (Arg) residue in position 52 of DQ alpha-chain. Individuals with two DQB1 (non-Asp) alleles and two DQA1(Arg) alleles had the highest relative risk for disease: they constituted approximately 40% of IDDM patients compared with 0% of control subjects. Heterozygosis at either residue 57 of DQB1 or residue 52 of DQA1 was sufficient to abrogate statistical significance for disease association, although 47% of IDDM patients were included in these two groups compared with 21% of normal control subjects. On the other hand, the presence of two DQB1 alleles with Asp in position 57 was sufficient to confer resistance to disease irrespective of the DQA1 genotype. CONCLUSIONS: The results demonstrate that the complete HLA-DQ genotype, more than a single DQB1 or DQA1 locus, should be determined to estimate the highest risk for disease. Screening a population for preventive purposes and/or early signs of IDDM should then take advantage of this result, and "susceptible homozygous" individuals should be followed very closely and considered the first group of choice for possible new therapeutic trials.
Alleles↗
HLA-DQB1 typing of north east Italian IDDM patients using amplified DNA, oligonucleotide probes and a rapid DNA-enzyme immunoassay (DEIA).
We report on HLA-DQB1 typing in IDDM patients of north east Italian region using an enzymatic method based on the detection of hybridization reaction between PCR amplified DNA from whole blood and allele specific oligonucleotides by an antibody directed against double stranded DNA (DNA-enzyme immunoassay or DEIA). The method is reliable, simple and sensitive as the classical radioactive method with the advantage of using a universal non radioactive detection reagent. Nineteen families, each including one subject with juvenile insulin-dependent diabetes mellitus (IDDM) were analyzed. A strong association between absence of an aspartic acid (Asp) in position 57 of DQB1 beta chain in homozygous conditions and susceptibility to IDDM was found. In contrast with some previous observations, however, no significant association was found between Asp/non-Asp heterozygous genotype and IDDM. No patients were found with an homozygous Asp/Asp genotype, known to be protective in caucasoid population. Of particular interest was the DQB1 allelic distribution in our population sample. The non-Asp allele most frequently found in IDDM subjects was the DQB1 0201 allele and this finding was statistically significant (Pc value < 0.05, relative risk = 5.01). No significant association was found for any other allele including the DQB1 0302 (Pc value = not significant although with relative risk = 3.28) previously reported as the most frequent allele in IDDM caucasoid patients.
Adolescent↗
Susceptibility of human-mouse T cell hybrids to HIV-productive infection.
Interspecies human x mouse cell hybrids were used to investigate the genetic basis of human permissivity to HTLV-IIIB infection. T cell hybrids between the mouse BW 51.47 T lymphoma line and normal, PHA-IL-2 activated, human peripheral mononuclear cells (PBMCs) were generated. These hybrids preferentially segregated human chromosomes, as assessed by phenotype and karyotype analysis. Viral integration occurred only in those hybrids expressing CD4+ at the cell surface. However, infectious progeny production was demonstrated only in two of the three CD4+ hybrids tested. By segregation analysis, we could correlate the absence of human chromosomes 1, 3, and 9 with the lack of infectious viral progeny.
Animals↗
Detection of human chromosomes in somatic cell hybrids by PCR analysis.
The detection of human chromosomes in somatic cell hybrids is usually made by chromosomal analysis, Southern blot analysis with human probes, and starch-gel electrophoresis of isoenzymes. We describe here a new, quick, and very efficient method to detect human chromosomes in somatic cell hybrids between human and rodent (rat and mouse) cells. The method is based on the polymerase chain reaction to promote amplification of human DNA, using primers derived from localized genes or DNA fragments from each human chromosome.
Animals↗
Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12.
In this study we describe the generation and characterization of interspecies somatic cell hybrids between human activated mature T cells and mouse BW5147 thymoma cells. A preferential segregation of human chromosomes was observed in the hybrids. Phenotypic analysis of two hybrids and their clones demonstrated coexpression of CD4 and CD69 antigens in the same cells. Segregation analysis of an informative family of hybrids followed by molecular and karyotype studies clearly demonstrated that the locus encoding CD69 antigen mapped to human chromosome 12. Although the expression of CD69 antigen is an early event after T-lymphocyte activation and rapidly declines in absence of exogenous stimuli, in the hybrids described in this study the expression was constitutive, similarly to what was previously found in early thymocyte precursors and mature thymocytes. In this respect it was important to note that the behavior of the hybrids in culture strongly suggested a dominant influence of the thymus-derived mouse tumor cell genome in controlling the constitutive expression of human CD69. These hybrids may thus provide a system to study the genetic and molecular mechanisms controlling the expression and function of this activation antigen.
Antigens, CD↗
Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.
The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.
Animals↗
Cell lineage-specific and developmental stage-specific controls of MHC class-II-antigen expression.
In this report we present evidence and we review data from our laboratory which indicate the genetic complexity of regulatory mechanisms controlling MHC class-II-gene expression. The MHC class-II genes can be expressed in 2 ways: in a constitutive fashion, as in B cells, and in an inducible fashion, as in macrophages, endothelial cells and certain tumors. In both cases the regulatory controls are mainly exerted at transcriptional level as a result of interactions between cis-acting regulatory DNA elements and trans-acting factors. The constitutive class-II-gene expression in B cells is under the control of developmentally regulated trans-acting factors with activator function and encoded by a series of genes, the AIR genes, one of which has been mapped in the mouse on chromosome 16. Interestingly, these regulatory mechanisms are conserved across species for at least 70 million years, because murine AIR-gene products can complement AIR gene defects of human B-cell mutants. The constitutive B-cell phenotype behaves as a dominant trait up to the plasma cell stage in which class-II-gene expression is lost because of the activation of suppressor factors which repress transcription and which, in turn, behave as a dominant trait in somatic cell hybrids between B cells and plasma cells. Thus positive and negative signals regulating class-II-gene expression may behave as dominant or recessive traits, depending upon the particular developmental stage of the cell in which they operate. The mechanisms controlling class-II expression in inducible cells are distinct from those mediating constitutive expression. Indeed, induction of these genes is not sufficient to complement AIR-gene defects in hybrids between macrophages and class-II-negative mutant B cells. In contrast, constitutive expression is dominant in hybrids between class-II-positive B cells and macrophages, suggesting that in uninduced cells class-II-gene activation does not take place more because of lack of activator factors than because of the presence of constitutive transcriptional suppressors. On the basis of these results, we propose a model for developmentally controlled MHC class-II-gene expression during ontogeny.
Animals↗
The molecular biology of MHC genes.
Antigenic peptides become associated with major histocompatibility complex (MHC) class I and class II surface antigens, are then presented to T cells and thereby elicit an antigen-specific cellular or humoral immune response. MHC molecules are genetically heterogeneous and polymorphic; their structure is therefore relevant to modulation of the immune system. The selective pressure resulting from this modulation is in turn the main driving force for the evolution of the complex genetic system. The density of MHC molecules on the cell surface is another parameter that influences immune responsiveness. The study of the evolution and regulation of MHC genes is, therefore, of great interest. These and other themes were discussed at the Third IIGB Workshop* which was recently held in Capri.
Animals↗
Inducible and constitutive MHC class II gene expression. Distinct tissue-specific genetic controls.
B cells express MHC class II Ag in a constitutive fashion, whereas macrophages can do so only after induction by a variety of exogenous stimuli. In this study we describe interspecies somatic cell hybrids between the human B cell Raji and the murine macrophage cell P388 D1. This murine cell line does not express detectable levels of class II mRNA. Phenotypic, molecular, and karyotype analysis of a series of hybrids showed that murine macrophage class II genes can be expressed in a constitutive fashion under the control of the human B cell genome. This event is the consequence of de novo accumulation of class II specific mRNA and thus probably reflects activation of transcription. In certain cases the amount of murine class II Ag expressed on the surface of the hybrid cell was significantly higher than the one observed in the parental macrophage cells after induction with IFN-gamma and was not further modified by treatment with the murine lymphokine. Reversion from a murine class II-positive to class II-negative cell surface phenotype in the hybrids correlated with reduced expression of human markers and more important with segregation of human chromosomes. Interestingly, in this case certain hybrids still expressed detectable levels of murine class II mRNA and increased levels of murine invariant chain mRNA when compared with parental P388 D1 murine macrophage cells. These results indicate that constitutive class II gene expression behaves as a dominant trait in B cell x macrophage somatic cell hybrids. Possible mechanisms responsible of the different control of class II gene expression during cell type differentiation are discussed.
Animals↗
Loss of a DNA-protein complex correlates with extinguished major histocompatibility complex class II expression in a human B cell.
An E beta DNA protein complex termed complex A, whose binding activity has recently been shown to correlate with both constitutive and regulated class II expression in murine cell lines, is also present in a human B cell, Raji. The DNA involved in complex A, which includes three previously defined transcriptional motifs, W, X, and Y, is a cis-acting transcription element in Raji cells. Both complex A binding activity and transcriptional activity of its target sequence are absent in an Ia- mutant subclone of Raji, RJ 2.2.5. This cell line, whose defect is complemented by a locus on mouse chromosome 16, reexpresses both class II and complex A upon transfection with mouse genomic DNA. We suggest that factors that form complex A or that regulate complex A formation account for the molecular lesion in this cell line.
Animals↗