Major Histocompatibility Gene Research Articles

Inhibition of IFN-γ induction of class II MHC genes by cAMP and prostaglandins

Triggering of the cyclic AMP (cAMP) signal transduction pathway inhibits the interferon γ (IFN-γ) -mediated induction of class II major histocompatibility (MHC) genes. We have investigated the mechanism of the inhibition of IFN-γ induction of the murine Aα class II MHC gene by cAMP and E series prostaglandins (PGEs). 151 base pairs of the Aα promoter were sufficient to confer positive regulation by IFN-γ and negative regulation by cAMP which accurately mirrored the regulation of the endogenous Aα gene. cAMP also inhibited the IFN-γ activation of the Fcγ receptor I (FcγRI) gene promoter, an “early” promoter which is activated immediately after treatment of cells with IFN-γ. PGEs, which cause an elevation in intracellular cAMP, inhibited the induction of the Aα promoter, and inhibition was greater in the presence of tumor necrosis factor α (TNFα). A mutational analysis of the Aα promoter showed that all four conserved class II promoter elements, the S, X1, X2, and Y boxes, play a role in mediating Aα promoter activation by IFN-γ. Mutations in these elements did not diminish the cAMP inhibition of promoter activation by IFN-γ. Thus, conserved class II promoter sequences which mediate most known examples of positive and negative regulation, including cAMP inhibition of constitutive class II expression, do not mediate cAMP inhibition of IFN-γ activation of the Aα promoter. We suggest that this inhibition may be mediated by a novel class II promoter element or by disruption of an early step in the IFN-γ signal transduction pathway.

Gene transfer in vivo with DNA-liposome complexes: lack of autoimmunity and gonadal localization.

Direct gene transfer into localized arterial segments can be performed in vivo by transfection with DNA-liposome complexes. This technique holds promise for the treatment of human diseases, including malignancy and cardiovascular disorders. We have previously characterized the potential toxicity of this form of treatment in mice in vivo (Stewart et al., 1992). In this report, we examined two issues relevant to long-term expression of foreign recombinant genes: (i) the potential for autoimmune damage to major organs and (ii) DNA localization in gonadal tissue. Autoimmunity and toxicity of allogeneic major histocompatibility (MHC) gene transfer was assessed in mice after induction of an immune response to a recombinant murine class I MHC gene by direct gene transfer in vivo. Histological examination of brain, heart, lung, liver, kidney, spleen, and skeletal muscle revealed no clinically significant immunopathology or organ damage. The toxicity of gene delivery by DNA liposomes was also analyzed in pigs and rabbits in vivo. No histopathology was observed following the introduction of plasmids encoding several different gene products, and analysis of serum following DNA liposome delivery revealed no abnormalities of serum biochemical parameters. The potential for transfer of recombinant DNA into testes and ovary in animals was evaluated by the polymerase chain reaction. Although evidence of recombinant plasmid was consistently observed in transfected, but not untransfected, arterial sites and occasionally in lung, kidney, spleen, and liver, no plasmid DNA was detected in testes or ovary. These studies suggest that uptake of recombinant DNA following direct gene transfer by liposomal transfection in major organs is not associated with autoimmunity, toxicity, or gonadal localization.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel downstream regulatory element of the mouse H-2Kb class I major histocompatibility gene.

The H-2Kb gene equipped with a minimal promoter (5' deletion up to -61) was fully expressed in transfected fibroblasts, but inactive in transfected embryonal carcinoma cells. A strong transcriptional regulatory element (H2DRE) was identified when a fragment spanning the second exon and second intron was used to activate transient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in mouse Ltk- or NIH3T3 fibroblasts. Its activity was twice that of a construct where the CAT gene was driven by the H-2Kb 5' enhancer region (H2TF1/KBF1 site) and comparable to that of pRSVCAT construct carrying the strong Rous sarcoma virus LTR enhancer. In accord with regulated transcriptional activity of the intact H-2Kb gene, the H2DRE did not activate the CAT expression in P19 mouse embryonal carcinoma cells. The H2DRE did not function as a typical enhancer since its activity was strongly position dependent. Consistent with its anticipated role in transcription regulation, H2DRE displayed more than five target sites for specifically interacting nuclear factors, two of them being present in H-2 positive fibroblasts, but not in H-2 negative teratocarcinoma cells. None of them was cross-competed by sequences of the 5' enhancer. The results of deletion experiments show that H2DRE is the only regulatory region that can activate transcription from the 5' enhancerless H-2Kb gene in mouse L fibroblasts.

Endowing T cells with antibody specificity using chimeric T cell receptors.

T cells recognize antigen in the form of a peptide associated with a cell surface molecule encoded by the major histocompatibility gene complex (MHC). The elaborate requirements for the T cell receptor (TCR)-antigen interaction stand in contrast to the simple and defined nature of the antigenic determinants recognized by antibodies. The similarity in the molecular structure and gene organization between antibodies and the TCR has prompted attempts to interchange the antigen-binding, variable regions of these molecules. To this end, chimeric TCR (cTCR) genes, composed of the variable domains of antibodies linked to TCR constant regions, have been used to confer antibody-type specificity on T cells. cTCR-expressing T cells respond to stimulator cells as well as to immobilized antigen in an MHC unrestricted and independent manner. The antibody-like specificity of the resulting T cells has been exploited, using defined ligands, to elucidate the physicochemical parameters that govern TCR-mediated signaling, and to provide a useful experimental system to study the role of MHC and cell-adhesion/accessory molecules in T cell activation. The successful expression of such cTCR in transgenic mice opens new avenues to explore the role of the MHC in T cell development and maturation. Eventually, chimeric receptors specific to tumor or viral antigens might be used for in vivo targeting of T cells in the framework of immuno- and gene therapy.

Temporal order of DNA replication in the H-2 major histocompatibility complex of the mouse.

As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.

Temporal order of DNA replication in the H-2 major histocompatibility complex of the mouse.

As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.

Peptide binding to class I molecules of the major histocompatibility complex on the surface of living target cells.

Molecules encoded by the class I major histocompatibility genes bind short (nonameric) peptides produced by intracellular proteolysis of antigens. These complexes formed intracellularly are then expressed on membranes of target cells and recognized by the antigen receptor of cytolytic T cells. No binding of externally added peptides could so far be monitored directly on the antigen presenting cells, although cytotoxicity experiments and indirect binding assays provided evidence for its existence. Here we report experiments where specific binding to class I molecules, of externally added peptides, has been monitored on living cells. N-terminal biotin-labelled Kd-restricted peptides (residues 147-155, residues 147-158, and an analogue lacking the arginine at position 156, derived from the sequence of the influenza A virus nucleoprotein) were incubated with murine H-2Kd mastocytoma cells (line P815) at 4 degrees C. The binding on surface of live, intact cells was then demonstrated fluorometrically via the interaction of a streptavidin-phycoerythrin conjugate with the biotin-labelled peptides. Thus, this binding does not involve processing, and its specificity in terms of peptide structure was established by competition with the respective unmodified peptides. The specificity of binding to class I molecules was demonstrated by blocking experiments using monoclonal antibodies specific for H-2Kd. Finally, a correlation was observed between the results of peptide binding measurements and those of cytotoxicity assays.

Mouse hepatitis virus nucleocapsid protein-specific cytotoxic T lymphocytes are L d restricted and specific for the carboxy terminus

Infection of mice with the JHM strain of mouse hepatitis virus (MHV) results in an acute encephalomyelitis associated with primary demyelination of the central nervous system. Efforts at understanding the components of the immune response in the development of chronic MHV-induced demyelination have implicated the antibody response and both the CD4 + and CD8 + T cell responses. In this report, we demonstrate that Balb/c (H-2°) mice immunized with the JHM (JHMV) strain of MHV develop a CD8 + cytotoxic T lymphocyte (CTL) response. One population of these virus-specific CTL recognize the nucleocapsid (N) protein. Recombinant vaccinia viruses expressing either the entire N protein or carboxy-terminal deletions were used to determine the number and location of the epitope(s) recognized. The CTLs were found to recognize a peptide contained within the carboxy-terminal 149 amino acids of the N protein. Analysis of infected cell lines expressing transfected major histocompatibility genes demonstrated that the anti-N protein CTLs were restricted exclusively to the L° molecule. These data provide the first definition of a MHV-specific CTL response directed to a viral protein and suggest that the anti-N protein CTL response is one potential mechanism used by the host to clear JHMV from the central nervous system.

Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.

Familial testicular cancer: a report of the UK family register, estimation of risk and an HLA class 1 sib-pair analysis

Forty-two families with two or more cases of testicular cancer have been reported to the UK Register for Familial Testicular Cancer, comprising two pairs of identical twins, 27 sets of other brothers (25 pairs, two triples), nine father-son pairs, two pairs of first cousins and two uncle-nephew pairs. In total 91 testicular tumours are described in 86 individuals (42 (46%) pure seminoma, 49 (54%) other germ cell tumours). The median age at diagnosis in these patients was significantly younger than that in a comparable series of non-familial patients (29 c.f. 32.5 years, P less than 0.01). In a case-control comparison of 794 testicular cancer patients, eight patients (1.0%) had a brother and four patients (0.5%) had a father with a previous diagnosis of testicular cancer at the time of their own diagnosis (and these families are all included in this report). Two out of 794 controls (0.3%) had a first degree relative with testicular cancer. The cumulative risk to a brother of a patient for developing testicular cancer by the age of 50 years was estimated to be 2.2% (95% C.I. 0.6-3.8%) which results in a relative risk of 9.8 (95% C.I. 2.8-16.7) in comparison with the general population. HLA Class I typing of 21 affected sib-pairs demonstrated four (19%) sharing two haplotypes, 13 pairs (62%) sharing one and four pairs (19%) sharing none. This did not differ significantly from the expected proportions of 25%/50%/25%. It is unlikely, therefore, that there is a major gene associated with testicular cancer predisposition within or closely linked to the major histocompatibility gene complex on chromosome 6.

Immunobiology of pregnancy

This report presents new findings on two conditions that permit genetically disparate tissues to coexist during pregnancy: (a) regulation of major histocompatibility genes in placental trophoblast, and (b) synthesis of uterine and placental polypeptide growth factors with immunosuppressive properties. Recent experiments examining inter-relationships between these two protective mechanisms are cited, and potential explanations for trophoblast resistance to factors with MHC-inducing properties are proposed.

Insulin-dependent diabetes mellitus

Type I diabetes occurs as a result of T-cell-mediated beta-cell destruction. Several candidate antigens have been described recently, including glutamic acid decarboxylase, heat shock protein 65 and peripherin. Restricted T-cell receptor V beta gene usage in non-obese diabetic mice has been suggested but not yet proven. In addition to major histocompatibility genes, several non-H-2 predisposing genes have been mapped on chromosomes 1, 3 and 11.

Allelic Variation in the TNF‐β Gene Does Not Explain the Low TNF‐β Response in Patients With Primary Biliary Cirrhosis

Autoimmune disorders in humans are often associated with particular alleles of major histocompatibility genes. However, the chronic inflammatory liver disease primary biliary cirrhosis (PBC) has not been found to be correlated with certain haplotypes so far. Interestingly, an impaired production of tumour necrosis factor beta (TNF-beta) upon mitogen stimulation was observed for PBC patients, especially in the immunologically active stages of the disease. Furthermore, the identification of alleles of the TNF-beta gene which differ in one unique amino acid, and in the production of TNF-beta after phytohaemagglutinin stimulation, has prompted the idea of a possible linkage between the impaired TNF-beta response in PBC and the genetic prevalence of a certain TNF haplotype. We report here a rapid method for typing the TNFB*1 and TNFB*2 genes by a standard polymerase chain reaction. PBC patients (n = 60) as well as randomized healthy controls (n = 179) of the Munich area were studied for the occurrence of the TNF alleles. No deviation was found in the PBC collective (0.7) for the TNFB*2 distribution when compared with the control (0.67).

Interferon-β specifically inhibits interferon-γ-induced class II major histocompatibility complex gene transcription in a human astrocytoma cell line

We established cultures of human astrocytes and astrocytoma cells from surgical specimens, to study regulation of class II major histocompatibility (MHC) complex antigen expression by interferons. Using these cultures we previously showed that expression of the class II MHC determinant HLA-DR could be induced by interferon-γ and this induction was inhibited by interferon-β. In this report, we extend these observations by showing that the inhibitory effect of interferon-β on interferon-γ induction of the class II MHC gene HLA-DRα was exerted at the transcriptional level, as documented by nuclear run-on experiments and confirmed with blot hybridization analysis. Astrocyte expression of intercellular adhesion molecule-1 (ICAM-1) was induced efficiently by interferon-γ, but not in interferon-β, and induction of ICAM-1 expression by interferon-γ could not be impaired by interferon-β, suggesting that the suppressive effect on induction of HLA-DR was relatively gene-specific. Furthermore, interferon-β did not antagonize interferon-γ induction of HLA-DR expression in human monocytes, suggesting that the inhibition observed in astrocytes was relatively tissue-specific.

Cis- and Trans-Repression of Class I Major Histocompatibility Gene Expression in Abelson Virus-Transformed Murine Leukemia

Numerous tumor cell lines of leukemic origin are known to modulate cell surface expression of major histocompatibility complex (MHC) class I antigens resulting in alterations in their immune detection and tumorigenicity. We have been studying the mechanisms responsible for attenuation of MHC class I gene expression in an H-2 heterozygous (H-2b x H-2d) Abelson-Murine leukemia virus (A-MuLV)-transformed leukemic cell line (designated R8). Here we report that treatment of the R8 cell line with the protein synthesis inhibitor cycloheximide (CHX) increased H-2Kb steady-state messenger RNA (mRNA) levels several fold. The induced H-2Kb mRNA transcripts were functional, as demonstrated by their ability to be translated into immunoprecipitable H-2Kb alloantigen. H-2Kb null variants derived from the R8 cell line were shown to be the product of both cis- and trans-acting mechanisms, insomuch as the treatment of R8-derived H-2Kb non-expressor lines with CHX re-established expression of H-2Kb mRNA to the same extent as transfection of the variant cell line with the wild-type H-2Kb gene. Such findings indicate that downregulation of MHC class I gene expression is constitutive for the R8 leukemic cell line, a phenomenon that may be related to the immature pre-B-cell phenotype of this A-MuLV transformant.

Cis- and trans-repression of class I major histocompatibility gene expression in Abelson virus-transformed murine leukemia

Numerous tumor cell lines of leukemic origin are known to modulate cell surface expression of major histocompatibility complex (MHC) class I antigens resulting in alterations in their immune detection and tumorigenicity. We have been studying the mechanisms responsible for attenuation of MHC class I gene expression in an H-2 heterozygous (H-2b x H-2d) Abelson-Murine leukemia virus (A-MuLV)-transformed leukemic cell line (designated R8). Here we report that treatment of the R8 cell line with the protein synthesis inhibitor cycloheximide (CHX) increased H-2Kb steady-state messenger RNA (mRNA) levels several fold. The induced H-2Kb mRNA transcripts were functional, as demonstrated by their ability to be translated into immunoprecipitable H-2Kb alloantigen. H-2Kb null variants derived from the R8 cell line were shown to be the product of both cis- and trans-acting mechanisms, insomuch as the treatment of R8-derived H-2Kb non-expressor lines with CHX re-established expression of H-2Kb mRNA to the same extent as transfection of the variant cell line with the wild-type H-2Kb gene. Such findings indicate that downregulation of MHC class I gene expression is constitutive for the R8 leukemic cell line, a phenomenon that may be related to the immature pre-B-cell phenotype of this A-MuLV transformant.

Human X-box-binding protein 1 is required for the transcription of a subset of human class II major histocompatibility genes and forms a heterodimer with c-fos.

A complementary DNA encoding a member of the leucine-zipper class of proteins (human X-box-binding protein, hXBP-1) that binds to the 3' end of the conserved X box (X2) of the HLA-DRA major histocompatibility complex gene was recently described. Further gel-retardation analysis has demonstrated that hXBP-1 also binds to HLA-DPB X2 but not to other X2 sequences. Transient transfection of a mammalian expression vector with the hXBP-1 cDNA inserted in the antisense orientation represses the surface expression of HLA-DR and HLA-DP in Raji cells. Cotransfection of the antisense hXBP-1 vector with a HLA-DRA/chloramphenicol acetyltransferase (but not a HLA-DQB/chloramphenicol acetyltransferase) reporter plasmid decreases chloramphenicol acetyltransferase activity in Raji cells and in gamma-interferon-treated HeLa cells relative to cells cotransfected with a control antisense vector. Moreover, hXBP-1 is shown to form a stable heterodimer with the product of the c-fos protooncogene. These data suggest that the hXBP-1 c-fos heterodimer is critical for the transcription of a subset of the human class II major histocompatibility complex genes and that the regulatory mechanisms for the different class II genes are distinct.

The Specificity and Regulation of T-Cell Responsiveness to Allergens

CD4+ T lymphocytes initiate and regulate both the specific and nonspecific effector mechanisms of allergic immune responses. The definition of immunogenic components within allergens has allowed the specificity of the T-cell repertoire to be examined, and the refinement of their molecular structure is now facilitating the mapping of functional epitopes. Nevertheless, the immunological mechanisms that distinguish between atopic and nonatopic states remain unclear; as knowledge of the complex network of cytokines in the regulation of immune responses unfolds, this problem may be resolved. Population genetics indicates that susceptibility to allergic disease is partly determined by products of the major histocompatibility gene complex. The use of molecular genetic probes and monoclonal T cells demonstrates the functional importance of both the HLA-DRAB1 and -DRAB3 gene products in T-cell recognition of allergen. Collectively, this information has made it possible to develop in vitro models examining the modulation of responsiveness to allergens.

Identification of an interferon-γ-responsive element of a class II major histocompatibility gene in rat type 1 astrocytes

Interferon-γ(IFN-γ) has been shown to induce class II major histocompatibility complex (MHC) antigens on several cell types. Previous analysis of cell lines including a glioblastoma multiforme line by our laboratory has mapped an IFN-γ-responsive element to the upstream −141 to −109 base pair (bp) region of the DRA promoter. Using deletion mutants, this report shows that this same general region (−135 to −109 bp) is important for IFN-γ induction in two other human glioma lines and more importantly in primary astrocytes. We have confirmed that this regulatory region of the HLA-DRA gene is necessary for IFN-γ inducibility in astrocytes using a substitution mutant. Sequences beyond −135 bp do not appear to have any additional positive or negative elements.

The effect of swine leukocyte antigen haplotype on birth and weaning weights in miniature pigs and the role of statistical analysis in this estimation.

Miniature pigs of seven swine leukocyte antigen (SLA) genotypes were examined for birth and weaning weights to determine the influence of major histocompatibility (MHC) and background genes on these traits. Effects of different statistical analyses on the magnitude of these associations also were examined. Data on 154 piglets from 32 litters were analyzed by least squares using a linear model that included effects of SLA, litter, and sex of piglet. In these miniature pigs, SLA genotype had little influence on birth and weaning weight; litter, which includes effects of background genes, had a significant effect on both birth weight (P less than or equal to .0001) and weaning (P less than or equal to .005) weight. When litter effects were not accounted for, SLA haplotype appeared to influence both birth (P less than or equal to .001) and weaning (P less than or equal to .10) weights. Use of simple group means, as opposed to least squares means, for comparisons of genotypes yielded misleading results. Simple means indicated that piglets with the aa genotype were heavier (P less than or equal to .01) at birth than those with the cd genotype; in contrast, least squares means showed that cd piglets were slightly heavier at birth (P less than or equal to .10). Heterosis was not observed for birth or weaning weights among piglets of various genotypes, nor did one vs two copies of any haplotype influence these traits.