Constitutive Expression Of MHC Class Research Articles

IFN-λ Enhances Constitutive Expression of MHC Class I Molecules on Thymic Epithelial Cells.

Regulation of MHC class I (MHC I) expression has been studied almost exclusively in hematolymphoid cells. We report that thymic epithelial cells (TECs), particularly the medullary TECs, constitutively express up to 100-fold more cell surface MHC I proteins than epithelial cells (ECs) from the skin, colon, and lung. Differential abundance of cell surface MHC I in primary ECs is regulated via transcription of MHC I and of genes implicated in the generation of MHC I-binding peptides. Superior MHC I expression in TECs is unaffected by deletion of Ifnar1 or Ifngr1, but is lessened by deletion of Aire, Ifnlr1, Stat1, or Nlrc5, and is driven mainly by type III IFN produced by medullary TECs. Ifnlr1 -/- mice show impaired negative selection of CD8 thymocytes and, at 9 mo of age, present autoimmune manifestations. Our study shows unanticipated variation in MHC I expression by ECs from various sites and provides compelling evidence that superior expression of MHC I in TECs is crucial for proper thymocyte education.

Attempted Depletion of Passenger Leukocytes by Irradiation in Pigs

Allograft/xenograft rejection is associated with “passenger leukocyte” migration from the organ into recipient lymph nodes. In Study 1, we attempted to deplete leukocytes from potential kidney “donor” pigs, using two regimens of total body irradiation. A dose of 700 cGy was administered, followed by either 800 cGy (“low-dose”) or 1,300 cGy (“high dose”) with the kidneys shielded. Neither regimen was entirely successful in depleting all leukocytes, although remaining T and 8 cell numbers were negligible. Study 2 was aimed at providing an indication of whether near-complete depletion of leukocytes had any major impact on kidney allograft survival. In non-immunosuppressed recipient pigs, survival of a kidney from a donor that received high-dose irradiation was compared with that of a kidney taken from a non-irradiated donor. Kidney graft survival was 9 and 7 days, respectively, suggesting that depletion had little impact on graft survival. The lack of effect may have been related to (i) inadequate depletion of passenger leukocytes, thus not preventing a direct T cell response, (ii) the presence of dead or dying leukocytes (antigens), thus not preventing an indirect T cell response, or (iii) constitutive expression of MHC class II and B7 molecules on the porcine vascular endothelium, activating recipient T cells.

Down-Regulation of MHC Class II Expression through Inhibition of CIITA Transcription by Lytic Transactivator Zta during Epstein-Barr Virus Reactivation

The presentation of peptides to T cells by MHC class II molecules is of critical importance in specific recognition to a pathogen by the immune system. The level of MHC class II directly influences T lymphocyte activation. The aim of this study was to identify the possible mechanisms of the down-regulation of MHC class II expression by Zta during EBV lytic cycle. The data in the present study demonstrated that ectopic expression of Zta can strongly inhibit the constitutive expression of MHC class II and CIITA in Raji cells. The negative effect of Zta on the CIITA promoter activity was also observed. Scrutiny of the DNA sequence of CIITA promoter III revealed the presence of two Zta-response element (ZRE) motifs that have complete homology to ZREs in the DR and left-hand side duplicated sequence promoters of EBV. By chromatin immunoprecipitation assays, the binding of Zta to the ZRE(221) in the CIITA promoter was verified. Site-directed mutagenesis of three conserved nucleotides of the ZRE(221) substantially disrupted Zta-mediated inhibition of the CIITA promoter activity. Oligonucleotide pull-down assay showed that mutation of the ZRE(221) dramatically abolished Zta binding. Analysis of the Zta mutant lacking DNA binding domain revealed that the DNA-binding activity of Zta is required for the trans repression of CIITA. The expression of HLA-DRalpha and CIITA was restored by Zta gene silencing. The data indicate that Zta may act as an inhibitor of the MHC class II pathway, suppressing CIITA transcription and thus interfering with the expression of MHC class II molecules.

Cellular responses to human chondrocytes: absence of allogeneic responses in the presence of HLA-DR and ICAM-1

To assess the accessory cell function of human articular chondrocytes, we assessed the ability of human chondrocytes to stimulate allogeneic peripheral blood mononuclear cells (PBMC) and to support phytohaemagglutinin (PHA)-induced proliferation of highly purified T cells. We also examined the surface expression of HLA-DR and ICAM-1 on the chondrocytes both unstimulated and stimulated with cytokines in vitro. Chondrocytes failed to stimulate allogeneic PBMC despite the constitutive expression of MHC class I molecules and the cytokine-induced expression of class II molecules but were able to support T cell proliferation to PHA, IFN-gamma and to a limited extent, IL-1 beta, induced class II expression on chondrocytes. ICAM-1 was present on 94-99% of freshly isolated cells; this declined with culture (17-59%; P < 0.005) but was readily induced by IFN-gamma, IL-1 beta, and tumour necrosis factor-alpha. Alloreactivity and, presumably, autoreactivity to chondrocytes requires factors in addition to the surface expression of DR and ICAM-1. However the presence of these molecules suggests a capacity for cell-cell interactions in inflammatory sites such as the cartilage pannus junction.

Human small intestinal epithelial cells constitutively express the key elements for antigen processing and the production of exosomes

In humans, the small intestinal epithelial cells (IEC) have a high constitutive expression of MHC class II (MHC II), and contains lysosomes. The IEC also contains MHC II rich multivesicular compartments and has been shown to produce exosomes. This suggests a role for the IEC in antigen processing and presentation either directly or indirectly by the production of exosomes. However, the presence and localisation in the IEC of other key molecules involved in this process has not been studied previously. In the present work, we have investigated small intestinal biopsies from healthy adults and the HT29 IEC cell line with monoclonal antibodies against molecules involved in the antigen processing/presenting systems and molecules typically found on exosomes derived from professional APCs and IECs. Immunohistology was performed to study the expression and localisation of MHC II (HLA-DR), HLA-DM, MHC I (HLA-ABC), CD1d, Invariant chain, Lamp-1, CD68, CD63, B7.1, B7.2, ICAM-1, Cathepsin D/S/L and the IEC specific marker A33 in the IECs. We found that the IECs from the biopsies constitutively express MHC II, HLA-DM, MHC I, Invariant chain, Lamp-1, CD 68, CD63 and A33, and these markers were also found in the IFN-g treated HT-29 cells. All these molecules were found apically in the IECs of the biopsies, localised mainly in vesicular structures. Interestingly, in the baso-latereral area of the IEC, only MHC II, MHC I, Lamp 1, CD68, CD63 and A33 were found and also here with vesicular staining pattern which matches the molecules previously found on exosomes derived professional APCs and human IEC lines. CD1d, B7, ICAM-1, CD9 and cathepsin D and L were absent in the IEC compartment, but cathepsin S showed a relatively weak staining in the apical part of the IEC. The staining pattern and the morphological localisation of these markers suggest a prominent antigen processing/loading and trafficking compartment, and a possible baso-lateral release of exosomes in the normal human IEC.

Expression of major histocompatibility complex class I molecules on the different cell types in multiple sclerosis lesions.

Multiple sclerosis is considered to be an immune-mediated disease of the central nervous system, characterized by chronic inflammation, primary demyelination and axonal damage. The mechanisms of demyelination and axonal injury are heterogeneous and complex. One possible mechanism is direct damage of oligodendrocytes and neurons by Class I MHC restricted cytotoxic T-cells. In this study we analyzed the expression of functional MHC class I molecule complex, consisting of alpha-chain and beta2-microglobulin, in a large sample of human autopsy material, containing 10 cases of acute MS, 10 cases of chronic active MS, 10 cases of chronic inactive MS and 21 controls. To examine the expression of MHC class I and II molecules on the different cell-types in brain, we used quantitative immunohistochemical techniques, double staining and confocal laser microscopy scans on paraffin embedded sections. We found constitutive expression of MHC class I molecule on microglia and endothelial cells. A hierarchical up-regulation of MHC class I was present on astrocytes, oligodendrocytes, neurons and axons, depending upon the severity of the disease and the activity of the lesions. MHC class II molecules were expressed on microglia and macrophages, but not on astrocytes. These data indicate that in MS lesions all cells of the central nervous system are potential targets for Class I MHC restricted cytotoxic T-cells.

Isoforms of the class II transactivator protein.

The class II transactivator (CIITA) controls both the constitutive and IFN-gamma inducible expression of HLA-D genes. In addition, through the squelching of another transactivator CREB-binding protein, CIITA was more recently shown to have a wider cellular function, including cell cycle control or cellular response to IFN-gamma and IL-4. However, due to its low expression level, its analysis mainly relies on the study of recombinant overexpressed forms of the protein. We report here the analysis of native CIITA in various cell types. We first show the precise timing of CIITA protein expression in a fibroblast cell line in response to IFN-gamma. This expression is observed 2 h after the cytokine addition with a peak of expression ranging from 16 to 24 h. We next show the existence of two major isoforms of the CIITA protein differentially expressed in fibroblast, B lymphocyte or melanoma cell lines. We present the first demonstration that these isoforms originate from alternative translation initiation codons. We finally show that CIITA isoforms translocate to the nucleus with an apparently similar efficiency. Our data therefore demonstrate the existence of CIITA isoforms whose respective ratio depends on the cell type examined. However, we present evidence for a modulation of this ratio in a melanoma cell line with an abnormal constitutive expression of MHC class II molecules.

TGF-β1 and IFN-γ cross-regulate antigen presentation to CD4 T cells by macrophages

We studied the interaction of transforming growth factor-beta1 (TGF-beta1) and interferon-gamma (IFN-gamma) in regulating Ag presentation in macrophages. TGF-beta1 blocked, and IFN-gamma enhanced Ag presentation of two T cell epitopes from the group A streptococcal M protein processed from viable Streptococcus pyogenes. Consistent with the functional data, TGF-beta1 reduced the constitutive expression of MHC class II transactivator (CIITA), MHC class II (MHC-II), invariant chain, and DO mRNA, whereas IFN-gamma up-regulated the expression of CIITA and MHC-II mRNA without affecting invariant chain or DO mRNA. However, neither cytokine affected DM mRNA expression. Treatment of macrophages with the two cytokines in combination showed that TGF-beta1 down-regulated IFN-gamma-mediated enhancement of antigen presentation and inhibited IFN-gamma-inducible CIITA and MHC-II class II mRNA expression. The effect of TGF-beta1 on Ag presentation was shown to be independent of the surface expression of CD80, CD86, or CD40 costimulatory molecules by flow cytometry. Our results show that TGF-beta1 and IFN-gamma cross-regulate Ag presentation by influencing the transcription of several genes associated with antigen presentation function, which may represent an important mechanism limiting T cell activation during an immune response.

Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii.

Toxoplasma gondii is able to invade phagocytic cells of the monocyte-macrophage lineage and replicates within a parasitophorous vacuole. Since macrophages may activate specific T lymphocytes by presenting pathogen-derived antigens in association with molecules of the MHC, we investigated the in vitro expression of host cell molecules involved in antigen processing and presentation before and during infection of murine bone marrow-derived macrophages (BMM) with T. gondii. Fifty-one hours after addition of T. gondii tachyzoites at different parasite-to-host ratios, up-regulation of total MHC class II molecules by interferon-gamma (IFN-gamma) was dose-dependently abrogated in up to 50% of macrophages compared with uninfected control cultures. Quantitative analyses by flow cytometry revealed that the IFN-gamma-induced surface expression of class II antigens as well as the IFN-gamma-induced upregulation of class I molecules was significantly decreased in T. gondii-infected macrophage cultures compared with uninfected controls. However, the constitutive expression of MHC class I antigens was not altered after parasitic infection, and infected BMM remained clearly positive for these molecules. After infection of macrophages preactivated with IFN-gamma for 48 h, T. gondii also actively down-regulated an already established expression of MHC class II molecules. Furthermore, kinetic analysis revealed that the reduction in intracellular and plasma membrane-bound class II molecules started approximately 20 h after infection. While MHC class II antigens were most prominently reduced in parasite-positive host cells, culture supernatant from T. gondii-infected BMM cultures also significantly inhibited expression of these molecules in uninfected macrophages. However, down-regulation of MHC class II molecules was not mediated by an increased production of prostaglandin E2, IL-10, transforming growth factor-beta or nitric oxide by infected BMM compared with uninfected controls. Our data indicate that intracellular T. gondii interferes with the MHC class I and class II antigen presentation pathway of murine macrophages and this may be an important strategy for evasion from the host's immune response and for intracellular survival of the parasite.

Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

In melanoma cell lines, two different patterns of MHC class II expression have been described, either an IFN gamma-inducible expression of HLA-DR and HLA-DP, with a faint or null expression of HLA-DQ, resembling that described for melanocytes, or a constitutive expression, i.e., IFN-gamma independent, of all three HLA-D isotypes. As this latter phenotype has been associated with a more rapid progression of melanoma tumors, we have analyzed in different melanoma cell lines the molecular mechanisms leading to this abnormal pattern of MHC class II expression. In agreement with the evidence of a coordinate transcription of the HLA-D genes in these cell lines, we have shown the constitutive expression of CIITA (class II transactivator) transcripts, CIITA being known as the master switch of MHC class II expression. Unexpectedly, these transcripts initiate from promoter III of the CIITA gene, a promoter that is mainly used constitutively in B lymphocytes. This expression was further shown to occur through factor(s) acting on the enhancer located upstream of CIITA promoter III, which was previously described in epithelioid cells as an IFN-gamma-response sequence. The hypothesis of a general abnormality of the IFN-gamma transduction pathway was dismissed. Constitutive transcription of CIITA from promoter III having been observed in unrelated melanoma cell lines, we propose the hypothesis that this phenomenon might not be a random event, but could be linked to the neoplasic state of the melanoma cells.

Transduction of a murine dominant negative activation transcription factor 1 increases cell surface expression of the class I MHC on a human epidermoid tumor cell line.

The transcription of the MHC class I genes is regulated by interaction of cis-elements, located in the 5' genomic flanking regions, with sequence-specific trans-factors. We have identified a cis-regulatory element, 5'-TGACGCG-3', of the H-2D(d) gene. This cyclic adenosine-3',5'-monophosphate regulatory element (CRE)-like sequence, named H-2 binding factor 1 (H-2 BF1) binding motif, is highly conserved among species. In addition, we found that homo- and heterodimers of activation transcription factor 1 (ATF-1) and CRE binding protein (CREB) associate with the H-2 BF1 binding motif and activate transcription of the H-2D(d) gene. Here we demonstrate that a homologue of ATF-1, originally isolated and designated ATF-1DN, acts as a dominant repressor, blocking the ability of wild-type ATF-1 and CREB to bind to the H-2 BF1 probe in electrophoretic mobility shift assays (EMSA). We have utilized this molecule to analyze the participation of the H-2 BF1 complexes, consisting of the H-2 BF1 binding motif and ATF-1/CREB trans-factors, in the physiological regulation of MHC class I expression in tissue culture cells. A human epidermoid carcinoma cell line, A431, was transfected with ATF-1DN and clones expressing the gene transcripts were selected. When analyzed in the EMSA, nuclear proteins prepared from these clones exhibited a decreased shift of the H-2 BF1 probe corresponding to the levels of the ATF-1DN gene expression. Additionally, MHC class I expression of cells with reduced H-2 BF1 activity was significantly higher than in control cells lacking ATF-1DN. These findings indicate that in these carcinoma cells, the H-2 BF1 complexes negatively regulate the constitutive expression of MHC class I.

Effect of isolation techniques, in vitro culture and IFNγ treatment on the constitutive expression of MHC Class I and Class II molecules on goat neutrophils

Previous studies on the ability of human neutrophils to synthesize cytokines and express MHC Class I and inducible Class II molecules have suggested a possible role of these cells as accessory or antigen presenting cells (APC). There is no information available to date concerning this aspect in ruminants. Therefore, as the first step of these investigations, we have studied the cell surface expression of MHC molecules on goat neutrophils. We show that goat neutrophils can be distinguished from eosinophils with monoclonal antibody (MoAb) ILA-24 which recognizes cattle monocytes and neutrophils. Goat neutrophils constitutively express MHC Class II molecules. However, cell surface expression of MHC Class I and Class II molecules is dramatically reduced on neutrophils purified by density gradient centrifugation in comparison to neutrophils obtained from whole blood after lysis of erythrocytes. Also, the level of expression of MHC Class I antigens is seasonal and donor-dependent and rapidly decreases after in vitro culture despite negligible necrosis and apoptosis of neutrophils. Although treatment with IFNγ partially prevents the loss of MHC Class I molecules on neutrophils, it fails to induce MHC Class II antigens. Implications of these results for further studies on the potential role of neutrophils as APC are discussed.

Overexpression of chicken interferon regulatory factor-1 (Ch-IRF-1) induces constitutive expression of MHC class I antigens but does not confer virus resistance to a permanent chicken fibroblast cell line.

The chicken fibroblast cell line C32 has been transfected with the chicken homolog (Ch-IRF-1) of the mammalian transcription factor IRF-1. Stable transfectants were generated, constitutively overexpressing Ch-IRF-1 mRNA and protein. Cells overexpressing Ch-IRF-1 showed enhanced constitutive expression of MHC class I (B-F, beta-microglobulin) antigens. With increasing number of passages cells with normal B-F IV surface antigen expression accumulated. In the revertants, the amount of Ch-IRF-1 mRNA was reduced. Overexpression of Ch-IRF-1 had no effect on the constitutive expression and the induction by chicken interferon type-I and type-II (Ch-IFN) of guanylate-binding protein (GBP). Susceptibility to vesicular stomatitis virus, sindbis virus, Newcastle disease virus and vaccinia virus was not altered by overexpression of Ch-IRF-1. An antiviral state could be induced against all viruses tested by similar amounts of Ch-IFN type I in clone 20-18 expressing Ch-IRF-1 and cells transfected with empty vector.

Role of endothelial cells in allograft rejection.

The immunological properties of endothelial cells suggest they perform a pivotal role in acute and chronic rejection following solid organ transplantation. Their constitutive expression of MHC class II molecules (which initiate allograft rejection by activating CD4 T cells) and accessory molecules allows them to present foreign antigen by both the direct and indirect route to the recipient's immune system. The costimulatory molecules used by endothelial cells appear to differ from those used by traditional antigen-presenting cells such as B cells and dendritic cells. Release of non-HLA antigens from damaged endothelial cells results in a chronic antibody response--possibly contributing to graft vasculopathy and chronic rejection. Further understanding of the factors that regulate MHC class II and accessory molecule expression on endothelial cells could lead to novel strategies of therapeutic intervention.

IFN-γ-Stimulated Enhancement of MHC Class II Antigen Expression by the Human Mast Cell Line HMC-1

The expression of MHC class II molecules by human mast cells has been reported in immunohistochemical surveys of inflammatory conditions, such as in tuberculin hypersensitivity. While these data suggest that human mast cells may act as antigen-presenting cells under inflammatory conditions, the induction of class II antigens on human mast cells has not been examined. In this study, we determined the effects of the inflammatory cytokines IFN-γ and IL-4 on the expression of class II antigens HLA-DR, -DP, and -DQ by the human mast cell line HMC-1. HMC-1 cells were incubated with or without 1000 U/ml recombinant human IFN-γ (rhIFN-γ) and IL-4 (rhIL-4) for 72 hr and analyzed for expression of MHC class II antigens by direct immunofluorescence and flow cytometry. HMC-1 cells expressed significant levels of HLA-DR and moderate levels of HLA-DP and -DQ at baseline and when cultured without exogenous cytokines. Stimulation by rhIFN-γ for 72 hr significantly increased the levels of HLA-DR and -DP expression but did not affect levels of HLA-DQ. Stimulation by rhIL-4 for 72 hr had minimal effect on expression of class II molecules, but induced a significant difference in levels of ICAM-1 (CD54) expression, indicating that this cytokine is involved instead in the control of certain accessory molecules. Our data showing constitutive expression of MHC class II molecules on HMC-1 cells and upregulation of that expression by rhIFN-γ suggest that human mast cells function as antigen-presenting cells at sites where inflammatory cytokines are present.

Isolation and characterization of a unique natural killer cell inhibitory factor present in the anterior chamber of the eye.

The eye is a classic example of an immunologically privileged organ. Immune privilege in the anterior chamber is a multifaceted phenomenon that may have evolved to protect delicate ocular tissues from immune-mediated damage. The anterior chamber of the eye is lined with corneal endothelial cells, which are terminally differentiated cells that are incapable of regeneration. Since corneal endothelial cells are crucial for maintaining corneal clarity, damage to these cells leads to impairment of vision and, in some cases, blindness. The absence of constitutive expression of MHC class I molecules on corneal endothelial cells makes them potential targets for NK cell-mediated cytolysis. The aqueous humor (AH) that fills the anterior chamber of the eye and bathes the corneal endothelial surface contains numerous immunomodulatory molecules, such as TGF-beta, which may protect the corneal endothelium from NK cell-mediated damage. Functionally significant levels of TGF-beta are present in AH. In this study, we demonstrate that AH inhibits NK cell-mediated cytotoxicity in vitro, but does not affect cytotoxic T lymphocyte-mediated lysis. The inhibitory effect was immediate and occurred without preincubation of NK cells with aqueous humor. The inhibitory factor could not be neutralized by Abs to TGF-beta, but was abrogated by treating AH with proteases. Inhibition was not simply due to lysis of NK cells, since AH did not alter NK cell viability. Initial purification using gel filtration chromatography showed that the activity was restricted to a single elution peak. Analysis of the active fractions by gel electrophoresis indicated that the inhibitory factor was a single protein of approximately 10 kilodaltons. The NK inhibitory factor might be an important modulator for protecting the corneal endothelium from unbridled NK cell-mediated injury and, thus, might be instrumental in preserving vision.

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8 +CD45RC − subset and lyse MHC class II + tumor cells

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II +) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II + tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II + human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50–250 μg SEA per animal with a peak in response 48–72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCRαβ + CD4 − CD8 + CD45RC − cell population. MHC class II − colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-γ (IFN-γ) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II + colon carcinomas revealed a peak in sensitivity to lysis at 10–100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarial and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.

Detection and regulation of rat thyroid MHC class II (RT1.D) transcripts.

We have reexamined 1B-6 cells, a cloned line of FRTL-5 rat thyroid cells in order to determine whether such cells demonstrate constitutive MHC class II gene expression. A new homologous cDNA probe for the RT1.D alpha-chain region was developed and proved significantly more sensitive at detecting specific mRNA levels in 1B-6 cells than a human class II alpha-chain specific probe used in previous studies. Recombinant rat gamma-interferon induced MHC class II (RT1.D) antigen at concentrations as low as 1 U/ml (in the presence of bovine TSH), as detected by both alpha-chain specific mRNA transcripts and surface antigen expression. There was no evidence for constitutive expression of MHC class II transcripts in proliferating 1B-6 cells in the absence of gamma-interferon, even when bovine TSH was present. These studies add further evidence to our hypothesis that MHC class II antigen expression in thyroid cells is secondary to external stimulating factors.