MHC regulation in neural cells. Distribution of peripheral and internal beta 2-microglobulin and class I molecules in human neuroblastoma cell lines

mAb were used in an immunocytochemical assay to examine beta 2-microglobulin (b2-m) and class I MHC expression in human neuroblastoma cell lines. In lines with weak class I expression among the whole population, under ordinary assay conditions, strong b2-m and class I expression were concentrated in a small subpopulation. In positive cells, Ag was not restricted to any part of the cell body or processes. Strong expression was not required for establishment of any morphologic form or any type of cell contact. These findings complement studies in other experimental systems, where a nonimmunologic role for class I or b2-m in neural cell growth was not revealed. When the microscopic assay was modified to reveal Ag within the internal membrane system, b2-m was detected in every neuroblastoma cell. Most often, the Ag appeared as a ring around the nucleus, or in a punctate distribution in the juxtanuclear area. Internal expression of HLA chains and class I molecules was more difficult to detect, possibly reflecting a normal excess of b2-m. These findings increase understanding of MHC regulation in neural cell lines. They provide the technical and conceptual background for examination of internal MHC Ag in neural tissue.     

Distribution of beta 2-microglobulin in olfactory epithelium: a proliferating neuroepithelium not protected by a blood-tissue barrier

The olfactory neuroepithelium is unique in adult vertebrates in that bipolar sensory neurons are constantly dying and being replaced. The sensory neurons are also unusual because they are directly exposed to the external environment via their dendritic processes in the nasal cavity. Surveillance of this tissue by major histocompatibility complex (MHC) class I-restricted cytotoxic T cells would presumably serve as an important means of defense against foreign pathogens. Although adult brain shows a lack of class I molecules, it has not been reported if either proliferating neurons or sensory neurons in olfactory neuroepithelium also lack class I. To examine olfactory neuroepithelium, an antiserum against beta 2-microglobulin (beta 2-m), the invariant light chain associated with all class I molecules, was employed as a general probe in an immunocytochemical assay. beta 2-m was detected in columnar respiratory epithelium, blood vessel walls, and a small population of interstitial cells in the lamina propria, but no cell in the olfactory neuroepithelium stained for beta 2-m. Parallel patterns were obtained in the vomeronasal organ. These results suggest that lack of beta 2-m, and presumably class I, may be a general phenotype of neuronal cells regardless of their mitotic state or exposure to environmental antigens.     

Monoclonal antibody analysis of MHC expression in human brain biopsies: tissue ranging from "histologically normal" to that showing different levels of glial tumor involvement

The expression of class I and class II MHC products in human brain was studied. Radioimmunoassay confirmed weak expression of HLA-A,B,C and beta 2-microglobulin (beta 2-m) in brain extract. Quantitative inhibition assay showed brain had 1/70 as much activity as spleen, per microgram of extract protein. Immunoblot assay confirmed that HLA chains and beta 2-m were present in the brain extract. Class II was not detected. Microscopic analysis was performed on eight brain biopsies. The histologic appearance ranged from "apparently normal," to the presence of reactive astrocytes, to the presence of glial tumor. In every case, HLA-A,B,C and beta 2-m activity was concentrated at blood vessel walls. Small and medium-sized vessels were uniformly stained. Cell body staining was not seen in neurons, glia, oligodendrocytes, microglia, reactive astrocytes, or the majority of glial tumor cells. Class II activity was seen in occasional cell bodies in both grey matter and white matter in the microscopic assays. These cells had the morphologic appearance of microglia or reactive astrocytes. Occasional blood vessels also showed class II activity. Unlike the class I activity, the class II blood vessel stain was often discontinuous. More class II+ cell bodies were seen in tumor-associated tissue.     

Specific molecular interaction between the insulin receptor and a D product of MHC class I

The density of MHC class I was determined on a murine thymoma cell line (R1), an H-2 negative variant (R1E), and R1E-derived cell lines in which H-2 expression was restored by transfection of various MHC class I genes (Db, Kb, and truncated Db) and/or a beta-2-microglobulin gene (beta 2-m; B2). Appreciable MHC class I expression was found on R1 cells and on the variants in which MHC class I expression was restored by transfection of Db/beta 2-m or Kb/beta 2-m genes. Only approximately 20% difference was observed between the number of Db molecules and Kb molecules on the R1E/B2/Db and on R1E/B2/Kb, respectively. However, specific insulin binding was significantly different between these lines. By using a computer assisted curve fitting program, the insulin binding data for R1 and R1E/B2/Db cell lines best fitted a two-site model (K approximately 6 x 10(-9) M for high-affinity sites and a 2 to 3 x 10(-7) M for low-affinity sites), whereas all other lines only expressed one type of insulin binding site. These sites were unrelated to IGF-I and IGF-II receptors. Cross-linking of 125I-labeled insulin demonstrated specific binding of the ligand to a Mr approximately 130,000 dalton band in all lines. In the R1E/B2/Db cells, insulin also cross-linked to cell membrane molecules with Mr approximately 48,000 and approximately 60,000 Da, which were identified by immunoprecipitation to be the H chain of MHC class I and the heavy chain of MHC class I plus beta 2-m, respectively. It is concluded that the insulin receptors in the cell membrane interact specifically with D-products of MHC class I and that class I molecules of MHC may have a crucial role in insulin receptor expression. This may reflect a more general nonimmunologic role of MHC class I.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.     

Regulation of MHC expression in vivo. II. IFN-alpha/beta inducers and recombinant IFN-alpha modulate MHC antigen expression in mouse tissues

We examined the effect of type I IFN inducers and rIFN-alpha on MHC expression in mouse tissues in vivo. MHC expression was assessed in a radiolabeled mAb binding assay and by indirect immunoperoxidase staining of tissue sections. polyI:C, an inducer of IFN-alpha/beta, induced large increases in class I MHC in many tissues, with little effect on class II expression. In the kidney, which was studied in detail, polyI:C increased class I expression from day 1 to day 6, localized in glomeruli, tubules, and arterial endothelium. Renal class II MHC was less affected but tended to be decreased at days 3 to 6, corresponding to diminished staining of class II-positive interstitial cells. polyI:C increased renal class I MHC in nude mice and mice with severe combined immunodeficiency, and in mice treated with cyclosporine or mAb against IFN-gamma. The effects of influenza virus resembled those of polyI:C. However, a potent T cell stimulus, allogeneic ascites tumor cells, induced markedly different MHC changes, with massive and sustained increases in class I and II, presumably due to IFN-gamma release, which was inhibited by cyclosporine or by mAb against IFN-gamma. The effect of polyI:C was largely simulated by rIFN-alpha, whereas the effect of allogeneic cells was simulated by rIFN-gamma. Thus, rIFN-alpha and its inducers in vivo produce a sustained increase in renal class I expression in kidney and other tissues, sometimes with changes in class II expression. Such effects could be relevant to the immune modulatory actions of IFN, and to the immunologic consequences of viral infections.     

Expression of MHC class I, MHC class II, and cancer germline antigens in neuroblastoma

Background: Neuroblastoma is the most common solid extracranial tumor in childhood, still with poor survival rates for metastatic disease. Neuroblastoma cells are of neuroectodermal origin and express a number of cancer germline (CG) antigens. These CG antigens may represent a potential target for immunotherapy such as peptide-based vaccination strategies. Objective: The purpose of this study was to analyze the presence of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 on an mRNA and protein level and to determine the expression of MHC class I and MHC class II antigens within the same tumor specimens. Methods: A total of 68 tumors were available for RT-PCR, and 19/68 tumors were available for immunohistochemical (IHC) analysis of MAGE-A1, MAGE-A3/A6, and NY-ESO-1. In parallel, the same tumors were stained with a panel of antibodies for MHC class I and MHC class II molecules. Results: Screening of 68 tumor specimens by RT-PCR revealed expression of MAGE-A1 in 44%, MAGE-A3/A6 in 21%, and NY-ESO-1 in 28% of cases. Immunohistochemistry for CG antigens of selected tumors showed good agreement between protein and gene expression. However, staining revealed a heterogeneous expression of CG antigens. None of the selected tumors showed MHC class I or MHC class II expression. Conclusions: mRNA expression of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 is congruent with the protein expression as determined by immunohistochemistry. The heterogeneous CG-antigen expression and the lack of MHC class I and II molecules may have implications for T-cell–mediated immunotherapy in neuroblastoma.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.     

Invasive equine trophoblast expresses conventional class I major histocompatibility complex antigens

Monoclonal antibodies and alloantisera were used in an indirect immunohistochemical assay to determine the expression of class I and class II Major Histocompatibility Complex (MHC) antigens by equine placental cells and the endometrial tissues at the fetal-maternal interface. MHC class I antigens were expressed at high density on the surface of the trophoblast cells of the chorionic girdle at days 32-36, just prior to their invasion of the endometrium. The mature gonadotrophin-secreting cells of the endometrial cups, which are derived from the chorionic girdle cells, had greatly reduced levels of MHC class I antigen expression while no MHC class I antigens were detectable on the non-invasive trophoblast cells of the allantochorion, except in small isolated patches. MHC class I antigens immunoprecipitated from chorionic girdle cells with either monoclonal antibodies or alloantisera had a relative molecular mass of 44,000, which was identical to that of MHC class I antigens precipitated from lymphocytes with the same reagents. MHC class II antigens were not detected on any trophoblast cells, although they were expressed at high levels by the endometrial glandular and lumenal epithelium immediately bordering the endometrial cups. MHC class I antigens were also expressed at high levels by endometrial tissues in the area of the cups. The high level of MHC class I antigen expression by endometrial glands within and bordering the cups was in sharp contrast to the greatly reduced class I antigen expression by the mature endometrial cup cells themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Edmonston measles virus prevents increased cell surface expression of peptide-loaded major histocompatibility complex class II proteins in human peripheral monocytes

Gamma interferon (IFN-gamma) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-alpha/beta can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-alpha/beta and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-alpha/beta. Infection of purified monocytes with Edmonston B MV resulted in an apparent increase in cell surface expression of HLA-A, -B, and -C class I proteins, but it had no effect on the expression of HLA-DR class II proteins. MV-infected purified monocytes expressed IFN-alpha/beta, but no measurable IFN-gamma expression was detected in supernatant fluids. Class II protein expression could be enhanced by coculture of purified monocytes with uninfected peripheral blood mononuclear cell (PBMC) supernatant. MV infection of PBMCs also did not affect expression of class II proteins, but the expression of HLA-A, -B, and -C class I proteins was increased two- to threefold in most donor cells. A direct role for IFN-alpha/beta suppression of MHC class II protein expression was not evident in monocytes since MV suppressed class II protein expression in the absence of IFN-alpha/beta. Taken together, these data suggest that MV interferes with the expression of peptide-loaded class II complexes, an effect that may potentially alter CD4(+)-T-cell proliferation and the cell-mediated immune responses that they help to regulate.     

The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Ethanol: an enhancer of major histocompatibility complex antigen expression

Ethanol enhances expression of cell surface class I major histocompatibility complex (MHC) antigens in a variety of cell lines; up to an eightfold increase is observed in an embryonic cell line. In ethanol-treated L cells, increased cell surface expression of MHC antigens occurs with a concomitant increase in steady-state RNA levels. This effect is promoter dependent and restricted, because not all gene products are elevated. The effective ethanol concentration (1%) is physiologically attainable, leading to speculations about the role of elevated MHC antigens in alcohol-related diseases.     

Transfection of beta 2-microglobulin restores IFN-mediated protection from natural killer cell lysis in YAC-1 lymphoma variants

A beta 2-microglobulin (beta 2m)-deficient variant of YAC-1, A.H-2-, was transfected with a genomic beta 2m clone. Transfected cells were used to investigate the role of beta 2m in IFN-induced protection from NK cell lysis. IFN-gamma treatment of the NK-sensitive murine YAC-1 lymphoma results in reduced sensitivity to NK cell-mediated lysis in parallel with increased expression of its constitutively low MHC class I expression. It was previously shown that the A.H-2- variant had lost both these capacities, although it retained other responses to IFN-gamma. Here beta 2m transfection restored the YAC-1 phenotype with respect to an inducible expression of MHC class I molecules and a concomitant protection from NK cell lysis after treatment with IFN-gamma. In the absence of IFN-gamma the NK sensitivity of the transfectants did not differ significantly from A.H-2-. A similar protection from NK cell lysis, in parallel with enhanced MHC class I expression, was observed for in vivo-passaged beta 2m transfectants whereas no protection was found for in vivo-passaged A.H-2- cells. The present study provides evidence that the IFN-gamma-mediated protection from NK cell lysis is dependent on beta 2m expression in the YAC-1 lymphoma. Restoration of MHC class I assembly, transport, and concomitantly an IFN-gamma augmentable cell surface expression of MHC class I molecules is a possible explanation for the effect of beta 2m.     

Coexpression of the human HLA-A2 or HLA-B7 heavy chain gene and human beta 2-microglobulin gene in L cells

L cells expressing human HLA-A2 or HLA-B7 class I antigen heavy chains are not recognized by human cytotoxic T lymphocytes directed at HLA-A2 or HLA-B7 antigens. To test whether the absence of human beta 2-m was the cause of the lack of recognition by the human cytotoxic T lymphocytes, coexpression of the human beta 2-m gene and the HLA-A2 or HLA-B7 heavy chain in L cells ("double transfectants") was obtained. In addition, L cells expressing HLA-A2 or HLA-B7 antigens in association with human beta 2-m were obtained by an exchange reaction, in which human beta 2-m from serum replaced the endogenous murine beta 2-m. Both types of transfectant cells were used in 51Cr-release assays and cold target inhibition assays for human cytotoxic T cell clones which were directed at HLA-A2 or HLA-B7. Neither human CTL clones nor a mixture of CTL specific for HLA-A2 and HLA-B7 were able to recognize these cells. Several alternative explanations for these observations are discussed.     

Expression of invariant chain (CD 74) and major histocompatibility complex (MHC) class II antigens in the human fetus.

During the initiation of an immune response, antigen-presenting cells employ MHC class II antigens as key molecules to present small peptides to CD4-positive lymphocytes. The invariant chain (Ii; CD74) plays a critical role in this process by influencing the expression and peptide loading of the MHC class II molecules. Therefore, coordinate expression of these molecules is believed to play an important role in antigen presentation. This study explores the expression of these molecules in fetal tissues. Formalin-fixed, paraffin-embedded multi-organ tissue blocks from aborted fetuses (age range 7-22 weeks) were immunostained for Ii/CD74 and MHC class II antigens using commercially available monoclonal antibodies for Ii/CD74 (LN2) and MHC class II antigens (LN3), respectively. Coordinate staining for Ii/CD74 and MHC class II antigens was seen in the skin, proximal renal tubules, tips of small intestinal mucosa, and cells of the reticuloendothelial system, including the spleen and thymus. Expression of Ii/CD74, but not of MHC class II antigens, was seen in pulmonary alveolar epithelium in all cases and in testicular Leydig cells (11 of 11 testes examined). The distribution and intensity of staining did not change significantly with age. In conclusion, this study describes distribution of Ii/CD74 and MHC class II antigens in human fetal tissues. Coordinate expression of Ii/CD74 and MHC class II antigens was identified in most fetal tissues, but there were also notable exceptions. In all cases this took the form of expression of Ii/CD74 in the absence of MHC class II expression. Discordance was particularly striking in pulmonary alveolar epithelium and testicular Leydig cells. This suggests that the Ii/CD74 molecule has functional roles in addition to its role in antigen presentation.     

Human NK cells differ more in their KIR2DL1-dependent thresholds for HLA-Cw6-mediated inhibition than in their maximal killing capacity

In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-γ secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels) required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.     

Induction of class I and class II MHC antigen expression on murine bone marrow-derived macrophages by IL-4 (B cell stimulatory factor 1)

We have studied the effects of IL-4 (B cell stimulatory factor 1) on the expression of MHC gene products in normal bone marrow-derived macrophages, peritoneal macrophages, and the myelomonocytic cell line WEHI-3. Using both IL-4-containing T cell supernatant and rIL-4, we have observed significant induction of both class I and class II MHC surface expression (about 1.5- to 4-fold increase) in 2-, 3-, and 4-day cultures of bone marrow-derived macrophages. This induction was also apparent at the mRNA level as assessed by Northern blot analysis using A beta, E alpha, and class I probes. Kinetic analysis revealed that induction of class II mRNA by IL-4 was slower than induction by IFN-gamma, requiring 48 h before a significant increase was noted. The magnitude of MHC induction by IL-4 was not as great as that seen with IFN-gamma, which was found to increase surface expression of MHC antigens two- to eightfold. IL-4 also differs from IFN-gamma in the repertoire of macrophages responsive to it. IL-4 was unable to induce class I or class II expression in either thioglycolate-elicited peritoneal macrophages or WEHI-3 cells whereas IFN-gamma induced MHC antigen expression on both cell types under the same conditions. These data demonstrate that IL-4 is capable of inducing both class I and class II MHC gene products in some, but not all, macrophages.     

Expression of MHC antigens on murine trophoblast and their modulation by interferon

Primary cultures of defined populations of mouse trophoblast, isolated from mature placentas, were analyzed for their MHC antigen expression and for the modulatory effect of interferon (IFN) by antibody- and complement-mediated cytotoxicity and flow cytofluorometry. The cells were obtained from placentas by enzymatic digestion, followed by Percoll gradient fractionation, and are large, fetally derived epithelial cells, which we previously characterized and identified as trophoblast cells. After 2 days in culture, a significant proportion of the trophoblast cells were susceptible to antibody- and complement-mediated lysis by anti-paternal strain alloantisera (40%) and, to a lesser degree, by an anti-class I monoclonal antibody (20%). Flow cytofluorometric analysis indicated that 20 to 50% of the cultured trophoblast cells expressed low levels of paternal strain class I antigens as compared to L cell fibroblasts. After culture for 48 hr with IFN-alpha/beta or IFN-gamma, the percent of class I-positive cells was increased to 68 to 76%, as was the mean fluorescence intensity, which correlated with the increased percent of antibody- and complement-mediated specific lysis (73%). No expression of class II MHC antigen by the cultured trophoblast cells was detected, even after culture in the presence of IFN-gamma. The cultured trophoblast cells, when tested for alkaline phosphatase (AP) activity, were composed of strongly positive and weakly positive subpopulations. An inverse correlation between strength of AP activity and the expression of H-2 was observed by double staining. These results indicate that trophoblast cells cultured in vitro are able to express paternal strain class I but not class II MHC antigens, as has been reported in vivo, and that this expression can be modulated by IFN. Further study of these cells should provide important clues for the understanding of materno-fetal coexistence in the face of MHC antigen differences.