Adenovirus E1A-mediated regulation of class I MHC expression.

Expression of class I MHC transplantation antigens has been shown to be reduced in baby rat kidney (BRK) cells transformed by highly oncogenic adenovirus type 12 (Ad12), as compared with untransformed cells and cells transformed by non-oncogenic Ad5. Here we show that this reduction of class I expression also occurs in a variety of other primary cell cultures transformed by Ad12, and that reduction of class I gene expression occurs for all class I loci. Transfection of Ad5E1 into class I-negative Ad12-transformed BRK cells leads to complete restoration of class I expression. Introduction of Ad12E1 into most class I-positive established cell lines does not result in suppression of class I expression. However, transfection of the Ad12E1A region into a class I-positive cell line which was immortalized by a mutant Ad12E1A region resulted in suppression of class I gene expression, implying that the suppression of class I activity in Ad12-transformed cells is due to an active switching-off process.     

Treatment of ovarian cancer cell lines with 5-aza-2′-deoxycytidine upregulates the expression of cancer-testis antigens and class I major histocompatibility complex-encoded molecules

Purpose  To test the hypothesis that decrease in DNA methylation will increase the expression of cancer-testis antigens (CTA) and class I major histocompatibility complex (MHC)-encoded molecules by ovarian cancer cells, and thus increase the ability of these cells to be recognized by antigen-reactive CD8⁺ T cells. Methods  Human ovarian cancer cell lines were cultured in the presence or absence of varying concentrations of the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC) for 3–7 days. The expression levels of 12 CTA genes were measured using the polymerase chain reaction. The protein expression levels of class I MHC molecules and MAGE-A1 were measured by flow cytometry. T cell reactivity was determined using interferon-γ ELISpot analysis. Results  DAC treatment of ovarian cancer cell lines increased the expression of 11 of 12 CTA genes tested including MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, NY-ESO-1, TAG-1, TAG-2a, TAG-2b, and TAG-2c. In contrast, DAC treatment decreased the already low expression of the MAGE-A2 gene by ovarian cancer cells, a finding not previously observed in cancers of any histological type. DAC treatment increases the expression of class I MHC molecules by the cells. These effects were time-dependent over a 7-day interval, and were dose-dependent up to 1–3 μM for CTA and up to 10 μM for class I MHC molecules. Each cell line tested had a unique pattern of gene upregulation after exposure to DAC. The enhanced expression levels increased the recognition of 2 of 3 antigens recognized by antigen-reactive CD8⁺ T cells. Conclusions  These results demonstrate the potential utility of combining DAC therapy with vaccine therapy in an attempt to induce the expression of antigens targeted by the vaccine, but they also demonstrate that care must be taken to target inducible antigens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distinct effect of forskolin and interferon-gamma on cell proliferation and regulation of histocompatibility antigen expression in hematopoietic cells

The adenylate cyclase activator, forskolin, was found to induce expression of class I and class II major histocompatibility complex antigens in a B precursor cell line, Reh, as well as in a B lymphoid cell line, Raji. No such effect was, however, observed when the promyelocytic cells line HL-60 was treated with either forskolin or the cAMP analogue 8-bromoadenosine cyclic monophosphate. As expected, all three cell lines showed reduced proliferation upon forskolin treatment. Forskolin induced expression of class I and class II major histocompatibility complex antigens in cell lines not affected by interferon-gamma and vice versa, indicating that cAMP is not involved in the regulation of histocompatibility antigens by interferon-gamma. We also compared the effect of interferon-gamma and 12-O-tetradecanoylphorbol 13-acetate on major histocompatibility complex class I and class II expression, and despite differences in the response on the tested cell lines, we can not at this point exclude the possibility that protein kinase C is involved in the action of interferon-gamma.     

Modulation of the tumorigenicity of human adenovirus-12-transformed cells by interferon

Human adenovirus-12-transformed cells express greatly reduced levels of the major histocompatibility complex class I antigens and are highly tumorigenic in syngeneic hosts. The finding that expression of a transfected class I gene is sufficient to abrogate their tumorigenicity underscores the importance of defining the conditions that will lead to derepression of endogenous class I genes in these cells. Brief treatment of Ad12-transformed cells with interferon results in the rapid but transient expression of class I antigens, and these interferon-treated cells have significantly reduced tumorigenicity in immunocompetent hosts. We have further demonstrated that subcutaneous administration of interferon, subsequent to the introduction of a tumorigenic dose of Ad12-transformed cells, results in complete protection against this tumor. The ability of interferon to "induce" class I gene expression may be an important modality in the treatment of a variety of spontaneous tumors that exhibit greatly reduced levels of class I antigens on their cell surface.     

Distinct effect of forskolin and interferon-γ on cell proliferation and regulation of histocompatibility antigen expression in hematopoietic cells

The adenylate cyclase activator, forskolin, was found to induce expression of class I and class II major histocompatibility complex antigens in a B precursor cell line, Reh, as well as in a B lymphoid cell line, Raji. No such effect was, however, observed when the promyelocytic cells line HL-60 was treated with either forskolin or the cAMP analogue 8-bromoadenosine cyclic monophosphate. As expected, all three cell lines showed reduced proliferation upon forskolin treatment. Forskolin induced expression of class I and class II major histocompatibility complex antigens in cell lines not affected by interferon-γ and vice versa, indicating that cAMP is not involved in the regulation of histocompatibility antigens by interferon-γ. We also compared the effect of interferon-γ and 12-O-tetradecanoylphorbol 13-acetate on major histocompatibility complex class I and class II expression, and despite differences in the response on the tested cell lines, we can not at this point exclude the possibility that protein kinase C is involved in the action of interferon-γ.     

Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure.

The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule. Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed. Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells. After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines. Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene. These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure.     

DNA-mediated transfer of major histocompatibility class II I-Ab and I-Abm12 genes into B lymphoma cells: molecular and functional analysis of introduced antigens

A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer.     

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.     

Establishment of cell lines from somite stage mouse embryos and expression of major histocompatibility class I genes in these cells

To study the regulation of MHC class I gene expression during embryonic development, we have characterized a number of clonal cell lines derived from somite stage mouse embryos that were established with or without infection by several transforming retroviruses in combination with murine leukemia viruses. Unlike embryonal carcinoma (EC) cells that have been used as a model for early embryos, the cell lines derived from somite stage embryos are negative for stage specific embryonic Ag-1 and do not appear to differentiate after retinoic acid treatment. Morphology varies from clone to clone and is distinct from that of F9 and other EC cells. In agreement with previous findings in in vivo embryos, expression of surface MHC class I antigen in 57 new clones is either undetectable or low (with variability). All of the clones respond to the addition of interferons and express MHC class I antigens at high levels, but the kinetics of mRNA accumulation vary considerably. To examine the basis of the generally low or absent MHC class I gene expression in these cells, we tested promoter activity of a MHC class I gene by CAT assay after transient DNA transfection. Regardless of the basal levels of mRNA or surface Ag, CAT activity directed by various portions of the 5' flanking region of the MHC class I gene was uniformly low. The cells showed neither the negative nor the positive regulation of MHC class I genes that had been noted respectively for EC cells and for cells expressing the Ag constitutively. The pattern seen in the new cell lines suggests that there is an intermediate stage in the developmental regulation of MHC class I gene expression that may operate during the middle to late stage of fetal development.     

Differential expression of the major histocompatibility antigen complex (MHC) on a series of Burkitt's lymphoma lines

We compared the expressions of class I and class II major histocompatibility antigen complex (MHC) on the surface of Jijoye and P3HR-1 cells of Burkitt's lymphoma sublines. Jijoye cells had a large amount of class I and class II MHC antigens, whereas these antigens were less expressed on P3HR-1 cells. On a subline of P3HR-1 K cells the expression of class I antigen markedly diminished and class II antigen was undetectable. On the other hand, Jijoye, P3HR-1, and P3HR-1 K cell lines were confirmed to be Epstein-Barr virus (EBV) nonproducer, low producer, and high producer, respectively. The chemical activation of EBV genome by treating P3HR-1 cells with 12-O-tetradecanoyl phorbol-13 acetate (TPA) and n-butyrate resulted in inhibition of the expression of class I and II antigens, while the addition of retinoic acid, an inhibitor of virus replication, blocked the decrease in the MHC antigen expression. These findings suggested that there might be an inverse correlation between the virus production and the expression of class I and II MHC antigens.