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.     

Changes in NF-kappa B and ISGF3 DNA binding activities are responsible for differences in MHC and beta-IFN gene expression in Ad5- versus Ad12-transformed cells.

Changes in MHC class I expression are frequently observed in tumors, which represents at least one mechanism by which tumor cells escape immune surveillance. MHC class I expression is often suppressed in type 12 adenovirus (Ad12)-transformed rodent cells, but is highly induced in Ad5-transformed cells. This difference helps to explain why Ad12 but not Ad5 can induce tumors in immunocompetent syngeneic rats. In this report we demonstrate that only Ad5- but not Ad12-transformed rodent fibroblasts constitutively express beta-IFN which results in ISGF3 factor induction, and stimulation of MHC class I expression. Furthermore, we demonstrate that in contrast to Ad12-transformed cells, Ad5-transformed cells show constitutive levels of nuclear NF-kappa B-like DNA binding activity. This is of particular interest since both the beta-IFN and the MHC class I promoters contain an NF-kappa B DNA binding site. Thus, high levels of MHC class I expression in Ad5-transformed cells are due to a combinatorial stimulation of two cis-regulatory sequences of the MHC class I promoter: the NF-kappa B binding site and the interferon stimulated response element (ISRE), which binds the ISGF3 factor complex. The failure of Ad12-transformed cells to activate this pathway explains their low levels of MHC class I expression and their greater oncogenicity.     

Driving adenovirus type 12-transformed BALB/c mouse cells to express high levels of class I major histocompatibility complex proteins enhances, rather than abrogates, their tumorigenicity.

The tumorigenicity of adenovirus type 12 (Ad12)-transformed cells has been attributed to the low levels of class I major histocompatibility complex (MHC) protein expression by these cells. These levels of class I proteins are thought to be below the threshold critical for cytotoxic T-lymphocyte recognition, a process that may be involved in tumor cell immunosurveillance. We have used gene transfer experiments to investigate the role played by class I protein expression in the tumorigenicity of Ad12-transformed BALB/c mouse cells in naive, syngeneic adult mice. Our Ad12-transformed mouse cells were tumorigenic in adult mice and were similar to other Ad12-transformed mammalian cells in that they expressed low levels of class I MHC mRNA and cell surface proteins. Despite these low levels of expression, the cells were highly immunogenic in syngeneic mice and were rejected as allografts by allogeneic mice. Transfection of genomic H-2Dd or H-2Ld fragments into these cells produced a variety of cell clones that expressed increased levels of cell surface class I proteins. These cells expressing high levels of class I protein were up to 16-fold more tumorigenic than the parental cells in syngeneic adult mice. Thus, by quantitative assays, the tumorigenicity of Ad12-transformed BALB/c mouse cells is not functionally related to the low levels of class I MHC proteins they express. The increased tumorigenicity expressed by H-2Dd- and H-2Ld-transfected cells was not detected in BALB/c nu/nu mice, suggesting that a thymus-dependent mechanism that is not mediated by evasion of cytotoxic T-lymphocyte recognition could contribute to the difference in tumorigenicity of Ad12-transformed BALB/c mouse cells that express low and high levels of class I MHC proteins.     

Downregulation of MHC class I expression due to interference with p105-NF kappa B1 processing by Ad12E1A.

We have previously demonstrated that expression of major histocompatibility complex (MHC) class I genes is repressed in baby rat kidney cells transformed by early region 1 of oncogenic adenovirus type 12 (Ad12E1). Reduced expression of MHC class I antigens contributes to the escape of Ad12-transformed cells from T-cell-mediated immune surveillance and to tumour induction. In this study, we show that repression of MHC class I expression by Ad12E1A is mediated via the H2TF1 element of the MHC class I promoter. This element binds NF kappa B and KBF1, two factors which play a major role in the regulation of MHC class I expression in vivo. In extracts from Ad12E1-transformed cells, binding of KBF1 and NF kappa B to the H2TF1 element is decreased. This is caused by reduced production of p50-NF kappa B1, the 50 kDa subunit shared by KBF1 and NF kappa B, due to interference with p105-NF kappa B1 processing by Ad12-13S-E1A protein. Overexpression of the p105-NF kappa B1 cDNA, or of a truncated p105-NF kappa B1 cDNA that codes for p50-NF kappa B1, restores MHC class I expression in Ad12E1-transformed cells. These data demonstrate that downregulation of MHC class I expression in Ad12E1-transformed cells is due to interference with processing of p105-NF kappa B1 by the Ad12-13S-E1A protein.     

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.     

Cells transformed by adenovirus type 12 but not by type 5 are dependent on insulin or insulin-like growth factor I for their proliferation

We have investigated the responsiveness to growth factors (GFs) of primary baby rat kidney (BRK) cells transformed by the E1 region of adenovirus 5 or 12. The in vitro growth of non-oncogenic adenovirus 5-transformed BRK cells is largely independent of serum GFs, whereas growth of highly oncogenic adenovirus 12-transformed cells is strictly dependent on GFs present in serum. For the growth of adenovirus 12 E1-transformed BRK cells serum can be replaced by insulin or insulin-like growth factor-I but not by epidermal growth factor. To maintain the in vitro growth of adenovirus 12-transformed cells physiological levels of insulin-like growth factor-I, but not of insulin, are sufficient. Similar results have been found with adenovirus-transformed primary murine cells and with transformants of an established rat cell line, NRK 49F. This indicates that the observed GF responsiveness is not dependent of the cell type used but is determined by the serotype of the adenovirus-transforming region. Using hybrid E1 regions consisting of E1A of one serotype and E1B of the other, we show that the pattern of GF-responsiveness correlates with the origin of the E1A region. The differences in the GF-responsiveness of the adenovirus 5-transformed and adenovirus 12-transformed cells will be discussed in terms of the oncogenicity of these cells.     

Neuronal cells are deficient in loading peptides onto MHC class I molecules.

Virally infected neurons avoid destruction by cytotoxic T lymphocytes (CTLs) by failing to express major histocompatibility complex (MHC) class I molecules. Like neurons in vivo and in primary culture, the OBL21 neuronal cell line expressed barely detectable levels of MHC class I molecules. This correlated with very low levels of mRNAs for the MHC class I heavy chains (alpha C). OBL21 cells also fail to provide MHC class I molecules with the peptides necessary for their efficient assembly and transport to the cell surface. This function can be restored by treatment with interferon-gamma (IFN-gamma). The mRNA for peptide transporters HAM1 and HAM2 was not detectable in OBL21 neuronal cells, but was induced by IFN-gamma treatment. Hence, the ability of neurons to evade CTL-mediated killing results from expression at low levels of the MHC class I alpha C, the peptide transporters HAM1 and HAM2, and possibly other genes of the peptide-loading machinery.     

Selective MHC expression in tumours modulates adaptive and innate antitumour responses

Progress towards developing vaccines that can stimulate an immune response against growing tumours has involved the identification of the protein antigens associated with a given tumour type. Epitope mapping of tumour antigens for HLA class I- and class II-restricted binding motifs followed by immunization with these peptides has induced protective immunity in murine models against cancers expressing the antigen. MHC class I molecules presenting the appropriate peptides are necessary to provide the specific signals for recognition and killing by cytotoxic T cells (CTL). The principle mechanism of tumour escape is the loss, downregulation or alteration of HLA profiles that may render the target cell resistant to CTL lysis, even if the cell expresses the appropriate tumour antigen. In human tumours HLA loss may be as high as 50%, inferring that a reduction in protein levels might offer a survival advantage to the tumour cells. Alternatively, MHC loss may render tumour cells susceptible to natural killer cell-mediated lysis because they are known to act as ligands for killer inhibitory receptors (KIRs). We review the molecular features of MHC class I and class II antigens and discuss how surface MHC expression may be regulated upon cellular transformation. In addition, selective loss of MHC molecules may alter target tumour cell susceptibility to lymphocyte killing. The development of clinical immunotherapy will need to consider not only the expression of relevant CTL target MHC proteins, but also HLA inhibitory to NK and T cells. Received: 20 March 1999 / Accepted: 3 May 1999     

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.     

Complex interplay of activating and inhibitory signals received by Vgamma9Vdelta2 T cells revealed by target cell beta2-microglobulin knockdown

Tumor cells often escape immunosurveillance by down-regulating MHC class I molecule expression. For human Vgamma9Vdelta2 T cells, a major peripheral blood T cell subset with broad antitumor reactivity, this down-regulation can affect signals transmitted by both the inhibitory and the activating MHC class I and Ib-specific NK receptors (NKRs) that these lymphocytes frequently express. To assess the overall impact of MHC down-regulation on Vgamma9Vdelta2 T cell activation, we used stable beta(2)-microglobulin knockdown to generate tumor cells with a approximately 10-fold down-modulation of all MHC class I molecules. This down-modulation had little effect on T cell proliferation or cytokine production, but modified tumor cell killing efficiency. Ab-blocking studies identified ILT2 as an important inhibitor of tumor cell killing by Vgamma9Vdelta2 T cells. Down-modulation of MHC class I and Ib molecules severely reduced ILT2 inhibitory signaling, but still allowed signaling by activating CD94-based receptors. It also unveiled a frequent enhancing effect of NKG2D on tumor killing by Vgamma9Vdelta2 T cells. Current models suggest that activating NKRs have less affinity for their MHC ligands than homologous inhibitory NKRs. Our results show that, despite this, activating NKRs recognizing MHC class I molecules play an important role in the increased killing by Vgamma9Vdelta2 T cells of tumor cells with down-regulated MHC class I molecule expression, and suggest that these T cells will best lyse tumor cells combining MHC class I molecule expression down-regulation with up-regulated NKG2D ligand expression.     

The level of expression of adenovirus type 2 transforming genes governs sensitivity to nonspecific immune cytolysis and other phenotypic properties of adenovirus 2-simian virus 40-transformed cell hybrids.

Syrian hamster embryo cells transformed by adenovirus type 2 (Ad2) or simian virus 40 (SV40) differ markedly in morphology, tumorigenicity, and susceptibility to in vitro lysis by nonspecific cytotoxic cells. Hybrid cells formed by fusing Ad2- and SV40-transformed Syrian hamster embryo cells may express only SV40 T antigens or both SV40 and Ad2 T antigens. Hybrids that express only SV40 T antigens are indistinguishable from the nonhybrid SV40-transformed phenotype, whereas hybrid cells that express T antigens from both viruses closely resemble the nonhybrid parental Ad2-transformed phenotype. Because these hybrid cells have been useful in the study of neoplastic transformation, we determined the amount of viral antigens that they accumulate in an attempt to correlate the level of expression of the transforming viral genes with some of their phenotypic properties. Hybrid cells that expressed proteins from both viruses showed reduced levels of SV40 T antigens compared with those of hybrid cells that did not express Ad2 T antigens. We also found that the production of several cellular proteins that influence cytomorphology was inhibited in hybrid and nonhybrid cells that expressed Ad2 T antigens, and the repression of these cellular proteins correlated with a change in cytomorphology from fibroblastic to spherical. Finally, we showed that the susceptibility of our hybrid cells to in vitro lysis by natural killer cells and activated macrophages, two putative host-effector cells involved in defense against neoplasia, correlated closely with the level of expression of a 58,000-dalton Ad2 protein. The results reported here, together with the results of previous studies, indicate that the oncogenic potential of hybrid cells that express both Ad2 and SV40 antigens is extremely sensitive to Ad2 expression, whereas other phenotypic properties depend on Ad2 expression in a dose-dependent manner.     

Adenovirus E1A 12S protein induces DNA synthesis and proliferation in primary epithelial cells in both the presence and absence of serum.

Infection of primary baby rat kidney (BRK) cells with an adenovirus that carries an E1A 12S cDNA in place of the normal E1A region (adenovirus 5 [Ad5] 12S) resulted in the induction of cellular DNA synthesis and proliferation of the epithelial cells in the population, even in the absence of serum. Increased cellular DNA synthesis was first detectable by 12 h after infection and was maintained at a 10- to 20-fold higher level than in mock-infected cells. By 5 days after infection there was a 10-fold-greater number of 12S virus-infected BRK cells. These infected BRK cells retained many of their normal epithelial cell characteristics and were not transformed. The expression of the E1A 12S protein(s) occurred early after infection. There was no induction of adenoviral gene expression or viral DNA replication in these cells. The early effects of a fully transforming gene product(s) were also examined. The Ad5-simian virus 40 hybrid virus, Ad5.SVR4, in which the early region of simian virus 40 has replaced the E1 region of Ad5, was used to infect BRK cells. The kinetics of expression of the T antigens were similar to those of the 12S polypeptides. Infection with Ad5.SV4 also resulted in the induction of cellular DNA synthesis and cell proliferation at levels similar to those observed with the 12S virus. However, infection with Ad5.SVR4 resulted in cells that had lost some of their epithelial cell characteristics and were fully transformed. Thus, although the early cellular events induced by the two genes were similar, they did not yield the same final cellular phenotype.