Modulation of MHC antigen expression by viruses and oncogenes.

It is becoming increasingly clear that regulation of MHC antigen expression by viruses and oncogenes, leading to either immune evasion or autoimmunity, is widespread and important in disease. At a recent meeting*, which brought together workers interested in tumour immunology, viral infection and the MHC, a number of mechanisms for the regulation of MHC antigen expression were revealed and the importance of balanced expression of MHC gene products to effective immunity was underlined.

Role of oncogenes in the regulation of MHC antigen expression.

Class I and class II major histocompatibility complex (MHC) antigens are required for CD8+ cytotoxic T cells and CD4+ helper T-cells, respectively, to recognize foreign antigen. Regulating the levels of expression of these MHC antigens regulates the T-cell responses [1]. This regulation is mainly carried out by the interferons (IFN), which are produced in the disease state. Type I IFN (IFN alpha or IFN beta; collectively 'IFN alpha beta) up-regulates class I MHC and IFN gamma up-regulates class I and class II MHC. We and others [1-3] have shown that transfection of cells with a variety of oncogenes including ras and myc affects the level of MHC antigen expression. This and other data provide evidence for a scheme in which the signal transduction mechanisms whereby IFN up-regulates MHC antigens involve several (proto) oncogenes.

Reduced stimulation of helper T cells by Ki-ras transformed cells.

A number of viral genes and cellular oncogenes inhibit major histocompatibility complex (MHC) antigen expression at the cell surface. In the case of inhibition of class I MHC antigens by viral genes this results in a reduced recognition by antigen-specific cytotoxic T cells. The activated Ki-ras cellular oncogene carried by the Ki-murine sarcoma virus (Ki-MuSV) in contrast inhibits class II MHC (or Ia) antigen expression on transformed cells. We have studied how transformation with Ki-ras affects recognition by alloreactive helper T cells. We found that the Ki-ras inhibition of class II MHC antigen expression led to greatly reduced stimulation of alloreactive T cells to proliferate and to secrete interferon-gamma (IFN-gamma). These findings support our hypothesis that the ability of an oncogene to reduce class II MHC antigen expression is crucial to its ability to produce tumour cells.

Regulation of IFN-gamma-induced host cell MHC antigen expression by Kirsten MSV and MLV. I. Effects on class I antigen expression.

We have reported previously that the Kirsten murine sarcoma virus (Ki-MSV) that carries the v-Ki-ras oncogene prevents C3H10T 1/2 fibroblasts from being able to respond to interferon-gamma (IFN-gamma) with the expression of the class II major histocompatibility complex (MHC) antigen, H-2A. In this report we investigate further as to whether MSV or its parent virus Kirsten murine leukaemia virus (Ki-MLV) is able to reduce host class I MHC antigen expression. The results demonstrate that class I expression is diminished in MSV-infected cells over a time-course of 7 days after exposure to IFN-gamma and over a range of IFN-gamma concentrations. The optimal concentration of IFN-gamma for maximal class I expression remained unchanged. Cells infected with Ki-MLV, which failed to abolish the induction by IFN-gamma of class II antigens, also expressed lower levels of class I antigens, similar to those for cells infected with Ki-MSV, after exposure to IFN-gamma. It is likely therefore that the inhibition of class I induction is due to genetic material shared between the viruses, principally in the long terminal repeats (LTR), and hence that the mechanism of action is distinct from that responsible for the abolition of class II induction by Ki-MSV alone. Since class I antigens are required for CD8+ T cells (mainly cytotoxic T cells) to recognize (foreign) antigen this reduction in class I expression might lead to reduced visibility of infected cells to T cells and thus might contribute to the tumorigenicity of Ki-MSV-infected cells.

Regulation of IFN-gamma-induced host cell MHC antigen expression by Kirsten MSV and MLV. II. Effects on class II antigen expression.

We have reported previously that the Kirsten murine sarcoma virus (Ki-MSV), which carries the v-Ki-ras oncogene, prevents the induction of the class II MHC antigen H-2A and reduces the induction of class I MHC antigens by interferon-gamma (IFN-gamma) on C3H10T 1/2 fibroblasts. It is here shown that the abolition by the virus of H-2A expression extends also to class II antigen H-2E and that this is maintained for at least 7 days after IFN treatment. In addition no concentration of IFN-gamma tested, including supra-optimal concentrations for class I antigen expression, induced class II antigens on MSV-infected cells. Thus MSV inhibits the induction by IFN-gamma of class II MHC antigens by a mechanism other than via a change in kinetics of response to, or in the sensitivity of the cells to, IFN. The possibility that transformation by MSV could result in the (selective) outgrowth of cells unresponsive to IFN was refuted by the observation that clones of C3H10T 1/2, when infected with Ki-MSV, expressed no or dramatically reduced levels of H-2A or H-2E. One C3H10T 1/2 clone chosen for high class II expression, when transformed with Ki-MSV, did express low levels of class II antigens at optimal concentrations of IFN-gamma, suggesting that the degree of the reduction of class II expression varies with the cells that are infected. Comparison with mechanisms whereby other viruses inhibit MHC antigen display revealed an interesting possibility: IFN response sequences (IRS) identified in the virus genomes might act in trans to (down) regulate MHC antigen expression. This could be an important mechanism determining the tumourigenicity of, and immune evasion by, Ki-MSV and other viruses.

Inhibition of expression of cell-mediated immunity by a cell surface-binding antibody directed against T-cell helper factors.

In this study, the effects of a monoclonal antibody, AF3.44.4, which is directed against antigen-specific, T-cell-derived helper factors, were investigated in two models of T-cell-macrophage co-operation. In vitro, cultures of nylon-wool-enriched peritoneal cells from mice immune to Listeria monocytogenes (T cells), macrophages from normal mice and Listeria antigen produced an activity mitogenic for mouse thymocytes (TMF). When AF3.44.4 was added to such cultures, the production of this activity was inhibited in a dose-dependent fashion. Preincubation of T cells with AF3.44.4, followed by its removal, was not sufficient for the inhibitory effect to be manifested. In an in vivo system, when spleen cells from Listeria-immunized mice were transferred to unprimed mice, the latter were protected against subsequent challenge with Listeria. Treatment of spleen cells with AF3.44.4 prior to transfer did not affect their ability to confer resistance upon recipients. It is postulated that AF3.44.4 acts on a population of T cells that is involved in macrophage-T-cell co-operation in vitro and that this population may be different from that involved in macrophage T-cell co-operation in vivo.

Kirsten murine sarcoma virus abolishes interferon gamma-induced class II but not class I major histocompatibility antigen expression in a murine fibroblast line.

The effect of infecting fibroblasts with Kirsten murine sarcoma virus/murine leukemia virus (Ki-MSV/MLV) on constitutive and IFN-gamma-induced H-2 antigen expression was investigated. The fibroblasts used were two established cell lines (C3H10T1/2 and BALB/c3T3) and fresh embryo fibroblasts from C3H mice. Class I antigens were expressed constitutively by BALB/c3T3; infection with MLV, MSV or the two together had little effect on this constitutive expression. Class I antigens (H-2K, H-2D) were strongly induced on all three types of fibroblast by rIFN-gamma, and infection had little effect on this. None of the fibroblasts expressed constitutively detectable levels of class II antigen; however, C3H10T1/2 fibroblasts could be induced for both H-2A and H-2E by IFN-gamma. Infection of C3H10T1/2 with helper-free Ki-MSV, or MSV together with MLV, completely abolished this induction of class II antigens, while infection with MLV alone had little effect, implying that the abolition of class II induction was due to genomic regions of Ki-MSV not shared with Ki-MLV, probably the v-Ki-ras gene.

Rapid intracellular calcium changes in U937 monocyte cell line: transient increases in response to platelet-activating factor and chemotactic peptide but not interferon-gamma or lipopolysaccharide.

The dye fura-2, a potentially more sensitive successor to quin2 for measuring intracellular free calcium ion concentrations [(Ca2+]i), has been applied here to investigate the possible involvement of early changes in [Ca2+]i in the stimulation of the human monocyte-macrophage-like cell line U937. The calcium ionophores A23187 and ionomycin, known pharmacological stimuli for macrophages, were found to cause sharp rises in [Ca2+]i as expected. Responses analogous to those reported for a murine macrophage cell (J774) were obtained on stimulation of U937 cells with ATP which caused rapid, but transient, increases in [Ca2+]i (from resting levels of about 70 nM to peaks of about 200 mM). In addition to ATP, several agents known to activate macrophages were used as stimuli. In particular, platelet-activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was found to cause rapid, but transient, increases in [Ca2+]i (from resting levels of about 70 nM to peaks of about 400 nM) even at concentrations as low as 10(-10) M. This contrasts with responses to ATP that were markedly reduced at 10(-6) M compared with 10(-5) M or above, suggesting that PAF is a highly potent stimulus for intracellular calcium mobilization in macrophages. Similar responses were obtained with chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine). On the other hand, two agents known to be potent activators of macrophages, interferon gamma and lipopolysaccharide, had no rapid effect on [Ca2+]i. This may reflect differences in the kinetics of signal-response coupling or alternatively a different mechanism of action by-passing the need for rapid elevation of [Ca2+]i.

Infection of cultured murine brain cells by Semliki Forest virus: effects of interferon-alpha beta on viral replication, viral antigen display, major histocompatibility complex antigen display and lysis by cytotoxic T lymphocytes.

Primary brain cell cultures prepared from newborn mice were infected with Semliki Forest virus (SFV). The effects of interferon (IFN-alpha beta) treatment on SFV replication, SFV and major histocompatibility complex (MHC) class I antigen expression, and susceptibility to lysis by SFV-specific cytotoxic T lymphocytes (CTL) were determined. The IFN-alpha beta treatment prevented replication of SFV as determined by incorporation of [3H]uridine into SFV RNA and very markedly reduced the expression of SFV antigens on the cell surface as determined by lysis with antibody and complement or indirect immunofluorescence. However, IFN-alpha beta increased expression of MHC class I antigens, measured by indirect immunofluorescence and as assessed indirectly by susceptibility to killing by alloreactive T cell lines. SFV infection had no effect on MHC class I expression in either IFN-alpha beta-treated or -untreated cells. The infected IFN-alpha beta-untreated brain cells were susceptible to killing by the CTL at effector/target ratios in the range 3 to 30. The killing was MHC antigen-restricted, and uninfected cells were not killed. A target cell (YAC) highly susceptible to natural killer cell cytotoxicity was not killed by the CTL. IFN-alpha beta treatment prior to SFV infection resulted in an augmentation of lysis by the CTL, indicating that even where SFV antigen expression is reduced, in the context of enhanced MHC class I expression brain cells remain susceptible to CTL killing.

Augmentation of cell-mediated responses in vitro by a monoclonal anti-helper factor antibody.

Previous studies have shown that monoclonal antibody AF3.44.4 has specificity for a constant region determinant on mouse antigen-specific helper factors and that it also binds to cultured T cells with functional helper cell characteristics. The antibody synergizes with antigen to enhance in vitro antibody responses; here we demonstrate that it will also enhance cell-mediated responses in vitro such as in the generation of proliferating cells in mixed lymphocyte responses and in the generation of specific killer cells in cytotoxic T lymphocyte cultures. The mechanism of AF3.44.4-generated enhancement was investigated. Increased levels of the lymphokines IL-2 and BCDF were detected in supernatants of AF3.44.4-treated cultures but the antibody itself could not replace interleukin-2 (IL-2), and would not stimulate primed cells in the absence of antigen. This type of monoclonal antibody which augments immunological responses in an antigen-dependent fashion may provide a new class of immunostimulant and a new approach to augmenting the responses of weak immunogens.

A monoclonal antibody against antigen-specific helper factor augments T-cell help.

Antigen-specific molecules, commonly termed 'factors', have been shown to be released from helper and suppressor T cells. These factors mimic the activity of the cells that secrete them and there is much speculation about the relationship of antigen-specific factors to T-cell receptors for antigen. We have raised a variety of antisera in rabbits which were shown to react against conserved 'constant' determinants on either helper or suppressor factors independently of antigenic specificity or mouse strain of origin of the factor. In contrast, syngeneic mouse antisera were found to react with 'variable' factor determinants in an antigen-specific and mouse strain-dependent manner. These antisera thus define two regions on factor molecules, one 'variable' (related to antigen specificity) and the other 'constant' (related to function). However, potential contaminants in these antisera have limited their usefulness. Thus, we are now generating monoclonal antibodies against T-cell factors and report here the properties of a monoclonal antibody (AF3.44.4) which reacts with antigen-specific helper factors. This antibody also binds to helper T cells and, in the presence of antigen, augments helper cell induction in vitro, which, in turn, leads to enhanced antibody production in vitro. These characteristics suggest that AF3.44.4 recognizes a determinant shared by helper factor and the antigen receptor on helper T cells.