Glycosylphosphatidylinositol-anchored H-2Db molecules are defective in antigen processing and presentation to cytotoxic T lymphocytes.

Glycosylphosphatidylinositol-anchored (GPI)-Db molecules are defective in mediating cytotoxic T lymphocytes (CTL) lysis of transfected lymphoma cells, compared to their transmembrane (TM) counterpart. This defect is manifest when antigenic peptide must be processed and presented through the endogenous pathway. These same transfectants can be lysed by allospecific CTL, or by antigen-specific Db-restricted CTL when pulsed with appropriate exogenous synthetic peptide, demonstrating that they can bind and present peptide for CTL-mediated lympholysis. The defect apparently results from differences between GPI-Db and TM-Db assembly and transport, or from differences in membrane topology that affect CD8+ CTL recognition of major histocompatibility complex/peptide complex.

Expression of the MuLV-tumor-associated antigen is restricted to MuLV-transformed cells.

Immunization of mice with Moloney murine leukemia virus (MoMuLV) induces the generation of a population of CTL which recognizes a non-viral, tumor-associated antigen (TAA) expressed on MuLV-induced tumors. To determine whether this TAA could be used as a pre-leukemic or leukemic cell marker, CTL clones directed against Moloney viral and TAA antigen were used to analyze viral and TAA antigen expression on chronically infected and leukemic lymphoid cells obtained from mice inoculated neonatally with MoMuLV. Although both sets of cells could be recognized and lysed by viral antigen specific CTL, they are not recognized by TAA-specific CTL. Only after transformed cell lines were established from leukemic spleen cells could susceptibility to TAA-specific CTL be observed. Thus, the appearance of the MoMuLV-TAA was restricted to MoMuLV-transformed cells.

Immune response to Moloney murine leukemia virus nonviral, tumor-associated antigens fails to provide in vivo tumor protection.

Two distinct populations of CTL have previously been shown to be generated in lymphocyte cultures derived from the spleens of C57BL/6 mice that have rejected Moloney murine leukemia virus:Moloney sarcoma virus (MoMuLV:MSV)-induced tumors. One population is specific for MoMuLV viral Ag whereas the other appears to be directed against a nonviral, tumor-associated Ag (TAA). Using a virus-negative variant of the MoMuLV-induced lymphoma MBL-2 that has retained the expression of the MuLV:TAA, we attempted to further characterize the MuLV:TAA-specific CTL population. First, this same pattern of CTL reactivity was observed using a variety of immunization protocols indicating that the TAA-specific CTL population was not an artifact of the original immunization protocol but was a reproducible component of the MoMuLV CTL response. Moreover, CTL precursor frequency analysis indicates that the MuLV:TAA-specific CTL represent approximately 60% of the CTL detected in in vitro cytotoxicity assays. However, when the role of MuLV:TAA CTL in the in vivo rejection of MoMuLV-induced tumors was examined, no role for the MuLV:TAA-specific CTL response could be determined. Immunization protocols that had been shown to give rise to both CTL populations were capable of protecting mice from tumor development after a challenge with the parental MBL-2 tumor cell line but not the virus-negative variant MBLv cell line. In addition, immunization with the variant, shown to give rise to only MuLV:TAA-specific CTL capable of lysing both MBL-2 and MBLv in vitro, failed to protect mice from a tumor challenge of either cell type.

In vivo immune selection of a Moloney murine leukemia virus-induced tumor results in the loss of viral- but not tumor-associated antigens.

A protocol of in vivo immune selection has been used to isolate a variant of the Moloney murine leukemia virus (MuLV)-induced tumor MBL-2. Characterization of the tumor variant indicated that selection resulted in the isolation of a cell which is incapable of producing infectious virus and no longer capable of synthesizing viral proteins. Although the failure to express viral Ag has rendered the variant tumor cells resistant to lysis by CTL specific for MuLV viral Ag, the variant tumor cells retained their susceptibility to lysis by CTL which appear to be directed against an MuLV-induced tumor-associated Ag. The data indicate that the expression of nonviral tumor-associated Ag by MBL-2 is not dependent upon continued viral gene expression.

Mechanism of induction of class I major histocompatibility antigen expression by murine leukemia virus.

Alterations in expression of major histocompatibility complex (MHC) antigens on tumor cells clearly correlate with the tumorgenicity and metastatic potential of those cells. These changes in the biological behavior of the tumor cells are presumably secondary to resulting changes in their susceptibility to immune recognition and destruction. Murine leukemia viruses (MuLV) exert regulatory effects on class I genes of the MHC locus. MuLV infection results in substantial increases in cell surface expression of all three class I MHC antigens. These viral effects on MHC antigen expression profoundly influence immune-mediated interaction with the infected cells, as assessed by cytotoxic T lymphocyte recognition and killing. Control of class I MHC and beta-2 microglobulin genes by MuLV takes place via a trans-acting molecular mechanism. MuLV controls expression of widely separated endogenous cellular MHC genes, transfected xenogeneic class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to a bacterial reporter gene. These findings indicate that MuLV exerts its effects on MHC expression via a trans mechanism. The MuLV-responsive sequences on the MHC genes appear to lie within 1.2 kilobases upstream of the initiation codon for those genes.

Murine retroviruses control class I major histocompatibility antigen gene expression via a trans effect at the transcriptional level.

Moloney murine leukemia virus (M-MuLV) and Moloney murine sarcoma virus (M-MSV) exert a regulatory effect on the class I genes of the murine major histocompatibility complex (MHC). We have previously shown that M-MuLV infection of mouse fibroblasts results in a substantial increase in cell surface expression of H-2K, H-2D, and H-2L proteins, whereas M-MSV, upon coinfection of the same cells, is apparently able to override the MuLV-induced increase in H-2 expression. As a result of this modulation, immune recognition of the infected cells is profoundly altered. Our efforts have been directed toward elucidating the molecular basis for this phenomenon. We report here that stimulation of interferon production as a result of infection with MuLV does not occur and, therefore, is not the cause of MuLV-induced enhancement of MHC expression. Control of H-2 class I and beta 2-microglobulin gene expression by M-MuLV, and probably by M-MSV, takes place at the transcriptional level as indicated by nuclear runoff studies and analysis of steady-state mRNA levels. Our demonstration that M-MuLV controls expression of widely separated endogenous cellular genes (those coding for H-2D, H-2K, H-2L, and beta 2-microglobulin), transfected class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to sequences encoding a procaryotic enzyme, chloramphenicol acetyltransferase, suggests that M-MuLV exerts its effect in trans and not by proviral integration in the vicinity of the H-2 gene complex. Finally, we show that the sequences of at least one MHC gene, which are responsive to trans regulation by M-MuLV, lie within 1.2 kilobases upstream of the initiation codon for that gene.

The immune response to Moloney murine leukemia virus-induced tumors: induction of cytolytic T lymphocytes specific for both viral and tumor-associated antigens.

The specificity of CTL generated against tumors induced by murine leukemia viruses (MuLV) has been reported to parallel the expression of two serologically defined tumor cell surface antigens--the cross-reactive FMR antigen expressed on the surface of tumors induced by Friend, Moloney, and Rauscher MuLV, and the Gross cell surface antigen (GCSA) expressed on tumors induced by AKV/Gross MuLV. We examined the specificity of CTL generated against MuLV-induced tumors and identified two distinct patterns of reactivity. The first follows the traditional pattern of FMR vs GCSA reactivity as assessed on a panel of established MuLV-induced lymphomas. However, CTL exhibiting this pattern of reactivity are incapable of lysing MuLV-infected fibroblasts. CTL exhibiting the second pattern of reactivity are capable of lysing MuLV-induced lymphomas as well as MuLV-infected fibroblasts. In addition, these CTL exhibit extensive cross-reactivity between lymphomas and fibroblasts infected by both groups of MuLV. Our results suggest that CTL exhibiting the traditional FMR vs GCSA pattern of reactivity are directed against a tumor-associated antigen and not against virus-encoded antigens, and that CTL directed against MuLV-encoded antigens demonstrate extensive cross-reactivity, including the ability to lyse AKV-infected cells.

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.

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). X. Tumorigenic potential of mouse cells transformed by SV40 in high responder C57BL/6 mice and correlation with the persistence of SV40 TrAg, early proteins and sequences.

Primary mouse embryo fibroblasts of C57Bl/6 origin and cells derived from a tumor induced by polyoma virus in a C57Bl/6 mouse were transformed with SV40. The tumorigenic potential of these cells in normal adult and SV40-immunized mice was correlated with the synthesis of SV40 tumor antigens including the transplantation rejection antigen (TrAg) and with the presence of SV40 early region DNA sequences. Primary cells transformed by SV40 (B6/WT-3) induced tumors in immunocompetent adult syngeneic mice after adaptation in the immunosuppressed host. Passage of these tumor cells (B6/WT-3-T) through SV40-immunized mice resulted in the retention of both T and t antigens and TrAg. However, passage of SV40-transformed polyoma tumor cells through SV40-immunized immunocompetent adult mice but not in nonimmunized mice resulted in the loss of expression of SV40 tumor antigens including TrAg. This loss correlated with the loss of SV40 early region sequences from these double transformed cells. These results demonstrate that the establishment of in vitro SV40-transformed primary mouse cells into a tumor capable of progressive growth in high responder mice does not lead to the selection of variants which have lost the expression of early region DNA sequences.

Cytotoxic T lymphocyte recognition of transfected cells expressing a cloned retroviral gene.

The lysis of murine sarcoma virus-murine leukaemia virus (MSV-MuLV)-induced tumour cells by cytotoxic T lymphocytes (CTL) appears to require that an antigen specified by MSV-MuLV, or induced by the infection, be presented in association with class I major histocompatibility complex antigens. The viral proteins of the tumorigenic MuLV seem to be a part of the antigens recognized by these dually restricted anti-MuLV CTL, but the precise nature of the putative viral antigen(s) recognized by CTL is unknown. Studies using recombinant viruses have suggested that a product of the viral envelope gene (env gene), perhaps the glycoprotein gp70, is the viral antigen recognized by CTL. Attempts to use purified gp70 or anti-gp70 antibodies to block CTL recognition of retrovirus-induced tumour cells, however, have yielded contradictory results. To examine more closely the role of gp70 in the CTL response to MuLV infections, we have constructed murine cell lines which express only the env gene of the Moloney murine leukaemia virus (M-MuLV). We show here that BALB/c-3T3 cells expressing the M-MuLV envelope gene products on their cell surface are susceptible to lysis by M-MuLV-specific CTL.

Lyt phenotype of H-2b CTL effectors and precursors specific for the SV40 transplantation rejection antigen.

The immune effector cells mediating the in vitro immune response to the SV40 transplantation rejection antigen were characterized by using monoclonal antibody directed against lymphocyte differentiation (Lyt) antigens. Two distinct T lymphocyte populations were found to be responsible for the in vitro lysis of SV40-transformed cells, and Lyt 1+,2+ cytotoxic T lymphocyte (CTL) that is present in the spleens of SV40 immunized mice 9 days postimmunization and an Lyt 1-,2+ CTL that is generated by secondary in vitro stimulation of in vivo primed spleen cells. During secondary in vitro stimulation of SV40 immune spleen cells obtained 9 days postimmunization, a shift in the Lyt phenotype of the CTL from Lyt 1+,2+ to Lyt 1-,2+ is observed. Although the Lyt 1-,2+ CTL can be derived from Lyt 1-,2+ noncytotoxic memory cells, it is not known whether the Lyt 1+,2+ CTL differentiates into an Lyt 1-,2+ CTL during in vitro stimulation.