Detection and induction of equine infectious anemia virus-specific cytotoxic T-lymphocyte responses by use of recombinant retroviral vectors.

Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.

In vivo model for evaluation of species-specific virus vaccines.

It is difficult to evaluate the protective efficacy of species-specific viruses of humans and expensive companion animals where there is no non-human animal model. This study describes an in vivo model system which allows simultaneous operation of humoral, cell-mediated, interferon-like or other unidentified immunological defence mechanisms. There was evidence of in vivo inactivation of both enveloped and unenveloped DNA and RNA viruses including retrovirus mouse sarcoma virus/mouse leukaemia virus as evaluated by assay of the enzyme reverse transcriptase. This model will allow examination of vaccine efficacy in immunocompetent host animals while avoiding morbidity and/or mortality from virus infection in these animals.

Induction of Moloney murine sarcoma virus tolerance in adult mice by anti-CD4 monoclonal antibody treatment.

The role of CD4+ cells in the immune response to Moloney murine sarcoma/leukemia virus complex was studied in adult mice undergoing long-term CD4+ cell functional depletion by treatment with anti-CD4 H129.19 monoclonal antibody (immunoglobulin G2a, kappa). Adult mice given injections of Moloney murine sarcoma/leukemia virus complex 1 day or 2 wk after monoclonal antibody treatment died with progressing sarcomas at the inoculation site; mice challenged 4 wk after such treatment, when CD4+ cells recovered their functional activity, behaved like conventional mice; i.e., they spontaneously regressed the virus-induced sarcomas. Mice with progressing tumors did not generate virus-specific cytotoxic T-lymphocytes, despite a normal cytotoxic T-lymphocyte response to unrelated antigens, and they became virus carriers as demonstrated by Moloney murine leukemia virus antigen expression on their lymphoid cells a few days after virus injection. Moreover, the observed T-lymphocyte unresponsiveness was not due to the activity of specific suppressor T-cells. The findings indicate that the transient functional depletion of CD4+ cells at the time of virus administration provides appropriate environmental conditions for the spread of virus and facilitates tolerance induction.

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.

Induction of effective immunity to Moloney murine sarcoma virus using monoclonal anti-idiotypic antibody as immunogen.

We have isolated an anti-idiotypic mAb (RS1.1.3), which recognizes an idiotope present on several IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag. The binding of RS1.1.3 to idiotypic antibody could be inhibited by specific Ag. Intraperitoneal immunization of mice with purified RS1.1.3 antibody-induced effective immunity against Moloney murine sarcoma virus challenge. A single injection of RS1.1.3 7 days before virus challenge resulted in a 27% reduction in tumor load compared to non-immune control mice challenged with the same dose of virus, whereas multiple injections of RS1.1.3 before virus challenge resulted in a 75% reduction in tumor load. The protective effect of anti-idiotype immunization appeared to be T dependent, because immunization of athymic mice had no effect on their susceptibility to tumor virus challenge. Administration of the anti-idiotypic antibody after virus inoculation caused an increase in tumor load of nearly 50% compared to non-immune controls. BALB/c mice immunized with RS1.1.3 developed anti-anti-idiotypic antibodies, as well as M-MuLV Ag-specific antibodies. Analysis of sera from RS1.1.3-immune mice subsequently challenged with Moloney murine sarcoma virus indicated an inverse relationship between tumor load and M-MuLV-specific serum IgG titers induced by the RS1.1.3 immunization. These results indicate that anti-idiotypic mAb may be used as immunogen to induce Ag-specific antibody responses, and to cause effective immunity to a retro-virus-induced tumor.

Delayed hypersensitivity reactions to virus-augmented syngeneic and allogeneic tumor associated antigens.

Delayed hypersensitivity (DH) reactions to intact MSV-induced tumor cells or their crude membranes (CM) were studied across the H-2 histocompatibility barrier, using the radioisotopic footpad assay. Allogeneic as well as syngeneic tumor cells or their CM produced significant DH reactions in MSV-immune mice. CM from these cells after infection with influenza virus induced a stronger tumor-specific DH reaction in immune mice than did CM from uninfected cells. These results indicate that H-2 histocompatibility is not always required for induction of a cell-mediated immune response to tumor associated antigens and support the feasibility of a tumor specific skin test in humans using CM of autologous or allogeneic cultured human tumor cells.

Regulation of expression of T cell gamma chain, L3T4 and Ly-2 messages in Abelson/Moloney virus-transformed T cell lines.

Recent results have suggested that T cells may exist in two distinct pathways, one expressing alpha and beta chain of the T cell receptor genes with either or both of the cell surface markers CD4 and CD8, while the other is negative for these cell surface markers and expresses the T cell-specific gamma chain genes. The relationship between these two pathways is not known. In this study, we have examined a series of either Abelson virus or Moloney virus-derived T cell lines for their expression of these T cell receptor and cell surface marker genes. Results indicate that the Abelson T cell lines do not express the cell surface markers CD4 and CD8, but express relatively high levels of gamma chain transcripts. After culture of these cell lines with the phorbol ester phorbol myristate acetate and interleukin 2, a down-regulation of these gamma chain transcripts can be observed. More interestingly, we found that the Moloney virus-derived T cell lines, which express the cell surface markers CD4 and/CD8, contain high levels of alpha and beta chain T cell receptor transcripts but little or no gamma transcripts even though they have rearranged these latter genes. The gamma transcripts, however, can be induced to high levels after culture with phorbol myristate acetate and interleukin 2. In the process, the cell surface markers CD4 and CD8 and their transcripts were dramatically down-regulated resulting in cells with high levels of gamma chain transcripts and a CD4-CD8- phenotype. The regulation of expression of these genes is reversible. Taken together, these results indicate that the T cell receptor gamma chain genes and those of the cell surface markers CD4 CD8 can be regulated in vitro by external factors and it opens up the possibility of studying the regulatory sequences associated with these genes.

Monoclonal antibody against IFN-gamma inhibits Moloney murine sarcoma virus-specific cytotoxic T lymphocyte differentiation.

The role of autochthonous IFN- production was evaluated in immune reactions to Moloney murine sarcoma virus (M-MSV)-induced tumors which are characterized by spontaneous regression mainly caused by virus-specific CTL activity. A functional IFN- depletion, induced by repeated administration of mAb anti-IFN- at the site of virus inoculation, prevented tumor regression in M-MSV-injected mice. Moreover, this antibody inhibited in vitro both proliferation and differentiation of M-MSV-specific T lymphocytes obtained in bulk cultures, but not growth and lytic activity of the already differentiated virus-specific CTL clone CHM-14 stimulated with rIL-2 and relevant tumor Ag. In addition, in mice receiving mAb treatment the frequency of M-MSV-specific CTL precursors, evaluated by means of limiting dilution analysis, was strongly reduced in comparison with that of control mice injected only with virus. Because CTL secrete IFN- following antigenic stimulation, the possibility that non-T effector cells recruited by this lymphokine might mediate tumor regression was also considered. Adoptive immunotherapy experiments, performed in T cell-deficient (Tx + BM) and in sublethally irradiated mice, demonstrated that transfer of CHM-14 CTL clone, which secretes IFN-, was able to counteract M-MSV tumor growth despite the local mAb anti-IFN- treatment which may have prevented host cell recruitment. Moreover, repeated local rIFN- inoculations in Tx + BM mice did not counteract M-MSV tumor progression, thus confirming that other IFN- properties such as non-T cell recruitment, antiviral or anti-proliferative IFN- activities have little or no relevance when M-MSV-specific CTL are lacking. On the whole, these results indicate that in M-MSV-injected mice, tumor enhancement after mAb anti-IFN- treatment is principally caused by impaired differentiation of virus-specific CTL precursors.

Functional activity in vivo of effector T cell populations. III. Protection against Moloney murine sarcoma virus (M-MSV)-induced tumors in T cell deficient mice by the adoptive transfer of a M-MSV-specific cytolytic T lymphocyte clone.

The functional activity of Moloney murine sarcoma virus (M-MSV)-specific T lymphocytes in vivo was assayed by the i.v. injection of virus-specific T lymphocytes into T cell-deficient "B mice". Virus-specific T lymphocytes generated in mixed lymphocyte tumor cell cultures were transferred i.v. into syngeneic "B mice" injected simultaneously at a distant site with the virus. These experiments indicated that a low dose (1 X 10(6) cultured cells) of infused lymphocytes can afford protection. To define the T lymphocyte subpopulation which was active, Lyt-2+ lymphocytes were selected by "panning" on plastic petri dishes coated with anti-Lyt-2 monoclonal antibody, and Lyt-2- lymphocytes selected by treatment with anti-Lyt-2 monoclonal antibody and complement. The results indicated that a Lyt-2+ lymphocyte-enriched population was more efficient in conferring protection against M-MSV-induced tumors. To investigate if cytolytic T lymphocytes (CTL) alone had a protective effect, a M-MSV-specific CTL clone was transferred in the same model system. The results demonstrated that a M-MSV-specific CTL clone prevented M-MSV-induced tumor growth and also induced the destruction of syngeneic Moloney murine leukemia virus (M-MuLV)-induced MBL-2 leukemic cells in the peritoneal cavity. However, the cell dose required to obtain protection using a CTL clone was higher than that which was effective when mixed lymphocyte tumor cell culture cells were used. To assess the ability of the transferred cells to home and to repopulate the lymphoid organs of the "B mice", the frequency of virus-specific CTL precursors in the spleen was evaluated by limiting dilution analysis. The results indicated that lymphocytes from mixed lymphocyte tumor cell cultures can be recovered from the spleens of "B mice" injected i.v. 25 days earlier. On the contrary, following the transfer of an active CTL clone, a very low frequency (less than 1/200,000 cells) of virus-specific CTL precursors was present in the spleens of recipient animals. The same M-MSV-specific CTL clone did not yield protection against M-MSV-induced tumors or MBL-2 leukemic cells when injected i.v. into M-MuLV tolerant mice.

Morphology and in vivo growth characteristics of an atypical murine proliferative osseous lesion induced in vitro.

Mandibular condyles of late embryonic NMRI mice were used to study the effect of the FBR murine osteosarcoma virus in an in vitro tissue culture system. Chondroprogenitor cells and chondroblastic cells present in the condylar tissue normally undergo rapid differentiation in vitro which results in an advanced stage of bone formation. The infection of condyles with FBR murine osteosarcoma virus induced the transformation of bone progenitor cells and the formation of an atypical proliferative osseous lesion. In markedly disorganized tissue many spindle-like cells, giant cells, and pleomorphic cells were seen together with the formation of large bone spicules and the heavy mineralization of osteoid-like material and of the remaining cartilage. Fibroblast-like cells were found to penetrate from the perichondrial zone into the condylar mass and also into the underlying collagen sponge. The in vivo growth characteristics of FBR murine osteosarcoma virus-infected condyles after 3 days in culture were studied via s.c. transplantation into syngeneic mice. Control condyles developed normal trabecular bone, whereas the infected condyles induced a strong cellular response with the presence of atypical cells and newly formed connective tissue and bone in situ. These observations raise the possibility of a novel approach for further investigations related to numerous aspects of virus-induced osteosarcomagenesis.

The effect of interferon treatment of targets on susceptibility to cytotoxic T-lymphocyte killing: augmentation of allogeneic killing and virus-specific killing relative to viral antigen expression.

The effect on CTL lysis of treatment of CTL targets with IFNs has been investigated. Treatment of targets for alloreactive CTL with either IFN-alpha beta or IFN-gamma markedly augmented cytotoxicity. Cold competition experiments implied that CTL recognized the same target structure on both untreated and IFN-treated cells. This augmented lysis is presumably caused by IFN increasing expression of target MHC antigens. In the case of SFV-specific lysis of SFV-infected fibroblasts, IFN-alpha beta or IFN-gamma treatment somewhat reduced CTL lysis, but less so than Ab + C lysis which was abolished at moderate IFN concentrations; in the case of SFV-infected lymphoblastoid cells, CTL lysis remained the same or was slightly increased, whilst Ab + C lysis was reduced at moderate IFN concentrations and abolished at high IFN concentration; in the case of MSV/MLV-infected fibroblasts, CTL lysis was moderately increased whilst Ab + C lysis was decreased. IFN therefore increases virus-specific CTL cytotoxicity relative to viral antigen expression.

Detection and preliminary characterization of a transmissible agent inducing autoantibody against Golgi-antigen.

A transmissible agent inducing autoantibody has been found in association with tumours induced in STU mice with the progressor strain of Moloney sarcoma virus (Mo-MSV). The activity of the agent detectable in serum from tumour-bearing hosts was expressed by development of autoantibody against Golgi-associated evolutionary conserved antigen (Weiland et al. 1984). This agent was tentatively designated 'AGIA', anti-Golgi inducing agent, on account of its most remarkable biological activity. Concomittant with autoantibodies were cytotoxic antibodies reactive with a Mo-MSV non-producer transformant (Sac). Both antibody activities were regularly detectable 2 weeks after inoculation of the agent. At that time the antibody containing serum possessed an infectivity titre of approximately 10(7.5) ID50/ml. Signs of illness were not observed during this period. The antibody-inducing agent was lost from progressor Mo-MSV transformants during their first passage in culture. Neither murine embryo fibroblasts nor murine tumour cells were permissive for propagation of the agent in vitro.

In vitro cleavage of Pr65gag by the Moloney murine leukaemia virus proteolytic activity yields p30 whose NH2-terminal sequence is identical to virion p30.

In vitro cleavage of Gazdar murine sarcoma virus Pr65gag, which has all of the antigenic determinants of Moloney murine leukaemia virus Pr65gag, i.e. p15, p12, p30 and p10, by the Moloney murine leukaemia virus proteolytic activity yielded a p30 whose partial NH2-terminal sequence was identical to Moloney murine leukaemia virus. Both [3H]leucine-labelled and unlabelled Pr65gag were used to generate the cleaved p30.

Regulation of B cell lymphomagenesis by a malignant Qal+ inducer T cell clone.

A series of Thy-1.2+ Ly-1+ Qa-1+ malignant T cell clones have been isolated from murine sarcoma virus-murine leukemia-Moloney (MSV-MuLV-M)-induced B cell lymphomas or from MSV-MuLV-M-infected B6 mice. These T cell clones enhance both antigen-independent and -dependent lymphocyte differentiation and function. They also induce the differentiation of granulocytes and erythrocytes in the stem cell compartment, a function that parallels the immunopathology of the disease in vivo. The malignant T cell appears to sustain B lymphoma growth in vivo by releasing a factor (BCGF) that promotes B cell proliferation.

Immunity against Moloney sarcoma virus in H-2Db mutant bm14 mice. Unimpaired tumor immunity despite absence of a virus-specific cytotoxic T-cell response.

It was shown previously that B6.C-H-2bm14 (bm14) mice, carrying a mutation in the H-2Db locus, are unable to generate cytotoxic T cells (CTL) against Moloney murine sarcoma virus (M-MSV). We now report an analysis of tumor induction and regression kinetics and of immunity to the virus, following the injection of graded doses of M-MSV into C57BL/6 (B6) and bm14 mice. Contrary to expectation, bm14 mice showed slightly less tumors than wildtype B6 mice. Moreover, all bm14 mice that developed a tumor were able to reject this tumor, even after injection of the highest virus dose tested. From the spleen cells of bm14 mice that had rejected tumors, no secondary in vitro CTL responses could be generated, in contrast to strong CTL responses generated from B6 spleen cells. Although bm14 mice were unable to generate virus-specific CTL, they showed normal antibody and T-cell proliferative responses against Moloney virus, suggesting an intact T-helper-cell function. It is concluded that in bm14 mice, under the conditions tested, virus-specific CTL are not generated despite excellent tumor immunity. Therefore, this CTL response is not necessary for protection against M-MSV-induced tumors. Protection is likely to be mediated by a normal T-cell proliferative response.

Vaccination against strontium-90-induced bone tumors.

The thesis was tested that immunization against a murine osteosarcoma virus can reduce the incidence of bone tumors induced by 90Sr. C57BL/6J female mice (190) were divided into three sets of 2 groups. Each set consisted of a control group and an experimental group treated ip with 1.0 muCi 90Sr at 66 days of age. The three sets of groups received the following additional treatments: none (controls), 6 injections of Formalin-inactivated FBJ osteosarcoma virus (vaccinated group), or 6 injections of active FBJ virus (active virus controls). Only 1 bone tumor developed in a mouse not treated with 90Sr in the active virus controls. In 90Sr-treated mice, vaccination reduced bone tumor deaths during the first 600 days from 9 of 36 in controls to 1 of 33 in vaccinated mice (P less than .01), but bone tumor deaths during the entire life-span, 10 of 36 and 5 of 33, respectively, were not significantly different (P = .07). Thus the vaccination procedure delayed the development of bone tumors. In contrast, injection of active virus into 90Sr-treated mice increased the lifetime incidence of bone tumors from 10 of 36 in controls to 19 of 32 (P = .01).