Influence of T-lymphocyte deprivation on the antileukemic effect of bacillus calmette-guérin in vivo

Summary The effect in AKR mice of T-lymphocyte deprivation in vivo, obtained by adult thymectomy plus/minus whole-body irradiation and bone-marrow reconstitution, was studied in the growth of grafted leukemia cells obtained from spontaneous AKR leukemia. Both thymectomized mice and mice subjected to thymectomy, whole-body irradiation, and bone-marrow reconstitution (B) had a lower take-frequency of graft leukemia than conventional mice. Growth of graft leukemia was inhibited by BCG treatment both in thymectomized mice and in B mice. Concomitant with the increased inhibition of leukemia growth, an increased incidence of wasting-like disease was observed. In vitro cytotoxicity studies revealed that spleen lymphoid cells from nonleukemic mice were cytotoxic to AKR leukemia cells, to nonmalignant AKR fibroblasts, and to other nonmalignant cells with H-2^k haplotype. The activity of this self-directed cytotoxicity was most marked in AKR mice with wasting-like disease. The presence of autocytotoxic cells was frequently associated with a positive direct Coombs' test. Immunofluorescence studies showed, further, that the cytotoxic activity was independent of retrovirus antigens as tested by indirect immunofluorescence with anti-MuLV antibodies. Adult thymectomy of AKR mice confers an increased antitumoral immune potential, but also an increased risk of development of serious autoimmune disease.     

Characterization of alloimmunization-induced T lymphocytes reactive against AKR leukemia in vitro and correlation with graft-vs-leukemia activity in vivo

We have reported that immunization of H-2k mice with lymphoid cells from various allogeneic strains induced a population of cells that could eliminate first-passage spontaneous AKR leukemia from the spleens of immuno-suppressed AKR (H-2k) hosts. In the present study, we examined the nature of the cells responsible for this graft-vs-leukemia (GVL) reaction and compared them to cytolytic cells detected in vitro. Spleen cells from alloimmunized CBA/J (H-2k) mice were selectively depleted of various subpopulations by treatment with antibody and complement (C), then tested in vivo for GVL reactivity. Cell suspensions depleted of Thy-1.2+, Lyt-1+, or Lyt-2+ lymphocytes had no significant GVL reactivity, whereas suspensions depleted of NK-1.2+ cells retained GVL reactivity. The GVL-reactive cells persisted in H-2-compatible donor mice for up to 56 days. Lyt-1+2+ lymphocytes that were cytotoxic for cultured AKR leukemia cells in vitro could be detected in the spleens of alloimmunized H-2-compatible mice after expansion of the cells in T cell growth factor. Using quantitative limiting dilution cytotoxicity assays, we found that the frequency of leukemia-reactive cytotoxic lymphocytes (CL) in the spleen showed a direct correlation with the GVL efficacy of the cells in vivo. Alloimmunization was essential for induction of the GVL-reactive cell population. CL in alloimmunized mice consisted of heterogeneous cytotoxic specificities; i.e., some CL were leukemia-specific, others lysed only nonleukemic AKR target cells, and a third group mediated killing of both leukemic and nonleukemic target cells. The CL appeared to be H-2 restricted and specific for non-H-2 antigens shared by the AKR leukemia and the alloimmunizing cells.     

Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

Expression of CTL-defined, AKR/Gross retrovirus-associated tumor antigens by normal spleen cells: control by Fv-1, H-2, and proviral genes and effect on antiviral CTL generation

In previous studies we have characterized H-2-restricted cytolytic T lymphocytes (CTL) type specific for Gross cell surface antigen-positive tumor cells induced by AKR/Gross leukemia viruses. The generation of such CTL was shown to be controlled by at least three genetic loci including H-2 and Fv-1. The Fv-1n phenotype was able to negate positive immune response gene effects of the H-2b haplotype. Fv-1n-mediated inhibition appeared to operated by allowing the early expression by normal cells of N-ecotropic leukemia virus-related antigens recognized by the antiviral CTL, perhaps via tolerance induction. In the present study, the expression of CTL-defined viral antigens by normal cells is further considered. Possible gene dosage effects by H-2 as well as Fv-1 and the other virus-related (V) genes, including proviral structural loci, were examined by comparison of a panel of congenic and F1 mice. These experiments indicated that the quantitative level of expression of CTL-defined viral antigens was primarily controlled by the Fv-1 genotype. Gene dosage effects were also observed for the V genes and, in some situations, for H-2. The importance of the early display of viral antigens by normal cells was underscored by the inability of those mice to generate specific antiviral CTL responses. Even strains expressing low levels of viral antigens, such as responder X nonresponder (AKR.H-2b:Fv-1b X AKR.H-2b)F1 mice, failed to respond. These results are discussed with respect to the inability of mice of the AKR background to respond with specific antiviral CTL generation and in light of their high incidence of spontaneous leukemia.     

Genetic control of the induction of cytolytic T lymphocyte responses to AKR/Gross viral leukemias. II. Negative control by the Fv-1 locus in AKR mice of responder H-2b haplotype

To assess whether the presence of a responder H-2b haplotype would be sufficient to allow mice of nonresponder "high leukemic" phenotype to generate syngeneic anti-AKR/Gross virus cytolytic T lymphocytes (CTL), the AKR.H-2b strain was examined. Although capable of mounting vigorous apparent anti-minor histocompatibility-specific CTL responses, AKR.H-2b mice failed to produce anti-viral CTL after a variety of stimulation protocols. In contrast, the "doubly congenic" AKR.H-2b:Fv-1b strain was able to respond with substantial levels of H-2-restricted anti-AKR/Gross virus CTL activity. These results indicated that Fv-1n alleles could exert negative epistatic control over responder H-2b-encoded gene(s). Because the B6.Fv-1n congenic was also able to generate anti-viral CTL indistinguishable from the prototype B6 strain, however, it was apparent that other genes of AKR background were required for the Fv-1n-mediated inhibition in AKR.H-2b mice. The mechanism by which Fv-1 intereacted with other genes to override positive H-2b control appeared to be related to the expression of the CTL-defined, virus-associated antigens by normal AKR.H-2b cells. Thus, AKR.H-2b spleen cells but not thymus cells were able to stimulate the production of B6 anti-AKR/Gross virus CTL and were recognized as target cells by such anti-viral CTL. In contrast, both spleen cells and thymocytes from AKR.H-2b:Fv-1b mice were negative when tested as stimulator or target cells in these assays. In addition, AKR.H-2b but not AKR.H-2b:Fv-1b spleen cells were shown to display serologically defined gp70 determinants and the Gross cell surface antigen. Taking these data together, it appeared that the inhibition of anti-viral CTL responsiveness might be due to tolerance induced by the cell surface expression of virus-associated antigens by normal AKR.H-2b cells. Widespread display of viral antigens, in turn, may have been due to the permissive effects of Fv-1n on the spread of the early arising N-ecotropic, endogenous AKR leukemia virus controlled by other background genes. In this context, the implications of the multi-gene control of anti-AKR/Gross virus CTL production are discussed with respect to the induction of spontaneous leukemia in the high incidence AKR strain.     

Generation of anti-AKR/gross murine leukemia virus cytotoxic T lymphocytes (CTL). An analysis of precursor CTL frequencies in the AKR.H-2b and C57BL/6 mouse strains.

C57BL/6 mice, after immunization and secondary in vitro restimulation with AKR/Gross murine leukemia virus (MuLV)-induced tumors, generate AKR/Gross MuLV-specific CTL. After similar immunization protocols, AKR-H-2b mice fail to generate CTL specific for AKR/Gross MuLV. The basis for nonresponsiveness in AKR.H-2b mice is unknown, however, unlike C57BL/6 mice, AKR.H-2b mice carry endogenous proviruses and express N-ecotropic viral Ag. Thus, clonal deletion of pCTL populations due to the expression of AKR/Gross MuLV-like Ag is a likely mechanism for the nonresponsiveness. To determine if nonresponsiveness is due to clonal deletion, limiting dilution cultures were performed to assess the presence of pCTL specific for AKR/Gross MuLV. Our study demonstrates that the frequencies of pCTL specific for AKR/Gross MuLV are similar in both the responder C57BL/6 and nonresponder AKR.H-2b strains. The observation that normal levels of AKR/Gross MuLV-specific pCTL exist in AKR.H-2b mice, suggests that clonal deletion of pCTL is not responsible for the inability of AKR.H-2b mice to generate anti-AKR/Gross virus-specific CTL.     

Cell surface expression of cytotoxic T lymphocyte-defined, AKR/Gross leukemia virus-associated tumor antigens by normal AKR.H-2b splenic B cells

We previously described a system in which H-2Kb-restricted C57BL/6 (B6) cytotoxic T lymphocytes (CTL) could be raised that were specific for tumors, such as the thymic lymphoma AKR.H-2b SL1, that were induced by endogenous AKR/Gross murine leukemia virus and that expressed the Gross cell surface antigen. In this study, certain normal lymphoid cells from AKR.H-2b mice were also found to express target antigens defined by such anti-AKR/Gross virus CTL. AKR.H-2b spleen, but surprisingly not thymus, cells stimulated the production of anti-AKR/Gross virus CTL when employed at either the in vivo priming phase or the in vitro restimulation phase of anti-viral CTL induction. This selective stimulation by spleen vs thymus cells was not dependent on the age of the mice over the range (3 to 28 wk) tested. Both AKR.H-2b spleen and thymus cells, however, were able to stimulate the generation of H-2-restricted B6 anti-AKR minor histocompatibility (H) antigen-specific CTL. Thus, AKR.H-2b spleen cells appeared to display the same sets (minor H and virus-associated) of cell surface antigens recognized by CTL as the AKR.H-2b SL1 tumor, whereas AKR.H-2b thymocytes were selectively missing the virus-associated target antigens, a situation analogous to that of cl. 18-5, a variant subclone of AKR.H-2b SL1 insusceptible to anti-AKR/Gross virus CTL. Like AKR.H-2b thymocytes, neither AKR spleen cells or thymocytes nor B6.GIX + thymocytes were able to stimulate the generation of anti-AKR/Gross virus CTL from primed B6 responder cell populations. In contrast, both T cell-enriched and B cell-enriched preparations derived from AKR.H-2b spleen cells were able to stimulate at the in vitro phase of induction, although B cell-enriched preparations were considerably more efficient. The discordant results obtained with AKR.H-2b spleen cells vs thymocytes were confirmed and extended in experiments in which these cells were employed as target cells to directly assess the cell surface expression of virus-associated, CTL-defined antigens. Thus, AKR.H-2b spleen cells, but not thymocytes, were recognized by anti-AKR/Gross virus CTL when fresh normal cells were tested as unlabeled competitive inhibitors, or when mitogen blasts were tested as labeled targets. Fresh or lipopolysaccharide-stimulated B cell-enriched spleen cells were as efficiently recognized as unseparated spleen cell preparations. Unexpectedly, fresh or Lens culinaris hemagglutinin-stimulated T cell-enriched spleen cell preparations, although susceptible to anti-minor H CTL, were almost as poor as targets for anti-viral CTL as were thymocytes. Together, these results demonstrate the H-2-restricted expression of CTL-defined, endogenous, AKR/Gross virus-associated target antigens by normal AKR.H-2b splenic B cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of thymus subpopulations in "T" leukemia development.

Based on antigenic properties of the cell surface of mouse thymocytes, spontaneous (AKR mice) and induced T leukemias (C57BL/6 mice) were shown to have characteristics of the minor thymus subpopulation, namely, low levels of θ and high levels of H-2. Leukemogenic agents (fractionated irradiation or inoculation of radiation leukemia virus) were shown to induce a transient or permanent change in thymus population patterns. Within several weeks following leukemogenic treatment there was a relative enrichment of thymocytes bearing low levels of θ and high levels of H-2 and partially resistant to hydrocortisone and capable of induceing a graft versus host response. Transplantation bioassays carried out indicated the lack of demonstrable leukemic cells in the thymus within several weeks following the leukemogenic treatment. Similar spontaneous age-related changes in the pattern of the nonleukemic AKR thymus, namely, increase in the high H-2 thymus subpopulation and a gradual decrease in the percentage of θ-bearing cells was observed from 5 months onwards. The relationship between the availability of certain thymus subpopulations and the ultimate overt leukemia development was indicated.     

Mechanism of nonresponsiveness to AKR/Gross leukemia virus in AKR.H-2b:Fv-1b mice. An analysis of precursor cytotoxic T lymphocyte frequencies in young versus moderately aged mice

As young adult AKR.H-2b:Fv-1b mice reach about 9 wk of age, they begin to develop a nonresponsiveness to AKR/Gross leukemia virus. Unlike young mice that are responders, moderately aged AKR.H-2b:Fv-1b mice, after immunization and secondary in vitro restimulation in bulk culture with AKR/Gross virus induced tumors, can not generate anti-AKR/Gross virus-specific CTL. The mechanism of conversion to nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice is not understood, but it is correlated with increased expression of endogenous ecotropic viral antigens. Our present investigation focuses on determining the frequency of anti-AKR/Gross virus precursor CTL in AKR.H-2b:Fv-1b mice as a function of age. This was achieved by performing limiting dilution cultures of immune spleen cells obtained from young and moderately aged AKR.H-2b:Fv-1b mice. Although spleen cells obtained from immune moderately aged mice can not differentiate in bulk cultures into anti-AKR/Gross virus-specific CTL, there was no evidence of substantially decreased frequencies of virus-specific precursor CTL, relative to precursor CTL frequencies observed in young responder AKR.H-2b:Fv-1b mice.     

Cell Surface Antigens Associated with Murine Leukemia Virus: Definition of the GL and GT Antigenic Systems

Two new serological specificities were identified on the surface of murine leukemia virus (MuLV)-infected cells by direct and absorption immunofluorescence tests. Both antigens were detected with antisera prepared in rats that were growing transplants of syngenic MuLV-induced leukemias. Antigen G_L was defined with the AKR leukemia K36 as the test cell; antigen G_T was defined with the W/Fu leukemia C58(NT)D as the test cell. G_L and G_T antigens were serologically and genetically independent of the MuLV-induced Gross and G_IX cell-surface antigens. G_L and G_T antigens were found in normal lymphoid cells of mice from high-leukemic strains, but not in lymphoid tissues of mice from most low-leukemic strains. Tumors and leukemias of mice of low-leukemic strains often were G_L and G_T positive. Similarly, infection of normal cells with MuLV resulted in expression of G_L and G_T. With ferritin-labeled antibody the G_L and G_T antigens were observed on virus-free segments of the cell surface. Genetically, G_L and G_T antigens were each controlled by two dominant unlinked genes in AKR mice; these same antigens were each controlled by three or more dominant unlinked genes in C58 mice. Penetrance of G_L and G_T regulatory genes was dependent upon the Fv-1 genotype of the host. Expression of G_L antigen was closely associated with virus production, whereas expression of G_T antigen was less closely associated.     

Induction of anti-AKR/gross virus cytolytic T lymphocytes in AKR.H-2b:Fv-1b congenic mice: age-dependent conversion to a nonresponder phenotype

Previously, we reported that the generation of cytolytic T lymphocytes (CTL) specific for syngeneic tumors induced by AKR/Gross leukemia viruses was under multi-gene control. Thus, although carrying the required immune response gene(s) encoded by the H-2b haplotype and characteristic of responder strains such as C57BL/6, AKR.H-2b congenic mice failed to mount antiviral CTL responses. Young adult AKR.H-2b:Fv-1b "doubly congenic" mice, however, were able to generate specific anti-AKR/Gross virus CTL activity. These results demonstrated that the positive effect of MHC-encoded immune response gene control could be overcome by the action of the Fv-1n allele. The responder status of the B6.Fv-1n congenic, however, indicated that this Fv-1n-mediated inhibition was dependent on the interaction of Fv-1n with another gene(s) encoded by the AKR background. The results of experiments performed with AKXL recombinant inbred mice further suggested that a single additional genetic locus, encoding the Akv-1 provirus, was necessary along with Fv-1n to cause inhibition of antiviral CTL generation. Here we show that the responsiveness of AKR.H-2b:Fv-1b mice is dependent on their age. Thus, with moderate aging these doubly congenic mice converted to a nonresponder status with respect to anti-AKR/Gross virus CTL production: 85% of mice less than or equal to 9 wk of age responded compared with 0% of mice greater than 9 wk old. As with nonresponder AKR.H-2b mice, an inverse correlation was observed between CTL responsiveness and the expression of CTL-defined viral antigens by normal cells. Namely, spleen cells from young AKR.H-2b:Fv-1b mice showed little or no expression of such viral antigens, whereas with moderate aging there was a steady increase in their display. These results are discussed with reference to possible mechanisms of unresponsiveness of AKR.H-2b vs moderately aged AKR.H-2b:Fv-1b mice, and with respect to the utility of this system as a model for naturally occurring retrovirus infections and the interactions of retroviruses with the immune system.     

Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells.

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.     

AKR.H-2b lymphocytes inhibit the secondary in vitro cytotoxic T-lymphocyte response of primed responder cells to AKR/Gross murine leukemia virus-induced tumor cell stimulation.

We have previously shown that AKR.H-2b congenic mice, though carrying the responder H-2b major histocompatibility complex haplotype, are unable to generate secondary cytolytic T-lymphocyte (CTL) responses specific for AKR/Gross murine leukemia virus (MuLV). Our published work has shown that this nonresponsive state is specific and not due to clonal deletion or irreversible functional inactivation of antiviral CTL precursors. In the present study, an alternative mechanism based on the presence of inhibitory AKR.H-2b cells was examined. Irradiated or mitomycin C-treated AKR.H-2b spleen cells function as in vitro stimulator cells in the generation of C57BL/6 (B6) anti-AKR/Gross virus CTL, consistent with their expression of viral antigens. In contrast, untreated viable AKR.H-2b spleen cells functioned very poorly as stimulators in vitro. Viable AKR.H-2b spleen cells were also able to cause dramatic (up to > or = 25-fold) inhibition of antiviral CTL responses stimulated in vitro by standard AKR/Gross MuLV-induced tumor cells. This inhibition was specific: AKR.H-2b modulator spleen cells did not inhibit allogeneic major histocompatibility complex-specific CTL production, even when a concurrent antiviral CTL response in the same culture well was inhibited by the modulator cells. These results and those of experiments in which either semipermeable membranes were used to separate AKR.H-2b modulator spleen cells from AKR/Gross MuLV-primed responder cells or the direct transfer of supernatants from wells where inhibition was demonstrated to wells where there was antiviral CTL responsiveness argued against a role for soluble factors as the cause of the inhibition. Rather, the inhibition was dependent on direct contact of AKR.H-2b cells in a dose-dependent manner with the responder cell population. Inhibition was shown not to be due to the ability of AKR.H-2b cells to function as unlabeled competitive target cells. Exogenous interleukin-2 added at the onset of the in vitro CTL-generating cultures partially restored the antiviral response that was decreased by AKR.H-2b spleen cells. Positive and negative cell selection studies and the development of inhibitory cell lines indicated that B lymphocytes and both CD4- CD8+ and CD4+ CD8- T lymphocytes from AKR.H-2b mice could inhibit the generation of AKR/Gross virus-specific CTL in vitro. AKR.H-2b macrophages were shown not to be required to demonstrate AKR/Gross MuLV-specific inhibition, however, confirming that the inhibition by T-cell (or B-cell)-depleted spleen populations was dependent on the enriched B-cell (T-cell) population per se.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD4-CD8+ T lymphocytes mediate AKR/gross murine leukemia virus nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice.

Previously we reported that as AKR.H-2b:Fv-1b mice become older than 9 wk of age they begin to specifically lose the ability to generate anti-AKR/Gross murine leukemia virus (MuLV) CTL responses after immunization and in vitro restimulation with cells expressing AKR/Gross MuLV-encoded Ag. Interestingly, the frequency of virus-specific precursor cytotoxic T lymphocytes (CTL) observed in moderately-aged AKR.H-2b:Fv-1b mice was not substantially decreased from that found in their young responder counterparts. To further investigate the mechanism(s) responsible for the inability of moderately-aged AKR.H-2b:Fv-1b mice to mount AKR/Gross MuLV-specific CTL responses, adoptive transfer experiments were performed in the present study. Transferring splenocytes from moderately-aged AKR.H-2b:Fv-1b donors into young AKR.H-2b:Fv-1b recipients resulted in inhibition of AKR/Gross MuLV-specific CTL responsiveness. Anti-Thy-1.1 plus complement depletion of T cells from the donor cell population before adoptive transfer resulted in a near complete restoration of AKR/Gross MuLV responsiveness of young recipient AKR.H-2b:Fv-1b mice suggesting that the inhibition observed in moderately aged mice was mediated by T lymphocytes. Additional experiments using depletion of T subsets before cell transfer demonstrated that inhibition of AKR/Gross MuLV-specific CTL responsiveness was mediated by a CD4-CD8+ T lymphocyte.     

SMX-1 virus-induced inhibition of ecotropic and recombinant proviral sequence amplification in thymuses of AKR mice

SMX-1 virus delays the appearance of spontaneous thymomas in AKR mice which have been inoculated as young adults by the intrathymic route. Analyses of high-molecular-weight thymus DNAs from SMX-1 virus-inoculated AKR mice indicated the absence of 3' recombinant proviral-cellular DNA junction fragments generated by EcoRI and PvuII digestion. An average of five recombinant proviral fragments were detected in DNAs from spontaneous thymomas that developed in medium-injected control mice. Preleukemic mice that amplify murine leukemia virus-related antigens on their thymocyte surface contained unintegrated proviruses in their thymus DNAs, and 2.3-kilobase EcoRI and 2.1-kilobase PvuII recombinant DNA fragments were detected.     

Host genetic determinants of neurological disease induced by Cas-Br-M murine leukemia virus.

Cas-Br-M is an ecotropic murine leukemia virus (MuLV) of wild-mouse origin that causes neurogenic hind-limb paralysis. By virtue of its N-tropism, the virus replicates well in tissues of mice bearing the n but not the b allele at the Fv-1 locus. To determine if different Fv-1n strains of mice were equally susceptible to virus-induced neurological disease, we inoculated NFS, C3H, DBA/2, CBA, AKR, C58, and NZB mice at birth with Cas-Br-M murine leukemia virus and observed them for the development of tremor and hind-limb paralysis. Three patterns of disease were observed: NFS and C3H mice developed disease within 3 months postinoculation; DBA/2 and CBA mice became affected between 8 and 15 months postinoculation; and no disease was observed in AKR, C58, or NZB mice up to 15 months after infection with Cas-Br-M murine leukemia virus. Studies of genetic crosses between intermediate-latency (DBA/2) or long-latency (AKR) strains with short-latency (NFS) strains showed that intermediate latency and long latency were semidominant traits determined by two or more interacting but independently assorting loci. These genes appear to determine the rate at which the virus replicates and at which viral gene products accumulate in the central nervous system.     

Leukemia prevention and long-term survival of AKR mice transplanted with MHC-matched or MHC-mismatched bone marrow

The current studies were designed to evaluate the effectiveness of marrow transplantation within and outside the major histocompatibility complex (MHC) on the long-term survival and occurrence of spontaneous leukemia in AKR mice. AKR mice, which were lethally irradiated and received MHC-matched marrow from CBA/J mice (CBA----AKR), never developed leukemia and were alive and remained healthy for up to 280 days post-transplant. These long-term surviving chimeras possessed substantial immune vigor when both cell-mediated and humoral responses were tested. Lethally irradiated AKR mice, which had received MHC-mismatched marrow (anti-Thy-1.2 treated or nontreated) from C57BL/6J mice (B6----AKR), never developed leukemia and survived up to 170 days post-transplant. However, both groups of these chimeras began dying 180 to 270 days post-transplant due to a disease process which could not be readily identified. Histological analysis of B6----AKR chimeras revealed severe lymphoid cell depletion in thymus and spleen; however, none of these chimeras exhibited classical features of acute graft versus host disease. Concanavalin A mitogenesis, primary antibody responses to sheep red blood cells and the production of interleukin 2 (IL-2) were suppressed in B6----AKR chimeras. IL-2 treatment of B6----AKR chimeras was shown to partially correct these deficiencies without stimulating mixed lymphocyte responsiveness to donor or host lymphocytes. These studies indicate that the use of MHC-mismatched marrow for the prevention of spontaneous AKR leukemia may rely on augmentative IL-2 therapy for complete immune reconstitution of leukemia-free chimeras.     

Antiretroviral cytolytic T-lymphocyte nonresponsiveness: FasL/Fas-mediated inhibition of CD4(+) and CD8(+) antiviral T cells by viral antigen-positive veto cells

C57BL/6 (H-2(b)) mice generate type-specific cytolytic T-lymphocyte (CTL) responses to an immunodominant Kb-restricted epitope, KSPWFTTL located in the membrane-spanning domain of p15TM of AKR/Gross murine leukemia viruses (MuLV). AKR.H-2(b) congenic mice, although carrying the responder H-2(b) major histocompatibility complex (MHC) haplotype, are low responders or nonresponders for AKR/Gross MuLV-specific CTL, apparently due to the presence of inhibitory AKR. H-2(b) cells. Despite their expression of viral antigens and Kb, untreated viable AKR.H-2(b) spleen cells cause dramatic inhibition of the C57BL/6 (B6) antiviral CTL response to in vitro stimulation with AKR/Gross MuLV-induced tumor cells. This inhibition is specific (AKR.H-2(b) modulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility antigen-specific CTL production), dependent on direct contact of AKR.H-2(b) cells in a dose-dependent manner with the responder cell population, and not due to soluble factors. Here, the mechanism of inhibition of the antiviral CTL response is shown to depend on Fas/Fas-ligand interactions, implying an apoptotic effect on B6 responder cells. Although B6.gld (FasL-) responders were as sensitive to inhibition by AKR.H-2(b) modulator cells as were B6 responders, B6.lpr (Fas-) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL stimulation cultures, relieved the inhibition caused by the AKR.H-2(b) cells if the primed responders were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2(b) cells express FasL. Because of the antigen specificity of the inhibition, these results collectively implicate a FasL/Fas interaction mechanism: viral antigen-positive AKR.H-2(b) cells expressing FasL inhibit antiviral T cells ("veto" them) when the AKR.H-2(b) cells are recognized. Consistent with this model, inhibition by AKR.H-2(b) modulator cells was MHC restricted, and resulted in approximately a 10- to 70-fold decrease in the in vitro expansion of pCTL/CTL. Both CD8(+) CTL and CD4(+) Th responder cells were susceptible to inhibition by FasL+ AKR.H-2(b) inhibitory cells as the basis for inhibition. The CTL response in the presence of inhibitory cells could be restored by several cytokines or agents that have been shown by others to interfere with activation-induced cell death (e.g. , interleukin-2 [IL-2], IL-15, transforming growth factor beta, lipopolysaccharide, 9-cis-retinoic acid) but not others (e.g., tumor necrosis factor alpha). These results raise the possibility that this type of inhibitory mechanism is generalized as a common strategy for retrovirus infected cells to evade immune T-cell recognition.     

Effect of bromocriptine on the ovarian cycle of two inbred strains of mice

Mice of the C3H/Sy (high incidence of spontaneous mammary cancer) and AKR/Sy (low incidence of spontaneous mammary cancer) inbred strains, which have different hormonal profiles, were injected daily with bromocriptine for 1 month. The treatment increased the duration of the ovarian cycle of the AKR/Sy mice, whereas that of the C3H/Sy mice was not affected. It is suggested that the effect of bromocriptine on the ovarian cycle depends on the concentrations of plasma progesterone reached in each strain of mouse.     

Characterization of the Fas ligand/Fas-dependent apoptosis of antiretroviral, class I MHC tetramer-defined, CD8+ CTL by in vivo retrovirus-infected cells

C57BL/6 (B6; H-2(b)) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8(+) CTL responses to the immunodominant K(b)-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2(b) congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2(b) spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2(b) cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2(b) veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8(+) CTL were identified by a labeled K(b)/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2(b) veto cell-mediated depletion of these CTL. A 65-93% reduction in the number of B6 K(b)/KSPWFTTL tetramer(+) CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2(b) veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2(b) veto cells) to block inhibition demonstrated that AKR.H-2(b) veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer(+) CTL cocultured with AKR.H-2(b) veto cells was annexin V positive and Fas(high), indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2(b) cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vbeta4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.