Variation of expression of histocompatibility antigens on tumor cells: absence of H-2Kk-gene products from a gross-virus-induced leukemia in BALB.K

The antigenic profile of the K-GV tumor of BALB.K origin, induced by Gross virus and maintained in vitro and in vivo, was investigated by serological and immunochemical methods and techniques of cell-mediated immunity. The H-2K^k-gene products were absent by several criteria: (1) monoclonal antibody and conventional alloantisera directed against the H-2K^k antigenic specificities were nonreactive by direct testing and by absorptions. (2) H-2K^k products could not be precipitated from glycoprotein or protein extracts of the radiolabeled K-GV tumor. (3) Cytotoxic effectors against H-2K^k produced by sensitization in vitro and in vivo failed to kill K-GV target cells. (4) The tumor could neither stimulate BALB.B congenic mice to produce cytotoxic effectors nor specific cytotoxic antibody against H-2K^k-gene products. In contrast, the H-2D^k antigen was readily detectable by all these criteria. These findings therefore describe a tumor which has selectively lost the H-2K-gene products. The K-GV tumor was able to generate Gross-virus-specific CTL, but had greatly reduced susceptibility to lysis by Gross-virus-specific CTL generated by H-2K expressing AKR (H-2 ^k) tumors. These findings have important implications for the associative recognition of tumor antigens and the immune surveillance of virally induced tumors.Abbreviations used in this paper MHS major histocompatibility system - LcH Lens culinaris hemagglutinin - SDS sodium dodecyl sulfate - CTL cytotoxic T lymphocytes - GCSA Gross-virus-induced cell-surface antigen - MuLV murine leukemia virus     

H-2-specific antibodies induced by injection of syngeneic leukemia cells

AKR/J mice immunized with several syngeneic leukemia cells contained antibodies in their sera which reacted with certain AKR leukemia cell lines, depending on their H-2 expression, and precipitated H-2K antigens from lysates of leukemia cells. Precipitation of H-2K was not due to virus-specific antibodies: it could not be blocked by prior absorption with H-2-negative leukemias, but was blocked by certain allogeneic lymphocytes. Tumor-specific H-2K antibodies did not react with H-2K from normal AKR lymphocytes either on the cell surface or after detergent solubilization; however, they did react with H-2K from mitogen-activated AKR and BALB.K lymphoblasts. Since both these latter cells were also lysed by AKR-Gross/MuLV-specific and H-2K^k-restricted cytotoxic T lymphocytes, we consider the possibility that antibodies detecting conformational alterations induced in H-2K^k molecules by viral association may be present in syngeneic AKR antileukemia sera.Abbreviations used in this paper GCSA Gross-virus-induced cell-surface antigen - MCF mink cell focus-forming virus - MuLV murine leukemia virus - Th T helper     

Cytolytic T lymphocyte-defined retroviral antigens on normal cells: Encoding by the Akv-1 proviral locus

We previously described the generation and specificity of H-2-restricted cytolytic lymphocytes (CTL) directed against tumors induced by AKR/Gross murine leukemia viruses (MuLV). Such anti-AKR/Gross virus CTL demonstrated type specificity; only tumors induced by endogenous MuLV that expressed the Gross cell-surface antigen were lysed. These CTL and their precursor also recognized normal spleen cells from AKR-H-2 ^b , but not AKR-H-2 ^b , Fv-1 ^b mice, however, suggesting that N-ecotropic, retrovirus-associated antigens were primarily involved. Here, expression of these CTL-defined retroviral antigens by H-2^b-positive AKR × C57L recombinant inbred strains was examined by using normal spleen cells as stimulators in the generation of specific anti-AKR/Gross virus CTL. Analysis of the strain distribution pattern of stimulation indicated that a single proviral locus, Akv-1, was primarily, if not entirely, responsible for CTL-defined retroviral antigen expression. The lack of correlation with two other well-defined proviral loci was interesting. Whereas Akv-3 is known to encode a defective virus, Akv-4 has been shown to code for an infectious virus thought to be very similar or identical to that of Akv-1. Although quantitative differences cannot be formally excluded, dose response experiments argued against this possibility and suggested that Akv-1 and Akv-4 may exhibit qualitative differences germane to antiviral CTL recognition.     

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.     

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)     

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.     

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)     

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.     

H-2-restricted cytolysis of L cells doubly transformed with a cloned H-2Kb gene and cloned retroviral DNA

We report the construction of target cells by the double transformation of mouse L cells with a cloned H-2Kb gene and molecular clones of Akv or Gross murine leukemia virus (MuLV) or the cloned gag gene of Akv MuLV. Cytolytic T lymphocytes (CTL) generated in BALB.B mice specific for Gross MuLV (closely related to Akv MuLV) kill these doubly transformed cells. This is a direct indication that a CTL subpopulation recognizes H-2Kb antigen in association with a viral antigen encoded by the gag gene of Gross MuLV.     

Analysis of intrathymic differentiation patterns during the course of AKR leukemogenesis

Thymocytes from AKR mice in different stages of leukemia development were analyzed with the fluorescence-activated cell sorter (FACS) using monoclonal antisera to Lyt-1, Lyt-2, Thy-1.1, H-2K^k, and Ia^k. In addition, the number of cells bearing receptors for peanut agglutinin (PNA) was assessed. The results were correlated with the expression of murine leukemia virus (MuLV) antigen. Thymocytes from late preleukemic and leukemic stages were found to have a phenotype characteristic of a more mature cell population in that there was an increase in the expression of determinants encoded within the K end of H-2^k and Ia^k. This was associated with a decrease in the number of thymocytes bearing receptors for PNA during the leukemic stage. Simultaneously, a shift from a Lyt-1⁺ 2⁺ thymocyte population to cells with varying expressions of Ly antigens was observed. Analysis of Lyt determinants on thymomas indicated that they could arise from cells bearing any of the different possible combinations of Ly phenotypes. The cell surface antigen changes occurred in temporal correlation with an increased expression of MuLV antigens.     

Anomalous reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leukemia viruses.

Certain alloantisera prepared in mice against H-2 region membrane antigens were found to be unexpectedly cytotoxic for murine sarcoma and leukemia cells in culture. This anomalous cytotoxicity was shown to be the result of antibody in these alloantisera directed against the p15 and gp70 envelope proteins of Mu LV which were present on the surface of the tumor target cells. Sera from aged unimmunized mice of strains used for the preparation of alloantisera also contained antibodies against MuLV protein p15 and gp70 that were cytotoxic for sarcoma and leukemia cells, which indicates that these antibodies occurred naturally in mice. These results independently confirm earlier findings of the widespread occurrence in mouse serum of antibodies reactive with MuLV. The presence of antibody against MuLV in mouse serum which can cause cytotoxic reactions with tumor cells points to the fact that particular caution should be used during the typing of murine sarcomas or leukemias for cell surface antigens, since mouse antisera may yield cytotoxicity (or other serologic reactions) based on anti-MuLV specificities, rather than on anticipated antigens.     

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.     

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.     

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.     

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.     

Identification of a viral antigen recognized by H-2-restricted cytolytic T lymphocytes on a murine leukemia virus-induced tumor

Monoclonal antibodies were produced against protein p30, a structural protein of murine leukemia viruses (MuLV) coded by the gag gene of MuLV. Three monoclonal antibodies of different isotypes (i.e., IgG-1, IgG-2a, and IgG-2b) were chosen for extensive analysis. These three antibodies bound to mouse tumor cells induced by Friend, Moloney, Rauscher, and Gross MuLV, but not to noninfected normal mouse spleen cells. The ability of these monoclonal antibodies to inhibit cytolytic T lymphocyte (CTL) activity by masking the antigens recognized by CTL on the target cell surface was studied in various CTL systems. It was found that the only CTL that were consistently inhibited in their lytic activity came from BALB.B (H-2b) mice immunized against syngeneic Gross MuLV-induced B.GV cells. These results thus showed that a subpopulation of BALB.B anti-Gross MuLV CTL recognized a Gross MuLV gag gene product expressed on the surface of B.GV cells.     

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.     

Mechanisms of leukemogenesis. I. Generation of autoreactive lymphocytes in response to a murine leukemia virus.

Examined in this paper is the capacity of 334C murine leukemia virus (MuLV) to stimulate the generation of virus-specific cytotoxic effector cells in mice of the C57BL/6 strain that are relatively resistant to Friend, Moloney, and Rauscher (FMR) MuLV-induced leukemia, and in BALB/c mice that are relatively susceptible to leukemia induced by FMR MuLV. Generation of cytotoxicity requires in vivo administration of the virus followed by in vitro culture of lymphoid cells from virus-injected animals. Lymphoid cells from MuLV-resistant C57BL/6 donors develop high levels of specific cytotoxicity after secondary in vitro stimulation with syngeneic MuLV-induced tumor cells. Cells derived from these same donors, cultured in the absence of MuLV-induced tumor cells, fail to exhibit cytotoxicity. Secondary in vitro stimulation of lymphocytes from MuLV-susceptible BALB/c animals results not only in generation of cytotoxic reactivity against syngeneic MuLV-induced tumor cells but also induces apparently autoreactive effector cells capable of lysing other H-2d tumor cells as well as normal peritoneal cells bearing H-2d antigens. Moreover, generation of cytotoxicity by BALB/c lymphocytes occurs whether or not MuLV-induced tumor cells are included in the secondary culture system.     

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.     

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.