Apoptosis of epitope-specific antiretroviral cytotoxic T lymphocytes via Fas ligand-Fas interactions.

C57BL/6 (B6; H-2b) mice are capable of mounting a vigorous AKR/Gross Murine Leukemia Virus (MuLV)-specific cytotoxic T lymphocyte (CTL) response to AKR/Gross MuLVs whereas AKR.H- 2b congenic mice, although carrying the responder H-2b major histocompatibility haplotype, are specifically nonresponsive. Furthermore, when viable AKR.H-2b spleen cells are cocultured with primed responder B6 antiviral precursor CTLs, the AKR.H-2b cells function as "veto" cells that actively mediate the inhibition by apoptosis of B6 antiviral CTL generation in a contact-dependent, MHC-restricted, and veto cell Fas ligand (FasL)/responder T cell Fas-dependent manner. In the present study we show that antigen-specific, antiviral CTLs that survive apoptotic inhibition by AKR.H-2b veto cells display a less activated cell surface phenotype, and are less able to bind specific MHC-peptide tetramers, including on a per-T cell receptor (TcR) basis. In addition, surviving antiviral CTLs also appeared to be functionally deficient, based on both their reduced ability to lyse specific target cells and to produce interferon (IFN)-gamma. Carboxyfluorescein diacetate succinimidyl ester staining confirmed that AKR/Gross MuLV-specific CTLs proliferated less extensively when AKR.H-2b veto cells were included in cocultures. AKR/Gross MuLV-specific effector CTLs as well as memory CTLs were each efficiently targeted for inhibition by AKR.H-2b veto cells. Attempts to enhance the quality of the priming by multiple in vivo immunizations did not alter the capacity of the AKR.H-2b cells to inhibit the antiviral CTL response. These results further characterize the nature of the interaction between veto cells and antiviral CTLs, and underscore the efficiency of veto cell-mediated inhibition of the CTL response.

Spontaneous in vivo retrovirus-infected T and B cells, but not dendritic cells, mediate antigen-specific Fas ligand/Fas-dependent apoptosis of anti-retroviral CTL.

C57BL/6 (H-2b), but not spontaneous virus-expressing AKR.H-2b congenic, mice generate retrovirus-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL. AKR.H-2b non-responsiveness is mediated by a peripheral tolerance mechanism. When co-cultured with primed B6 antiviral pCTL, AKR.H-2b splenocytes are recognized by the antiviral TcR as "veto" cells, which inhibit by an exquisitely virus-specific, MHC-restricted, veto cell FasL/responder T cell Fas, mediated apoptotic mechanism. Here, AKR.H-2b thymus, lymph node, and bone marrow cells are also shown to inhibit antiviral CTL generation. Purified AKR.H-2b CD4+ and CD8+ T cells, and B cells, served effectively as FasL-dependent veto cells. In contrast, AKR.H-2b dendritic cells (DC) did not efficiently veto antiviral CTL responses, despite expressing sufficient MHC class I/viral peptide complexes for TcR recognition. AKR.H-2b DC also expressed FasL mRNA and cell surface protein, albeit at a lower level than AKR.H-2b T and B cells. These findings suggest a fail-safe escape mechanism by virus-infected cells for escape from CTL-mediated immunity.

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.

A single amino acid variation within an immunodominant AKR/Gross MuLV cytotoxic T-lymphocyte epitope leads to a loss in immunogenicity.

C57BL/6 mice characteristically generate vigorous H-2K(b)-restricted cytotoxic T lymphocytes (CTL) directed against an immunodominant CTL epitope (KSPWFTTL) expressed by endogenous AKR/Gross murine leukemia viruses (MuLV). These AKR/Gross MuLV-specific CTL do not efficiently recognize tumor cells induced by Friend/Moloney/Rauscher (FMR) MuLV, which express the highly homologous peptide RSPWFTTL. In this report, we not only confirm the inefficient recognition of FMR tumors by AKR/Gross MuLV-specific CTL, but also demonstrate that RSPWFTTL is poorly immunogenic in C57BL/6 mice. To gain insight into the mechanism(s) contributing to the inefficient recognition of FMR MuLV-induced tumors, we examined the RSPWFTTL dissociation rate from H-2K(b) as well as the ability for RSPWFTTL to diminish CTL effector functions by T-cell antagonism. In contrast to immunogenic peptides, which form stable MHC class I-peptide complexes having slow dissociation rates, poorly immunogenic peptides characteristically have faster dissociation rates. On the basis of a cell-surface MHC class I peptide stabilization assay, the dissociation rate of RSP-WFTTL from H-2K(b) is characterized by a half-life that is nearly identical to the half-life of KSPWFTTL. In addition, we could find no evidence for antagonistic inhibition of AKR/Gross MuLV-specific CTL over a wide concentration range of RSPWFTTL. Analysis of the role of the transporter associated with antigen processing (TAP), by use of recombinant vaccinia and Sindbis viruses expressing a hydrophobic amino-terminal endoplasmic reticulum (ER) targeting sequence coupled to RSPWFTTL, indicated that RSPWFTTL cell-surface presentation can be dramatically enhanced when directly targeted into the ER.

Selective cytotoxicity of two rodent T cell lymphomas to rat yolk sac tumours involves a retroviral envelope protein expressed by the lymphoma.

A Gross virus induced rat T cell lymphoma G1-Tc1 and a Moloney virus induced mouse T cell lymphoma YAC-1 are shown to exert a strong cytotoxic activity against rat yolk sac tumours but not to various types of rat, mouse or human normal cells or tumour cell lines including carcinomas, sarcomas, lymphomas and gliomas. Both lymphomas are CD3+, CD4-, CD8- and T-cell receptor (TCR) alpha beta +. The cytotoxicity was not MHC restricted or dependent on the density of MHC class I of the target cells, and the mouse lymphoma killed the rat yolk sac tumour target. The cytotoxic action was fast and up to 80% specific killing was observed in 4-h 51Cr release assays. A rat B cell hybridoma was established from a Wistar/Furth (WF) rat immunized with the syngeneic lymphoma G1-Tc1 producing an immunoglobulin (Ig)G2c monoclonal antibody (MoAb) 1F2. This binds to the lymphomas G1-Tc1 and YAC-1 and also to a murine non-cytolytic Rauscher lymphoma RMA, but not to any other of several rat, mouse or human cell types tested. The 1F2 completely inhibited the killing of rat yolk sac tumours by the two cytolytic lymphomas, but did not interfere with the killing mediated by natural killer (NK) cells or cytolytic lymphokine-activated killer (LAK) cells. Immunochemical analysis of solubilized cell membranes of the lymphoma G1-Tc1 demonstrates that the 1F2 antibody recognizes an epitope on a retroviral gp 70 envelope protein. This indicates that a retroviral protein is involved in the lytic activity of the two lymphomas.

The role of proximal and distal sequence variations in the presentation of an immunodominant CTL epitope encoded by the ecotropic AK7 MuLV.

An emv-14-derived, replication-competent ecotropic murine leukemia virus [MuLV], designated AK7, was previously cloned from the AKXL-5 recombinant inbred mouse strain and partially characterized. While genetically encoding for an envelope-derived immunodominant CTL epitope [KSPWFTTL] located in the transmembrane region of p15TM, this virus, unlike the emv-11-derived virus AKR623, fails to be efficiently recognized by AKR/Gross MuLV-specific cytotoxic T lymphocytes [CTL]. AK7 thus provides the opportunity to study the role of retroviral sequence variations that are located outside of the immunodominant epitope as a mechanism of escape from CTL-mediated immune surveillance. In an attempt to identify which region[s] of the AK7 genome could account for its ability to evade efficient recognition by AKR/Gross MuLV-specific CTL, we have constructed recombinant murine retroviruses. The direct influence of a sequence variation twelve amino acids N-terminal to KSPWFTTL was explored with the use of chimeric viruses and determined not to significantly impair the presentation of KSPWFTTL to AKR/Gross MuLV-specific CTL. The long terminal repeat [LTR] derived from the AK7 virus, which possesses only one copy of the 99-base pair transcriptional enhancer in the U3 region, in contrast to AKR623 that possesses two copies of the tandem direct repeat enhancers, was also analyzed for its influence on the presentation of KSPWFTTL. Interestingly, our data indicate that the enhancer region derived from AK7 negatively influences the presentation of KSPWFTTL in the context of a recombinant AKR623 virus.

A shift in the requirement for CD4+ T cells in the generation of AKR/Gross MuLV-specific CTL in AKR.H-2b:Fv-1b mice occurs prior to the onset of age-dependent CTL nonresponsiveness.

In the current study, the elimination of CD4+ T cells from B6 mice, by treatment with anti-CD4 monoclonal antibody, had little effect on their ability to mount an AKR/Gross (MuLV)-specific CTL response. In contrast, for AKR.H-2b:Fv-1b mice, there was a shift as the mice aged from 5 to 7 weeks to a requirement for CD4+ T cells for AKR/Gross MuLV-specific CTL generation. When CD4+ T-cell-depleted AKR.H-2b:Fv-1b responder mice were immunized at 5 weeks of age they were able to elicit a strong anti-AKR/Gross MuLV CTL response. However, if the CD4+ T-cell depletion was done at 6 weeks and then the mice were primed in vivo, their antiviral CTL responsiveness was markedly decreased. Following CD4+ T-cell depletion at 7 weeks the mice were totally incapable of generating anti-AKR/Gross MuLV-specific CTL. AKR/Gross MuLV-specific CTL isolated from AKR.H-2b:Fv-1b mice recognized the class I-restricted immunodominant epitope (KSPWFTTL) and three subdominant epitopes, previously identified as CTL epitopes for B6 mice. Analysis of IL-2, IFN-gamma, IL-4, and IL-10 lymphokine profiles in supernates harvested from MLTC wells, and the results of supernate transfer experiments, suggested that the age-dependent shift to CD4+ T-cell dependence in AKR.H-2b:Fv-1b mice does not correlate with an obvious change in the in vitro lymphokine profiles. Experiments in which exogenous IL-2 was used to supplement in vitro cultures containing CD4+ T-cell-depleted 7-week responder mice suggested that the CD4+ T-cell requirement was at the in vivo priming stage of antiviral CTL generation. These data suggested a fundamental change in virus-specific CTL which correlates with slight aging in the AKR.H-2b:Fv-1b mouse strain. To our knowledge, this is the first report of a shift in the requirement for CD4+ T lymphocytes for the generation of virus-specific CTL over such a short period of time. Moreover, it is of interest that this shift in CD4+ T-cell-dependence by antiviral CTL occurs just prior to the onset of CTL nonresponsiveness in the AKR.H-2b:Fv-1b mouse strain.

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)

Impaired generation of anti-AKR/Gross murine leukemia virus cytotoxic T lymphocytes in mice experimentally infected with MuLV.

C57BL/6 (B6) and C57BL/6.Fv-1n (B6.Fv-1n) mice mount AKR/Gross murine leukemia virus (MuLV)-specific cytolytic T lymphocyte (CTL) responses following primary and secondary stimulation with AKR/Gross MuLV-induced tumor cells. In contrast, mice exposed to infectious virus rather than virus-infected cells generate little, if any, antiviral CTL activity. In this report, we show that inoculation of B6 or B6.Fv-1n mice with MuLV prior to priming with H-2-matched AKR/Gross virus antigen-positive tumor cells resulted in a profound inhibition of the virus-specific CTL response. Antiallogeneic major and minor histocompatibility antigen-specific CTL responses were not significantly diminished in MuLV-infected mice. The AKR/Gross MuLV-specific CTL response in B6 mice was inhibited by NB-tropic (SL3-3NB, Friend and Moloney), but not N-tropic (AKR623) MuLV, suggesting that productive infection of host cells was required. We were unable to inhibit the in vitro generation of virus-specific CTL by adding modulator cells from virus-infected mice to mixed lymphocyte-tumor cell cultures (MLTC) of spleen cells from uninfected animals. We also failed to augment CTL generation in MLTC from virus-infected animals by adding exogenous IL-2 or CD4+ lymphocytes from uninfected, tumor-primed mice. Taken together, the data suggested that the inhibition resulted from either a direct or an indirect effect on the in vivo priming of virus-specific CD8+ cells. It is therefore interesting that MuLV such as Friend and Moloney, which do not encode the immunodominant epitope recognized by anti-AKR/Gross MuLV CTL, are nonetheless able to specifically inhibit this response. These results demonstrate a potentially important mechanism by which retroviruses may escape CTL-mediated immunity.

Nonresponsiveness of AKR.H-2b congenic mice for anti-AKR/Gross MuLV CTL responses: involvement of inhibitory cells as defined by adoptive transfer experiments.

AKR.H-2b mice are unable to elicit AKR/Gross murine leukemia virus (MuLV)-specific cytolytic T lymphocyte (CTL) responses. The participation of inhibitory cells was addressed through adoptive transfer experiments utilizing young AKR.H-2b:Fv-1b congenic responder mice as the recipients of AKR.H-2b donor cells. The adoptive transfer of unfractionated viable splenocytes led to inhibition of virus-specific CTL responsiveness without affecting minor histocompatibility or allogeneic (H-2d)-specific CTL responses. Negative cell selection studies indicated that of the donor AKR.H-2b spleen cells that mediate specific inhibition, B lymphocytes, CD4-CD8+ and CD4+CD8- T lymphocytes, but not macrophages, even though they are viral antigen positive (as are B and T lymphocytes), were the cells responsible for the diminution of the generation of AKR/Gross virus-specific CTL by AKR.H-2b:Fv-1b mice. To evoke maximal inhibition, the adoptive transfer of AKR.H-2b cells had to be performed prior to in vivo priming with viral antigen. Anti-AKR/Gross MuLV nonresponsiveness of AKR.H-2b mice could not be overcome through utilization of exogenous IL-2 at either the priming or in vitro restimulation phases of CTL generation. These results illustrate the complex interaction between retroviruses and lymphocytes and are relevant to understanding how retrovirus-infected cells may not only escape immune surveillance themselves, but also may inhibit the cytolytic T cell response directed at other infected cells, such as tumor cells.

Major and minor Kb-restricted epitopes encoded by the endogenous ecotropic murine leukemia virus AKR623 that are recognized by anti-AKR/Gross MuLV CTL.

C57BL/6 mice can generate a type-specific and class IH-2Kb-restricted CTL response against histocompatible AKR/Gross murine leukemia virus (MuLV) cell surface antigen positive (GCSA+) tumor cells. These anti-AKR/Gross MuLV CTL are also known to lyse SC.Kb/623 target cells expressing the molecular MuLV clone AKR623 (derived from the endogenous ecotropic MuLV provirus emv-11). To help identify AKR623 viral epitopes recognized by these CTL, four chimeric proviruses were constructed from two parental plasmids, pAKR623 and pAK7. It has been shown that SC.Kb/7 fibroblast targets expressing the emv-14-derived molecular clone AK7 are only poorly lysed by anti-AKR/Gross MuLV CTL. Data from experiments employing SC.Kb cells infected with the chimeras as targets against anti-AKR/Gross MuLV CTL supported the location of a previously identified immunodominant epitope located within the viral p15E transmembrane envelope protein, peptide TM134-141 (KSP-WFTTL). Furthermore, the use of Kb-motif-defined AKR623 encoded peptides together with data obtained using the chimeric viruses allowed the identification of three additional anti-AKR/Gross MuLV CTL epitopes. Peptides representing these epitopes, MA125-132 (RSALY-PAL), RT142-149 (SHRWYTVL), and RT456-463 (RMTHYQAM), are characterized herein with respect to their ability to confer lysis upon EMV- target cells and to stimulate tumor primed splenocytes in vitro. The identification and characterization of these additional epitopes allow for a better understanding of both the CTL response against GCSA+ tumor cells and the dysfunctional CTL response against EMV-14 and AK7.

Lymphomagenesis in AKR.Fv-1b congenic mice.

In the AKR.Fv-1b congenic strain the Fv-1n allele of the AKR/J mice was substituted with the Fv-1b allele, thereby limiting viral replication and spread of the endogenous N-tropic murine leukemia virus. As a result of this genetic change AKR.Fv-1b mice develop a low spontaneous incidence (7%) of T-cell lymphomas and about 28% of Ly-1+ B-cell lymphomas are observed in old mice. Characteristic changes in thymus subpopulations of AKR/J mice (related to the formation of the dual tropic mink cell focus inducing (MCF) type virus in the thymus) were not observed in the thymus of AKR.Fv-1b mice. In contrast to the low susceptibility to spontaneous T-cell lymphoma development, these mice were highly sensitive to fractionated irradiation or to radiation leukemia virus (a mixture of N- and B-tropic viruses) induced T-cell lymphoma. Potential lymphoma cells (that would ultimately develop into Ly-1+ B-cell lymphomas) were demonstrated in bone marrow and spleens of 16-24-month-old mice. Analysis of the Ly-1+ IgM+ B-cell population in spleens of 18-month-old mice revealed a significant increase in this population (35% versus 2% in young spleens). The spontaneous Ly-1+ B-cell lymphoma incidence could be enhanced (up to 77%) by in vivo administration of anti-CD8 monoclonal antibody or IL-4 to 18-month-old mice. Virological analysis of T/B-cell lymphomas for class I MCF viruses indicated that Class I MCF development was tightly correlated with T-lymphoma development (except radiation induced tumors that showed no MCF provirus involvement). In contrast, Ly-1+ B-cell lymphoma development was independent of Class I MCF pathogenic virus involvement.

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.

Neonatal exposure to thymotropic gross murine leukemia virus induces virus-specific immunologic nonresponsiveness.

Neonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness. The specificity of this effect at the levels of both T and B cells was demonstrated by the ability of neonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction of immunologic nonresponsiveness to viruses.

Mechanism of escape of endogenous murine leukemia virus emv-14 from recognition by anti-AKR/Gross virus cytolytic T lymphocytes.

It was previously shown that spleen cells from endogenous ecotropic murine leukemia virus emv-14+ AKXL-5 mice fail to stimulate an anti-AKR/Gross virus cytolytic T-lymphocyte (CTL) response in a mixed lymphocyte culture with primed C57BL/6 responder spleen cells, whereas spleen cells from AKXL strains carrying the very similar emv-11 provirus do stimulate a response (Green and Graziano, Immunogenetics 23:106-110, 1986). We wished to determine whether the lack of response with AKXL-5 spleen cells was at the level of recognition between effector cell and target cell and whether the relevant mutation was within the emv-14 provirus. It is shown here that EMV-negative SC-1 fibroblast cells transfected with the major histocompatibility complex class I Kb gene and infected with virus isolated from the AKXL-5 strain (SC.Kb/5 cells) were not lysed by H-2b-restricted anti-AKR/Gross virus CTL. SC.Kb cells infected with virus isolated from emv-11+ strains, however, were efficiently lysed by anti-AKR/Gross virus CTL, indicating that there is nothing intrinsic to EMV-infected SC.Kb cells that would prevent them from being recognized and lysed efficiently by anti-AKR/Gross virus CTL. Analysis of virus expression for the infected SC.Kb cells by XC plaque assay and by flow cytometry indicated that emv-14 virus expression for SC.Kb/5 cells was not significantly different from that for emv-11-containing SC.Kb/9 or SC.Kb/21 cells. These data show that the mutation responsible for the lack of CTL recognition and lysis is at the level of recognition between target cell and effector cell. Furthermore, these data strongly suggest that the mutation is within the emv-14 genome. Flow cytometry experiments with monoclonal antibodies against a number of viral determinants indicated that there was no gross mutation detectable in the viral determinants analyzed. The data suggest that the relevant mutation may be a point mutation or a small insertion or deletion within a coding sequence that is critical for CTL recognition.

In vivo generation of antigenic variants of murine retroviruses.

Inoculation of adult BALB/c-H-2k (BALB.K) mice with both Gross murine leukemia virus (GV) and a biological clone derived from this virus resulted in the recovery of variant viruses which differ from GV with respect to the expression of specific epitopes associated with the env gene product, gp70. The loss of these epitopes correlated with the failure of antiserum raised in BALB.K mice against GV to neutralize variant virus although this antiserum neutralized GV. In contrast, BALB/c-H-2b (BALB.B) mice, immunized with GV, produced antibodies which neutralized both GV and the variant virus, indicating that BALB.B mice respond to epitopes distinct from those recognized by BALB.K mice. These results suggest that the selection of variant viruses resulting from in vivo passage may be related to the immunoselective pressures exerted in mice which express particular alleles of certain major histocompatibility complex (MHC)-linked genes.

Biochemical analysis of a novel H-2 class I-like glycoprotein expressed in five AKR-(Gross virus) derived spontaneous T cell leukemias.

The H-2 class I Ag profiles of five spontaneous AKR (H-2K) Gross virus leukemic cell lines were analyzed. A novel H-2 class I, "alloantigen"-like glycoprotein was immunoprecipitated and isolated from all the tumor cell lines using an H-2Dd-specific mAb 35-5-8. The novel Ag was also recognized in vitro by anti-H-2Dd-specific CTL. In addition, DNA from all the thymomas, but not the DNA from normal adult AKR thymic cells showed a transcribed gene detectable with an H-2Dd-specific oligonucleotide probe. The molecular profile of the novel antigen was further studied by two-dimensional gel electrophoresis and analyzed by a computer based image analyzer system and reverse-phase HPLC tryptic peptide mapping. Its molecular pattern was different from the syngeneic H-2Kk, H-2Dk, and the allogeneic H-2Dd gene products. The two-dimensional gel pattern of the novel H-2 class I molecule had a different overall structure reflected in isoelectric point, number, and distribution of polypeptide spots. The tryptic peptide map analysis showed six peaks exclusively identified with the novel Ag. The calculated degree of homology with the corresponding H-2Dd, H-Dk, and H-Kk peptides was 41, 56, and 51%, respectively. In addition, an unusual cell surface distribution of the novel Ag was observed in most of the leukemic lines. The removal of sialic acid residues by neuraminidase treatment facilitated the detection of the allodeterminants by anti-H-2Dd-specific mAb and CTL. Furthermore, we showed that in one AKR tumor line, 424, there is a close association of the novel Ag with the syngeneic class I molecules. Prior preclearance of the syngeneic class I molecules revealed the presence of the H-2Dd-like allospecificity. The genetic and molecular relationship between the expression of this novel class I-like glycoprotein and the recently sequenced Q5 gene is under current investigation.

Induction of anti-MuLV cytotoxic T lymphocytes in the AKR.H-2b and AKR.H-2b:Fv-1b mouse strains.

Following secondary in vitro sensitization with AKR/Gross virus-induced tumors, AKR.H-2b:Fv-1b mice develop cytotoxic T lymphocytes (CTL) specific for AKR/Gross viral antigens. It has recently been determined that the responder status of AKR.H-2b:Fv-1b to AKR/Gross virus declines with age. The nonresponsiveness observed in AKR.H-2b:Fv-1b is similar to that observed in AKR.H-2b mice which (regardless of age) does not develop anti-AKR/Gross virus CTL. It was of interest to determine the ability of these congenic mouse strains to respond to other murine leukemia viruses (MuLV). This was accomplished by immunizing AKR.H-2b and young or moderately aged AKR.H-2b:Fv-1b with Friend-Moloney-Rauscher (FMR) virus-induced tumors, and assessing the ability of anti-FMR CTL to develop following secondary in vitro stimulation. It was observed that both AKR.H-2b and AKR.H-2b:Fv-1b developed specific anti-FMR virus CTL. Similarly, following tumor challenge AKR.H-2b mice were unable to prevent the outgrowth of a syngeneic AKR/Gross virus-induced tumor, but were able to reject a syngeneic FMR virus-induced tumor.

Characterization of macrophage recognition and killing of SV40-transformed tumor cells that are "resistant" or "susceptible" to contact-mediated killing.

In this report we used the macrophage-"resistant" and -"susceptible" cell lines, F5m and F5b, to determine why AKR or AKR-like virus expression makes the F5m cell line more resistant to in vitro macrophage killing than the F5b cell line. We found that resistance to macrophage killing may be transmitted by an infectious AKR or AKR-like murine leukemia virus and that resistance was concomitant with virus expression as measured by the presence of AKR virus-specific 70 kDa glycoprotein. We report that macrophage cytotoxicity of these cell lines is dependent upon the direct contact between tumor cells and macrophages. In contrast, macrophage-mediated cytostasis occurred via soluble macrophage products and no differential susceptibility of F5b or F5m to macrophage-mediated cytostasis was observed. Macrophage binding of F5b was also significantly better than that of F5m. These data suggest that only the events that depend upon the close contact of macrophages and tumor cells will be affected by the expression of AKR or AKR-like virus. Therefore, the differences in susceptibility of F5m and F5b to direct macrophage-mediated cytotoxicity are apparently because the macrophage binding of F5m is less efficient than the binding of F5b.

Clonal heterogeneity of anti-AKR/gross leukemia virus cytotoxic T lymphocytes. Evidence for two distinct antigen systems.

AKR/Gross leukemia virus-induced tumor reactive cytotoxic T lymphocyte (CTL) clones were derived from C57BL/6 spleen cells. Analysis of their specificity pattern was performed by using a panel of target cells such as E male G2 and AKR.H-2bSL1 (susceptible tumors to polyclonal anti-AKR/Gross virus CTL), and cl. 18-5 and cl. 18-12 (insusceptible variant sublines derived from AKR.H-2bSL1). Several of these CTL clones were selected for further study. Lysis of Gross cell surface antigen-positive tumor cells by these clones was restricted by the H-2Kb molecule. The cell surface phenotype of these clones was Thy-1.2+, Lyt-2.2+, L3T4-, a phenotype consistent with that of polyclonal anti-AKR/Gross CTL, suggesting that they were of conventional CTL origin. According to their fine specificity pattern, the CTL clones were divided into two major groups (A and B) which were further subdivided into five and three subgroups, respectively. The specificity of group A clones was essentially the same as that of the standard polyclonal CTL population except for a variable level of natural killer-like activity by some of the CTL clones. That is, group A clones did not efficiently lyse the insusceptible variant tumors nor any of Friend-Moloney-Rauscher-positive tumors tested, but they showed strong lytic activity to susceptible tumors and iododeoxyuridine-treated insusceptible variants. Thus, their CTL activity appeared to be strictly directed to Gross cell surface antigen-positive tumors that are susceptible to polyclonal anti-AKR/Gross virus CTL. In contrast, group B clones could lyse both susceptible and insusceptible variant tumors and also a Friend virus-induced tumor (FBL3). Therefore, as defined by these CTL clones, at least two distinct antigenic systems (A and B), each with several antigenic determinants, appeared to be present. Because recent findings suggested that most of the polyclonal anti-AKR/Gross virus CTL activity appeared to be directed to N-ecotropic proviral determinants, we further investigated the nature of these two antigenic systems by use of additional target cells including lipopolysaccharide (LPS)-stimulated spleen cell blasts from AKXL recombinant inbred strains and retrovirus-infected fibroblasts. Group A clones could lyse all LPS blasts derived from AKXL recombinant inbred strains containing the AKV-1 proviral genome, but lysed only very insufficiently or did not lyse AKV-1-negative blasts containing the AKV-3 and/or AKV-4 provirus.(ABSTRACT TRUNCATED AT 400 WORDS)