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.

Effect of different promoters on immune responses elicited by HIV-1 gag/env multigenic DNA vaccine in Macaca mulatta and Macaca nemestrina.

pCMV-NL(Deltapol) and pAKV-NL(Deltapol) expressed human immunodeficiency virus type 1 (HIV-1) gag and env under the regulation of the human cytomegalovirus (CMV) immediate-early (IE) promoter/enhancer and the endogenous AKV murine leukemia viral long terminal repeat (LTR), respectively. Analysis of the immune responses elicited by direct DNA injection of pCMV-NL(Deltapol) and pAKV-NL(Deltapol) in macaques indicated that generation of the humoral and T-cell proliferative responses correlated directly with the promoter strength of the vaccine DNAs. In Macaca mulatta, pCMV-NL(Deltapol) generated stronger humoral responses and T-cell proliferative responses to Gag and Env using less DNA and fewer number of injections than pAKV-NL(Deltapol). Similarly, in Macaca nemestrina pCMV-NL(Deltapol) elicited high humoral responses, which persisted long-term and were boostable. Injection of large amounts of pAKV-NL(Deltapol), in general, failed to produce antibody levels comparable to pCMV-NL(Deltapol). However, injection of a control animal with large amounts of vector DNA produced a generalized enzyme-linked immunosorbent assay (ELISA) reactivity to HIV-1. The results indicated that generation of high immune responses to HIV-1 cannot be achieved by increasing the vaccine DNA dose and may require high protein expression from the DNA by including a strong promoter or by the use of other boosting agents. Furthermore, safety concerns may arise with increasing the DNA dose that could need additional investigation.

Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo.

The genetic information of retroviruses is retained within a dimeric RNA genome held together by intermolecular RNA-RNA interactions near the 5' ends. Coencapsidation of retrovirus-derived RNA molecules allows frequent template switching of the virus-encoded reverse transcriptase during DNA synthesis in newly infected cells. We have previously shown that template shifts within the 5' leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site specificity of recombination within the highly structured 5' leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA-RNA interaction, here mediated by palindromic loop sequences within the mature genomic RNA dimer, facilitates hotspot template switching during retroviral cDNA synthesis in vivo.

Prenatal transmission and pathogenicity of endogenous ecotropic murine leukemia virus Akv.

OBJECTIVE: Mouse strains carrying endogenous ecotropic murine leukemia viruses (MuLV) are capable of expressing infective virus throughout life. Risk of transplacental transmission of MuLV raises concerns of embryo infection and induction of pathogenic effects, and postnatal MuLV infection may lead to tumorigenesis. METHODS: Endogenous ecotropic MuLV-negative SWR/J embryos were implanted into Akv-infected viremic SWR/J mice, into spontaneously provirus-expressing AKR/J mice, and into noninfected SWR/J control mice; virus integration and virus expression were investigated at 14 days' gestation. Tumor development was monitored over 18 months. RESULTS: Of 111 embryos, 20 (18%) recovered from Akv-infected SWR/J mice, which had developed normally, were infected. New proviruses were detected in 10 of 111 (9%) embryos from Akv-infected SWR/J mice, and in 2 of 60 (3%) embryos from AKR/J mice; none expressed viral protein. Of 127 embryos recovered from Akv-infected SWR/J mice, 16 (13%) were dead; 4 of 5 (80%) were infected and expressed viral protein. Of 71 embryos from AKR/J mice, 11 (15%) were dead, and 2 of 2 had virus integration; virus expression was not detected. Numbers of dead embryos recovered from experimentally infected, viremic SWR/J mice and from spontaneously endogenous MuLV-expressing AKR/J mice were significantly higher, compared with numbers from nonviremic SWR/J control mice, and embryo lethality was significantly associated with prenatal provirus expression. Postnatal inoculation of Akv induced lymphoblastic lymphomas in 15 of 24 (61%) SWR/J mice within mean +/- SD latency of 14 +/- 2.4 months. Only 3 of 39 (8%) control mice developed lymphomas (P < 0.005). CONCLUSION: Embryos in MuLV-viremic dams are readily infected, and inappropriate prenatal expression of leukemogenic endogenous retroviruses may play a critical role in embryo lethality and decreased breeding performance in ecotropic provirus-positive mouse strains.

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.

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.

DNA recombination is sufficient for retroviral transduction.

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end. Both constructs proved to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.

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.

Akv murine leukemia virus enhances bone tumorigenesis in hMT-c-fos-LTR transgenic mice.

hMt-c-fos-LTR transgenic mice (U. Rüther, D. Komitowski, F. R. Schubert, and E. F. Wagner. Oncogene 4, 861-865, 1989) developed bone sarcomas in 20% (3/15) of females at 448 +/- 25 days and in 8% (1/12) of males at 523 days. After infection of newborns with Akv, an infectious retrovirus derived from the ecotropic provirus of the AKR mouse, 69% (20/28) of female animals and 83% (24/29) of males developed malignant fibrous-osseous tumors. The tumors in infected transgenics developed with higher frequency and a 200-days shorter mean tumor latency period. The hMt-c-fos-LTR transgene was expressed in all the fibrous-osseous tumors. They also showed newly integrated Akv proviruses, but in most tumors Akv was detected and expressed in only a small number of the tumor cells. Wild-type C3H mice infected with Akv developed benign osteomas with an incidence of 33% and a latency period of 474 days. The data indicate that Akv exerts distinct pathogenic effects on the skeleton. In hMt-c-fos-LTR transgenic mice, predisposed to bone sarcomagenesis, Akv acts synergistically with the fos transgene, resulting in the development of fibrous-osseous tumors.

Akv murine leukemia virus enhances lymphomagenesis in myc-kappa transgenic and in wild-type mice.

The contribution of endogenous retroviruses to the multistep process of lymphomagenesis was investigated in wild-type mice and in two different myc-kappa transgenic mouse lines by infection with Akv. This retrovirus is derived from the endogenous ecotropic provirus of the AKR mouse and was previously considered to be nonlymphomagenic. The mice of the two myc-k transgenic lines are predisposed to B-cell lymphomagenesis and were therefore considered to be more susceptible to Akv. For comparison, the same mouse strains were also infected with the exogenous Moloney murine leukemia virus (MoMuLV). Both MoMuLV and Akv increased the tumor incidence and shortened the tumor latency period in wild-type mice and in the transgenic mouse lines. The differences in pathogenicity, number of provirus integrations, and level of virus expression between MoMuLV and Akv indicate different mechanisms of lymphomagenesis: while MoMuLV induced tumors apparently by insertional mutagenesis involving common integration sites similar to previous reports, the enhancement of lymphomagenesis by Akv seems to be directed by other mechanisms.

Acquisition of immunogenicity by AKR leukemic cells following DNA-mediated gene transfer is associated with the reduction of constitutive reactive superoxide radicals.

We have employed the DNA-mediated gene transfer method to introduce the allogenic major-histocompatibility-complex(MHC)-class-I gene H-2Kb into the K36.16 tumor cells, H-2k, in order to generate tumor-specific immunity. The acquisition of immunogenicity by the H-2Kb-transformed clones following gene transfer is associated with the reduction of constitutive reactive superoxide radicals. When the levels of cellular superoxide for the H-2Kb-positive immunogenic clones were determined, they were significantly lower (30 to 60%) than that of the parental K36.16 tumor cells. This reduction of superoxide in the H-2Kb-transformed cells was associated with a significant increase in the level of Cu-Zn superoxide dismutase (SOD) and GPX I, together with a reduction in the DNA-binding form of the NF-kappa B transcription factor. The K36.16 parental tumor cells were also found to be relatively more resistant to the cytotoxic effects of hydrogen peroxide in vitro. To further support the role of superoxide anion radicals in tumorigenesis, in vivo depletion of glutathione promoted the tumorigenicity of the H-2Kb-transformed clones in (AKR/J x C57BL/6/J) F1 mice, whereas SOD was able to reduce their tumorigenicity. In addition, the presence of R-sulfoxine (BSO) in spleen-cell cultures in vitro abolished the ability of the immune lymphocytes to develop into tumor-specific cytotoxic T lymphocytes (CTL). These observations support the concept that oxidative processes in tumor cells may have a strong influence on the host response against tumors.

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.

A direct demonstration of recombination between an injected virus and endogenous viral sequences, resulting in the generation of mink cell focus-inducing viruses in AKR mice.

We analyzed viral recombination events that occur during the preleukemic period in AKR mice. We tagged a molecular chimera between the nonleukemogenic virus Akv and the leukemogenic mink cell focus-inducing (MCF) virus MCF 247 with an amber suppressor tRNA gene, supF. We injected the supF-tagged chimeric virus that contains all of the genes of MCF 247 except the envelope gene, which in turn is derived from Akv, into newborn AKR mice to evaluate its pathogenic potential. Approximately the same percentage of animals developed leukemia with similar latent periods when injected with either the tagged or nontagged virus. DNA from tumors induced in AKR mice by the tagged chimeric virus was analyzed by Southern blotting with the supF gene as a probe. One set of tumors contained the injected supF-tagged virus. Two kinds of supF-tagged proviruses were found in a second set of tumors. One group of supF-tagged viruses had a restriction map consistent with that of the injected virus, while the other group of proviruses had restriction maps that suggested that the proviruses had acquired an MCF virus-like envelope gene by recombination with endogenous viral sequences. These results demonstrate that injected viruses recombine in vivo with endogenous viral sequences. Furthermore, the progression to leukemia was accelerated in mice that develop tumors containing proviruses with an MCF virus env gene, emphasizing the importance of the role of the MCF virus env gene product in transformation.