Wild-derived inbred mice have a novel basis of susceptibility to polyomavirus-induced tumors.

Polyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard inbred strains, notably those bearing the H-2(k) haplotype. Susceptibility in these mice is conferred by an endogenous mouse mammary tumor virus superantigen (Mtv-7 sag) that acts to delete T cells required for polyomavirus-induced tumor immunosurveillance. In the present study we show that mice of two wild-derived inbred strains, PERA/Ei (PE) and CZECH II/Ei (CZ), are highly susceptible to polyomavirus but carry no detectable Mtv sag-related sequences and show no evidence of Vbeta deletion. C57BR/cdJ (BR) mice, which are H-2(k) but lack the endogenous Mtv-7, are highly resistant based on an effective anti-polyomavirus tumor immune response. When crossed with BR, both PE and CZ mice transmit their susceptibility in a dominant fashion, indicating a mechanism(s) that overrides the immune response of BR. Susceptibility in PE and CZ mice is not based on interference with antigen processing or presentation since cytotoxic T cells from BR can efficiently kill F(1)-derived tumor cells in vitro. The expected precursors of polyomavirus-specific cytotoxic T cells are present in both the wild inbred animals and their F(1) progeny. These findings indicate a novel basis of susceptibility that operates independently of endogenous superantigen and prevents the development of tumor immunity.

Reduction of otherwise remarkably stable virus-specific cytotoxic T lymphocyte memory by heterologous viral infections.

Experimental analyses of the acute cytotoxic T lymphocyte (CTL) response to viruses have focused on studying these infections in immunologically naive hosts. In the natural environment, however, viral CTL responses occur in hosts that are already immune to other infectious agents. To address which factors contribute to the maintenance and waning of immunological memory, the following study examined the frequencies of virus-specific CTL precursor cells (pCTL) not only using the usual experimental paradigm where mice undergo acute infections with a single virus, and in mice immune to a single virus, but also in immune mice after challenge with various heterologous viruses. As determined by limiting dilution assays, the pCTL frequency (p/f) per CD8+ T cell specific for lymphocytic choriomeningitis virus (LCMV), Pichinde virus (PV), or vaccinia virus (VV) increased during the acute infections, peaking at days 7-8 with frequencies as high as 1/27-1/74. Acute viral infections such as these elicit major expansions in the CD8+ T cell number, which has been reported to undergo apoptosis and decline after most of the viral antigen has been cleared. Although the decline in the total number of virus-specific pCTL after their peak in the acute infection was substantial, for all three viruses the virus-specific p/f per CD8+ T cell decreased only two- to fourfold and remained at these high levels with little fluctuation for well over a year. The ratios of the three immunodominant peptide-specific to total LCMV-specific clones remained unchanged between days 7 and 8 of acute infection and long-term memory, suggesting that the apoptotic events did not discriminate on the basis of T cell receptor specificity, but instead nonspecifically eliminated a large proportion of the activated T cells. However, when one to five heterologous viruses (LCMV, PV, VV, murine cytomegalovirus, and vesicular stomatitis virus) were sequentially introduced into this otherwise stable memory pool, the stability of the memory pool was disrupted. With each successive infection, after the immune system had returned to homeostasis, the memory p/f specific to viruses from earlier infections declined. Reductions in memory p/f were observed in all tested immunological compartments (spleen, peripheral blood, lymph nodes, and peritoneal cavity), and on average in the spleen revealed a 3 +/- 0.4-fold decrease in p/f after one additional viral infection and an 8.4 +/- 3-fold decrease after two additional viral infections. Thus, subsequent challenges with heterologous antigens, which themselves induce memory CTL, may contribute to the waning of CTL memory pool to earlier viruses as the immune system accommodates ever-increasing numbers of new memory cells within a limited lymphoid population. This demonstrates that virus infections do not occur in immunological isolation, and that CD8+ T cell responses are continually being modulated by other infectious agents.

Cross-reactivities in memory cytotoxic T lymphocyte recognition of heterologous viruses.

Analyses of the relationships between different viruses and viral proteins have focused on homologies between linear amino acid sequences, but cross-reactivities at the level of T cell recognition may not be dependent on a conserved linear sequence of several amino acids. The CTL response to Pichinde virus (PV) and vaccinia virus (VV) in C57BL/6 mice previously immunized with lymphocytic choriomeningitis virus (LCMV) included the reactivation of memory cytotoxic T lymphocyte (CTL) specific to LCMV. Limiting dilution assays (LDA) demonstrated that at least part of this reactivation of memory cells in LCMV-immune mice related to cross-reactivity at the clonal level, even though acute infections with these viruses in nonimmune mice elicited CTL responses that did not cross-react in conventional bulk CTL assays. Precursor CTL (pCTL) to LCMV were generated in splenic leukocytes from LCMV-immune mice acutely infected with PV or VV when stimulated in vitro with only the second virus but not with uninfected peritoneal exudate cells (PECs). Cytotoxicity mediated by LCMV-specific CTL clones activated by PV infection was greatly inhibited by anti-CD8 antibody, suggesting that these memory CTL clones recognizing LCMV-infected targets were of low affinity. LCMV-immune splenocytes stimulated in vitro with PV or VV demonstrated a low but significant precursor frequency (p/f) to the heterologous viruses, and splenocytes from PV- or VV-immune mice when stimulated in vitro against LCMV generated a low but significant p/f to LCMV. Short-term CTL clones cross-reactive between LCMV and PV were derived from splenic leukocytes from LCMV-immune mice acutely infected with PV. To distinguish whether the cross-reactivity was directed against a viral peptide or a virus-induced endogenous cellular neoantigen, we demonstrated that a pCTL frequency to PV about 1/4-1/7 that of the frequency to LCMV could be generated from LCMV-immune splenic leukocytes stimulated with the immunodominant LCMV NP peptide. A partially homologous PV peptide generated from the equivalent site to the LCMV NP peptide did not sensitize targets to lysis by either LCMV- or PV-specific CTLs, suggesting that the cross-reactivity in killing was not due to evolutionarily conserved equivalent sequences. Experiments also indicated that prior immunity to one virus could modulate future primary immune responses to a second virus. Elevated pCTL frequencies to PV were seen after acute PV infection of LCMV-immune mice, and elevated pCTL frequencies to LCMV were seen after acute LCMV infection of PV- and VV-immune mice.(ABSTRACT TRUNCATED AT 400 WORDS)

High frequency of cross-reactive cytotoxic T lymphocytes elicited during the virus-induced polyclonal cytotoxic T lymphocyte response.

Polyclonal stimulation of CD8+ cytotoxic T lymphocytes (CTL) occurs during infection with many viruses including those not known to transform CTL or encode superantigens. This polyclonal CTL response includes the generation of high levels of allospecific CTL directed against many class I haplotypes. In this report we investigated whether the allospecific CTL generated during an acute lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6 mice were stimulated specifically by antigen recognition or nonspecifically by polyclonal mechanisms possibly involving lymphokines or superantigens. An examination of the ability of different strains of mice to induce high levels of CTL specific for a given alloantigen showed that most, but not all, strains generated high levels of allospecific CTL, and that their abilities to generate them mapped genetically to the major histocompatibility complex locus, exclusive of the class II region. This indicated that the virus-induced allospecific CTL generation was independent of the class II allotype, and mice depleted of CD4+ cells generated allospecific CTL, indicating independence of class II-CD4+ cell interactions and resulting CD4+ cell-secreted lymphokines. FACS staining with a variety of V beta-binding antibodies did not show a superantigen-like depletion or enrichment of any tested V beta + subset during infection. Several experiments provided evidence in support of direct stimulation of CD8+ cells via the T cell receptor: (a) both virus- and allo-specific killing were enriched within a given V beta subpopulation; (b) relative CTL precursor frequencies against different class I alloantigens changed during the course of virus infection; (c) the relative levels of virus-induced, allospecific CTL-mediated lysis at day 8 after infection did not parallel the CTL precursor frequencies before infection; and (d) limiting dilution analyses of day 8 LCMV-infected spleen cells stimulated by virus-infected syngeneic peritoneal exudate cells (PEC) revealed not only the expected virus-specific CTL clones, but also a high frequency of clones that were cross-reactive with allogeneic and virus-infected syngeneic targets. In addition to the virus cross-reactive allospecific CTL clones, virus-infected PEC also stimulated the generation of some allospecific clones that did not lyse virus-infected fibroblasts. Surprisingly, LCMV-infected PEC were much more efficient at stimulating allospecific CTL clones from day 8 LCMV-infected splenocytes than were allogeneic stimulators. These results indicate that at least part of the polyclonal allospecific CTL response elicited by acute virus infection is a consequence of the selective expansion of many clones of allospecific CTL which cross-react with virus-infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)

CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection.

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C' or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C' depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells.

Virus-induced polyclonal cytotoxic T lymphocyte stimulation.

Infections with a variety of viruses (lymphocytic choriomeningitis (LCMV), murine cytomegalovirus, Pichinde virus, vaccinia virus) stimulated C57BL/6 mice to generate allospecific CTL coincidental with the generation of virus-specific CTL. In C57BL/6 (H-2b) mice, LCMV-induced CTL with reactivity against cells from mice bearing gene products of the d, f, k, p, q, and s but not the b MHC loci. Studies with congenic mouse strains indicated that the MHC loci coded for the target of the allospecific killer cells. The targets of the allospecific CTL were further identified as class I MHC Ag by three criteria: 1) target cells from congenic strains of mice differing from effector cells only in the expression of class I Ag were sensitive to lysis; 2) fibroblasts expressing low levels of class I Ag were resistant to lysis but were rendered sensitive after treatment with IFN-beta, which induced higher expression of class I Ag; and 3) antibody specific for class I Ag expressed on the target cell blocked killing. Studies with congenic mouse strains also suggested that the ability to generate high levels of the virus-induced allospecific killer cells was also under MHC regulation, as H-2b mice generated high levels and H-2k mice low levels of the allospecific CTL. Both C3H/St and C57BL/6 mice immunized against LCMV developed detectable LCMV-specific CTL when later challenged with either murine cytomegalovirus, Pichinde virus, or vaccinia virus, indicating that a virus infection can stimulate the reappearance of memory CTL. Cold target competition studies indicated no cross-reactivities between these viruses or allogeneic cells at the CTL level. Both the allospecific CTL and the reactivated LCMV-specific CTL were found in blast-size lymphocyte preparations. Spleen cells taken from LCMV-infected C57BL/6 mice 5 days post-infection spontaneously generated into allospecific and virus-specific CTL after 2 days of culture. The generation of both was dependent on the presence of supernatant factors produced only in the presence of L3T4+ cells. These factors activated allospecific CTL in spleen cells from virus-primed mice but not from control mice. We suggest that lymphokines produced as a consequence of virus infection may act to stimulate the proliferation and activation of CTL not specific to the challenge virus, resulting in a virus-induced polyclonal CTL stimulation.