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.

Mechanism of killing by virus-induced cytotoxic T lymphocytes elicited in vivo.

The mechanism of lysis by in vivo-induced cytotoxic T lymphocytes (CTL) was examined with virus-specific CTL from mice infected with lymphocytic choriomeningitis virus (LCMV). LCMV-induced T cells were shown to have greater than 10 times the serine esterase activity of T cells from normal mice, and high levels of serine esterase were located in the LCMV-induced CD8+ cell population. Serine esterase was also induced in purified T-cell preparations isolated from mice infected with other viruses (mouse hepatitis, Pichinde, and vaccinia). In contrast, the interferon inducer poly(I.C) only marginally enhanced serine esterase in T cells. Serine esterase activity was released from the LCMV-induced T cells upon incubation with syngeneic but not allogeneic LCMV-infected target cells. Both cytotoxicity and the release of serine esterase were calcium dependent. Serine esterase released from disrupted LCMV-induced T cells was in the form of the fast-sedimenting particles, suggesting its inclusion in granules. Competitive substrates for serine esterase blocked killing by LCMV-specific CTL, but serine esterase-containing granules isolated from LCMV-induced CTL, in contrast to granules isolated from a rat natural killer cell tumor line, did not display detectable hemolytic activity. Fragmentation of target cell DNA was observed during the lytic process mediated by LCMV-specific CTL, and the release of the DNA label [125I]iododeoxyuridine from target cells and the accompanying fragmentation of DNA also were calcium dependent. These data support the hypothesis that the mechanism of killing by in vivo-induced T cells involves a calcium-dependent secretion of serine esterase-containing granules and a target cell death by a process involving nuclear degradation and DNA fragmentation.

Acute, lethal, natural killer cell-resistant myeloproliferative disease induced by polyomavirus in severe combined immunodeficient mice.

Infection of severe combined immunodeficient mice, which lack T and B lymphocytes, with polyomavirus (PyV) induced an acute hematological disorder leading to the death of the mice by 2 weeks postinfection. The disease was characterized by a dramatic decrease in megakaryocytes, multiple hemorrhages, anemia, thrombocytopenia, splenomegaly, a massive myeloproliferation and splenic erythroproliferation with a defect in maturation of the myeloid elements similar to that in acute leukemia. This pathology in severe combined immunodeficient mice is very different from that of the well-characterized tumor profiles induced by PyV in normal newborn or nude mice. Viral T and capsid (VP1) antigens and viral genome were detected in some cells in the spleen, but not in the majority of the proliferating myeloid cells. This suggests that the myeloproliferation is induced by some indirect mechanism, such as secretion of growth factors or cytokines by virus-infected cells, rather than by direct transformation by PyV. Neither the spread of PyV, its replication in different organs, nor the pathogenesis or the time of death were altered by depleting natural killer cells in vivo by anti-natural killer cell antibodies. Analysis of the spleen leukocyte population indicated that the cells expressed high levels of class I major histocompatibility complex antigens and were resistant to lysis by activated natural killer cells.

Demonstration of the antiviral role of natural killer cells in vivo with a natural killer cell-specific monoclonal antibody (NK 1.1).

A monoclonal antibody (NK 1.1) to mouse natural killer (NK) cells selectively depleted NK cell activity in virus-infected mice without significantly depressing other immune functions, including the development of virus-specific cytotoxic T cells. NK cell depletion with this antibody resulted in markedly enhanced plaque-forming unit titers of some (murine cytomegalo, Pichinde) but not other (mouse hepatitis, lymphocytic choriomeningitis) viruses. This confirms that NK cells do play a role in regulating certain infections and shows that this antibody provides a convenient tool for examining the role of NK cells in viral infections.