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Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.

Lysis of virus-infected target cells by virus-specific cytotoxic T cells occurs where donors of T cells and targets share either H-2K or H-2D genes. The effect of four H-2K mutations on virus-induced antigens recognized by cytotoxic T cells from in vitro secondary response to infection was studied. B10.A(5R) cytotoxic T cells (which share the K end of H-2 with the mutant strains, except for the mutated gene(s)) efficiently killed virus-infected macrophage targets from mutant strains B6-H-2bg1 and B6-H-2bg2, were less effective against B6-H-2bh and did not appear to be cytotoxic for B6.C-H-2ba target cells. Conversely, B6-H-Ibg1 and B6-H-2bg2 cytotoxic T cells were more effective in killing virus-infected B10.A(5R) macrophages than B6-H-2bh and B6.C-H-2ba cytotoxic cells respectively. In addition, B6-H-2bg1 and B6-H-2bg2 cells appeared to be only slightly different from wild-type with respect to the interaction between virus-infected cells and T cells. The data obtained suggested that virus-induced antigenic patterns on infected B6.C-H-2ba (mutant) cells are more different antigenically from those on wild-type cells than are those on infected cells from the other mutants, B6-H-2bh, B6-H-2bg1 and B6-H-2bh2. This agrees with previous data using primary cytotoxic T cells and thus suggests that no detectable change in the affinity or specificity of cytotoxic T cell receptors occurs between primary and secondary responses to infection. These findings are also discussed in relation to the exclusion of T cells with receptors for H-2K determinants that are common to the mutants and wild-type, from the response to virus-infected self cells.

P-815 mastocytoma cells developed susceptibility to immune T-cell-mediated cytolysis shortly after infection by ectromelia virus. Intracellular viral replication and late protein synthesis seem to bu unnecessary events. Interference with early protein synthesis, however, inhibits the development of susceptibility to lysis. The important intracellular events necessary for subsequent cytolysis appear to occur within 1 hour of infection. Virus rendered non-infectious by ultraviolet irradiation but not by gamma irradiation is able to induce these changes. By determining the minimum and essential events of the infectious process which result in T-cell-mediated cytolysis, the task of establishing the molecular changes occurring in the target cell surface membrane necessary for immune T-cell recognition should be simplified.

Antiviral activity in vivo exerted by ectromelia virus-immune spleen cells transferred to ectromelia-infected recipients and cytotoxicity against virus-infected target cells in vitro were both properties of non-immunoglobulin (Ig)-bearing cells (which included T cells). Ig-bearing cells, including thymus-independent (B) cells and antibody-secreting cells, were much less active in vivo when injected alone and tended to block rather than amplify the effect triggered by T cells. Ig-bearing cells were also slightly active in vitro, possibly because some T cells have detectable Ig. Antiviral effects in cell transfer experiments were seen only when immune cell donors and infected recipients shared the same H-2 gene complex. These results are consistent with the hypothesis that the T cell response to ectromelia infection is directed against specific virus-induced change(s) in antigen(s), specified by gene(s) in the H-2 complex, which appear in virus-infected cells.

An in vitro method is described for primary induction of murine cytotoxic T cells against syngeneic cells infected with ectromelia or lymphocytic choriomeningitis (LCM) virus. Cytotoxicity was assayed by 51Cr release from macrophage or L929 target cells. Cytotoxic activity was sensitive to anti-theta and complement and was expressed only against target cells infected with the same virus and sharing H-2K or H-2D genes with the infected stimulator cells. The crucial factors in generating responses were mouse strain, responder: stimulator ratio, nature of infected stimulator cells, and presence of sufficient macrophages. C57BL/6 cells were less demanding than CBA/H and BALB/c cells. Under optimal conditions defined here, the in vitro response had similar kinetics and potency to the primary response in the spleen in vivo.

Peritoneal exudate cells from mice infected with ectromelia virus were cytotoxic for virus-infected target cells as measured in a 51Cr release assay. Cytotoxic activity seemed to be T cell-dependent as it was largely abolished by treatment with anti-theta serum and complement but was not impaired by macrophage depletion. The kinetics of development of cytotoxicity in the peritoneal cavity lagged behind spleen cytotoxicity by 1-2 days. Peak activity in peritoneal cells was present about 6 days after intravenous infection with virus. These studies suggest that macrophages present in the free peritoneal cell populations of ectromelia-infected mice are not cytotoxic for virus-infected target cells. The effect of macrophages in virus clearance is therefore likely to be due to phagocytic rather than cytotoxic effects.

Several B10 strains of mice, recombinant at the H-2 locus, have been shown to differ in their resistance to infection with ectromelia virus, a natural mouse pathogen. Of 10 strains, B10, B10.A(2R), B10.A(4R) and B10.D2 were the most resistant, while B10.G and B10.A(5R) were the most susceptible. Other strains were intermediate between these extremes. Several genes conferring resistance have been mapped to Db in B10.A(2R), Kk I-Ak I-Bk in B10.A, I-Jb in B10.A(2R) and to Dd in B10.T(6R). In general, death among susceptible strains was not a consequence of acute liver necrosis as in other non-B10 strains, and occurred randomly from 8-14 days after infection. The exact cause of death is unknown but is characterized by persisting high titers of virus in the spleen and sometimes the liver, despite an ongoing immune response indicated by strong cytotoxic T-cell activity detectable in the spleens of all mice. The most resistant B10 and B10.A(2R) strains cleared virus from the spleen and liver by 8 days after infection. Analysis of infection in chimeric mice indicates that H-2 genes, which determine susceptibility to virus persistence in the spleen, operate via radiosensitive cells of the lymphomyeloid system. This evidence, together with several examples of H-2-linked differences in cytotoxic T-cell responsiveness between resistant and susceptible strains, is consistent with the hypothesis that the mechanism by which H-2 genes control resistance to ectromelia virus in B10 strain mice is by their influence on the effectiveness of a cell-mediated immune response.

The anti-viral activity of secondary effector cells generated in vitro against ectromelia virus infection was investigated. Depending upon the order of administration of cells and virus, 2 X 10(6) cells significantly reduce virus titres in recipient mice. Mice injected with lymphocytic choriomeningitis (LCM) virus are not protected by secondary effector cells against ectromelia virus infection and vice versa. The cells conferring anti-viral activity are sensitive to anti-theta and complement treatment, and must share H-2K or H-2D region genes with the recipients in order for significant reduction of virus titres to occur. The possibility of exploiting this approach in clinical medicine by using T cell-mediated mechanisms against certain viral infections is briefly discussed.

An in vitro culture method was used to study secondary cell-mediated responses to ectromelia virus infection in mice. Infected, syngeneic spleen cells or peritoneal cells were efficient "stimulator" cells when cultured with "responder" cells obtained from mice infected with ectromelia 4-6 weeks previously. The kinetics of generation of cytotoxic cells in cultures were determined; a peak occurred on days 4-5. A separation procedure performed on the cytotoxic cells showed that activity was associated mainly with the Ig-negative subpopulation (T cell-rich) and that H-2 compatibility between cytotoxic cells and target cells was required. The secondary response was virus-specific, at the level of both induction and target cell lysis, at least so far as ectromelia and lymphocytic choriomeningitis (LCM) viruses are concerned. Seperation of responder cells prior to culture showed that a potent secondary response was generated with the Ig-negative (T cell-rich) subpopulation and only a weak response was observed when the responder cells were Ig-positive (rich in B cells). Infected stimulator cells did not appear to secrete significant amounts of soluble antigen into the medium over 4 days of culture. Thus, antigenic patterns effective in memory T cell stimulation may be largely associated with the surfaces of infected cells.Pretreatment of ectromelia virus with UV- or gamma-irradiation did not impair its ability to induce antigenic changes in stimulator cells. Stimulator cells treated with UV-or gamma-irradiated virus for 1 h and then immediately with pactamycin to inhibit further viral protein synthesis and replication were efficient stimulators, thus indicating that antigenic changes are induced very rapidly on the surface of stimulator cells after uptake of virus. These treatments are being used to further characterize the cellular requirements in the stimulator population.

The nature of infected stimulator cells in the in vitro secondary cytotoxic T cell response to ectromelia infection was investigated. It was found that macrophages were better stimulator cells than spleen cells. B cells (Ig-positive cells) were superior to T cells (Ig-negative cells) both on a relative proportion and on a cell-to-cell basis. Concanavalin A and lipopolysaccharide-stimulated lymphocytes were also effective stimulator cells but appeared to be slightly inferior to spleen cells. Spleen cells depleted of Ia-positive cells were markedly inferior to normal spleen cells as stimulators. It was also found that primary and secondary cytotoxic T cells were largely Ia-negative. These findings are discussed in relation to the likely events during T cell responses to infection in vivo.

The mechanism for innate resistance to ectromelia virus, which is controlled by a single gene in C57BL mice, was investigated. The cells or factors involved appear to be radioresistant and to impose an early barrier to viral penetration and spread via lymphatics or blood to the target organs, i.e., liver and spleen.

The role of adherent cells in an in vitro secondary response to ectromelia virus infection was investigated. Spleen cells from ectromelia-primed mice ("responder" cells) depleted of adherent cells by either carbonyl iron treatment, adherence to plastic or passage through cotton wool columns had a markedly decreased capacity to produce a secondary response, as indicated by decreased T cell-mediated cytotoxicity against virus-infected target cells, when cultured with virus-infected "stimulator" cells. The secondary response was restored by the addition of peritoneal cells from either normal or ectromelia-immune mice. Small numbers of peritoneal cells completely reconstituted the response within a certain dose range but larger numbers produced a marked inhibition of the response. Spleen cells were less effective in restoring the response. The peritoneal cells were not merely acting as additional, infected "stimulator" or antigen-presenting cells, since they could be added as late as 3 days after culture. Reconstituting activity was not affected by pretreatment with anti-theta serum and complement and cell separation studies showed that the activity was associated mainly with Ig-negative cells and that the active cell probably bears Ia antigens on its surface. These results indicate that the adherent cells involved are probably macrophages and that they act non-specifically to produce optimum conditions for the specific response of T cells.

Spleen cells and serum from mice immunized with ectromelia virus suppressed the immune response to infectious virus when transferred intravenously into recipient mice given an immunizing virus dose. The suppression was reflected in decreased cytotoxic T-cell activity directed against H-2 compatible virus-infected target cells in the spleens of recipients. Suppression was observed when immune cells or serum were transferred 1-2 h or 1 day after immunization of recipients, but not 2 days after, and was maximal when 6-day immune spleen cells were used as suppressor cells. H-2 compatibility between donor and recipient mice was necessary for suppression to be expressed. Use of recombinant mice showed that I-region compatibility was neither sufficient nor necessary, and that D-region compatibility was sufficient. Specificity of suppression was suggested by the finding that cells and serum from mice immunized with Listeria monocytogenes, a bacterium, had no suppressive activity on the antiviral response. Anti-theta treatment eliminated the ability of immune cells to suppress, and the suppressive effect was not markedly dose-dependent with respect to both cell dose and virus dose under the conditions employed. Virus levels in the spleens of recipients were significantly reduced after injection of immune cells. Adult thymectomy had no effect on the primary immune response to ectromelia virus infection, thus indicating no role for T1 cells in the suppressive mechanism. The results obtained therefore suggested that suppression in this system was due to effector T cells which triggered clearance of virus (and thus, of virus-induced antigens) necessary for the induction of precursors of effector T cells, and that this simple feed-back mechanism normally plays an important role in the regulation of the primary immune response to ectromelia infection at the level of precursor induction. The existence of other postinduction regulatory mechanisms, however, is unknown and under investigation.

Cell transfer experiments using mice with recombinant H-2 haplotypes were used to map the H-2 regions which must be shared by ectromelia-immune T-cell donors and virus-infected recipients for transfer of virus clearance mechanisms in the spleen. K- or D-region genes were necessary and sufficient; I-region genes were not involved. The remainder of the mouse genome could be varied widely without impairing the efficacy of T-cell antiviral function, provided either a K or a D region was shared in the donor-receipient combination. A mutation in a single genetic element of the K region of the H-2 complex abolished the antiviral effect of immune T-cell transfer in a donor-recipient combination which shared the K end.

Chimeras produced by reconstitution of 950 rads irradiated type A or type B host mice with (AXB)F1 fetal liver stem cells were examined in primary (in vivo) and secondary (in vitro) Tc-cell responses to ectromelia virus infection. Of 33 individual chimeras which gave primary responses, 26 produced significant specific lysis of infected targets of both A and B type, though host type targets were invariably lysed more efficiently (host bias). The other 7 chimeras gave lysis of infected host type targets only (absolute restriction). 12 individual chimeras were used in secondary responses. Nine showed host bias, and three showed absolute restriction. Whether an individual chimera showed host bias or absolute restriction seemed to be unrelated to whether the response was primary or secondary, to the time after reconstitution (ranging from 4 to 22 wk), to strain of mouse, or to the batch of fetal liver stem cells used.

Histological and immunofluorescence techniques showed that mononuclear cells invaded virus-infected foci in the livers of passively immunized mice within 10 hr of the receipt of immune spleen cells or hyperimmune serum; by 24 hr, marked destruction of virus antigens had occurred in these lesions. Immune cell transfer promoted denser packing of mononuclear cells in the foci and more efficient destruction of infectious material than immune serum. Similar liver lesions developed by the 6th day after sublethal, primary, subcutaneous infection in normal mice. In contrast, in mice with GVHR which were immunosuppressed but possessed hyperactive macrophages and unimpaired splenic interferon response, mononuclear cells did not invade liver lesions and the animals died. These results, together with data reported previously, indicated that mononuclear cell invasion of infected liver foci, triggered by CMI, was of key importance in recovery from primary mousepox. The roles of specifically sensitized lymphocytes and macrophages within lesions were not directly evaluated, but indirect evidence suggested that lymphocytes could cause no more than a halt in virus multiplication, and that macrophages were required for the inactivation of preformed virions. Possible augmentation of the efficiency of macrophages by locally-produced lymphocyte interferon, neutralizing antibody, or stimulation of their phagocytic and intracellular digestive capacity cannot be excluded.

Agglutination and immunofluorescence tests in vitro showed that the ATS used in these experiments cross-reacted with macrophages and RBC. However, ATS was not toxic in vivo, and small doses given subcutaneously depleted thymus-dependent areas of lymphoid tissues and selectively depressed blood lymphocyte counts without affecting other cell types in the blood. Furthermore, the function of littoral macrophages as indicated by the clearance of blood-borne virus and its subsequent behavior over a 48 hr period in the liver and spleen was not changed by ATS. Thus, the innate resistance of these vital target organs was not depressed. A similar regimen of subcutaneous ATS caused a highly significant increase in mortality from mousepox with an associated failure to control virus growth in the liver and spleen which was manifest by 6 days after infection. The interferon and neutralizing antibody responses were not impaired in ATS-treated mice, but the cell-mediated immune response was significantly suppressed. This evidence, and consideration of the timing of these host responses during the course of infection in relation to the control of virus growth in the liver and spleen, led to the conclusion that cell-mediated immunity probably contributed an essential acquired recovery mechanism. However, no evidence was obtained concerning the nature of this antiviral mechanism.

The following passive transfer experiments evaluated the contributions of the various host responses in recovery from mousepox. (a) Immune spleen cells transferred highly efficient antiviral activity, but preinfected recipients of these cells made no detectable splenic interferon or antibody in the 24 hr interval after cell transfer. (b) Passively administered interferon was ineffective. (c) Recipients of hyperimmune serum had much more antibody than recipients of immune spleen cells but significantly less antiviral activity. (d) Immune spleen cell populations with antiviral activity contained mediators of CMI to virus antigens. (e) The antiviral activity of immune spleen cells was specific; it was inhibited by in vitro treatment with ATS, anti-light chain serum, and anti-theta ascitic fluid, but not by removal of mononuclear phagocytes from the immune population. These results are interpreted to mean that recovery mechanisms conferred by immune spleen cells were triggered by specifically sensitized, thymus-derived lymphocytes, and that antibody and interferon responses were of less importance. A radiosensitive recipient component was necessary for the full expression of the antiviral activity of both immune cells and immune serum. It seemed likely that this component was the blood monocyte.