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SummaryNon-specific hyporesponsiveness of cytotoxic T cells to alphaviruses and alloantigens was induced in male mice by multiple, large injections, starting at birth, of gamma-inactivated alphavirus, stock-grown in outbred mouse brains. Offspring of matings between 2 out of 17 treated males and normal females also exhibited significant non-specific hyporesponsiveness (in comparison with normal control mice), without prior exposure to gamma-inactivated alphavirus stock.Australian Journal of Experimental Biology and Medical Science (1983) 61, 187–191; doi:10.1038/icb.1983.17

SummaryCells of (CBA/H × BALB/c) F1hybrid mice express CBA/H-derived H-2kantigens more weakly than do CBA/H ceils, but H-2dantigens are similarly expressed by F1and BALB/c cells. This was evident when F1hybrid macrophages were compared with CBA/H and BALB/c macrophage as targets for both alloreactive and H-2 restricted antiviral Tc cells. Quantitative absorption of anti-H-2Kkserum by spleen cells of F1or CBA/H mice also suggested about 3-fold less H-2Kkantigen on F1cells. With the use of two anti-H-2Kkmonoclonal antibodies. 30R3 and 27R9, the reduced expression of H-2Kkin this F1hybrid was further analysed in a two-stage radio-immunoassay employing the uptake of 125I-protein A to measure antibody binding. By a thermodynamic approach, estimates were made of the dissociation constant for antibody binding, and of the relative numbers of H-2 molecules expressed by both F1hybrid and CBA/H spleen cells. The results indicate that there is a two-fold reduction in the number of H-2Kkmolecules expressed on the surface of F1cells. Similar dissociation constants for F1and CBA/H cells indicated no detectable qualitative difference in their H-2Kkantigens with respect to sites recognized by 30R3 and 27R9.Australian Journal of Experimental Biology and Medical Science (1979) 57, 627–635; doi:10.1038/icb.1979.66

SummaryWhen virus-immune cytotoxic T cells were generated in secondary responses in vitro to syngeneic, infected stimulator cells, they only killed infected targets that shared H-2K or H-2D genes with the T cells and stimulator cells. H-2I region compatibility was neither sufficient nor necessary. The role of H-2K, H-2D and H-2I genes in the interaction between memory T cells and infected stimulator cells was then investigated. Initial attempts failed because of escape of virus into the responder population. Steps were taken to eliminate this problem; confirming their efficacy was the finding that when (CBA/H × BALB/c) F1memory T cells were stimulated by either parental type, the cytotoxic T cells generated only killed the target cell type syngeneic to the stimulator cell used to induce the response. Using recombinant strain macrophages as infected stimulator cells, it was further shown that K or D region sharing between stimulator and responder cells was sufficient to induce the response. I region homology, on the other hand, although stimulating high levels of 3H-thymidine incorporation, did not give rise to significant amounts of cytotoxic activity against virus-infected target cells. These results are compatible with the interpretation that the inductive signal to cytotoxic T cell precursors (memory T cells) in the in vitro secondary response is an antigenic pattern containing virus-induced and self H-2K or H-2D components; the response of the cytotoxic cells may be facilitated by a “helper”, DNA-synthesizing subclass which responds to antigenic patterns containing both virus-induced and I region-determined components.Evidence from cytotoxic T cell induction with semi-compatible infected macrophages shows that H-2K (or H-2D) antigens and viral antigen must be in the same cell membrane for efficient stimulation to occur. This suggests that both the antigens, and the T cell receptors for them, are closely associated in the infected cell and T cell membranes respectively.Immunology and Cell Biology (1977) 55, 549–559; doi:10.1038/icb.1977.54

SummaryThe 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 la-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.Immunology and Cell Biology (1977) 55, 539–547; doi:10.1038/icb.1977.53

SummaryThe 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 cither 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-θ 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.Australian Journal of Experimental Biology and Medical Science (1976) 54, 559–571; doi:10.1038/icb.1976.57

SummaryPeritoneal 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-θ 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.Australian Journal of Experimental Biology and Medical Science (1976) 54, 365–370; doi:10.1038/icb.1976.36

SummaryAn in vitro culture method was used to study secondary cellmediated 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. Separation 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 γ-irradiation did not impair its ability to induce antigenic changes in stimulator cells. Stimulator cells treated with UV- or γ-irradiated virus for 1 h and then immediately treated 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.Australian Journal of Experimental Biology and Medical Science (1976) 54, 253–264; doi:10.1038/icb.1976.26

The specificity of recognition of H-2 antigens by various subsets of Tc cells was investigated with respect to the two separate molecules known to be coded in the H-2D(d) region (a) D which carries the private specificity H-2.4 and (b) D' which carries the public specificity H-2.28. BALB/c.H-2(db) mutant mice express D but not D' on their cell surfaces, whereas wild-type BALB/c mice express both D and D'. H-2 restricted Tc cells specific for viral-plus- H-2D(d)-coded antigens on infected self cells, or minor H-plus-H-2D(d)-coded antigens on H-2-compatible cells apparently recognize D, but do not detectably recognize D. In contrast, BALB/c-H-2(db) anti-BALB/c Tc cell responses do recognize D' (the only known antigen which is not shared by mutant and wild-type); furthermore, D' is also detectably recognized by a significant proportion of the Tc cells that respond in MLR to H-2D(d)-coded antigens. In these latter responses, D' was recognized separately from D, i.e., the response was not "H-2 restricted". These results indicate that H-2 restricted Tc cell responses to modified-self cells are more specific for self H-2D(d)-coded antigens then are allogeneic Tc cell responses directed at the same antigens, in that haplotype-unique (private) specificity recognition (of the D molecule) exclusively occurs only in the former, not the latter case. The implications of this specificity of H-2 restricted responses for possible processes of somatic selection of anti-self recognition structures on progenitor Tc cells are briefly discussed.

The T(c)-cell response to ectromelia virus infection was studied in BALB/c-H-2(db) mice which carry a loss mutation in the H-2D region that results in the absence from cell surfaces of a molecule (D') bearing certain public H-2 specificities. When infected, these mice showed a poor response of T(c) cells that recognize H-2D(d) plus virus-specific determinants on infected macrophage targets, but gave a normal response to H-2K d plus virus-specific antigens. However, their own infected macrophages do display wild-type antigenic patterns involving virus and H-2D(d) since they were killed as efficiently as wild-type (BALB/c,H- 2(d))-infected cells by T(c) cells specific only for H-2D(d) plus viral antigens. When tested in vitro, infected BALB/c-H-2(db) cells stimulated a poor T(c)-cell response to H-2D plus virus-specific antigens, but stimulated a normal response (in comparison with infected BALB/c macrophages) to H-2K(d) plus viral antigens. Uninfected BALB/c-H-2(db) cells stimulated a normal T(c)-cell response to minor H antigens or trinitrophenyl in association with H-2D(d), thus suggesting that the defective response to infection may reside in a failure of the relevant H-2D(d) antigens of mutant cells to physically associate with viral antigens. Close association of viral and H-2D-coded molecules was also suggested by ability of specific anti-H-2K or -H-2D to partially block T(c)-cell-mediated lysis of infected targets. These results were interpreted to mean that H-2Dd-dependent, virus- immune T(c) cells recognized an antigenic pattern consisting of virus- specific and H-2D(d) determinants with the latter borne on an H-2D molecule carrying serologically-defined H-2D(d) private specificities. A second H-2D(d)-coded molecule (D') was not required for recognition and lysis by activated T(c) cells, but was apparently necessary for efficient stimulation of precursor T(c) cells, perhaps by promoting appropriate physical association of viral and H-2D(d) molecules.

SummaryMacropharge activation, as demonstrated by an increased ability to kill a bacterium (Listeria monocytogenes) occurred in virus-infected mice the phenomenon was present in the liver, spleen and peritoneum eight days after infection with lymphocytic choriomeningitis virus and in the liver and peritoneum eight days after ectromelia virus infection. The mechanisms of macropossible significance in viral infection are discussed.Australian Journal of Experimental Biology and Medical Science (1973) 51, 393–398; doi:10.1038/icb.1973.35