Studies on the induction and expression of T cell-mediated immunity. X. Inhibition by Lyt 2,3 antisera of cytotoxic T lymphocyte-mediated antigen-specific and -nonspecific cytotoxicity: evidence for the blocking of the binding between T lymphocytes and target cells and not the post-binding cytolytic steps.

Abstract

The role of murine cytotoxic T lymphocytes (CTL) cell surface alloantigens and the molecular interactions requisite for CTL-mediated target cell lysis were investigated with antisera directed against Lyt alloantigens in the absence of complement. The effect of Lyt antisera on antigen-nonspecific cytotoxic systems was examined in conjunction with antigen-specific cytotoxicity to delineate possible similarities or differences in their cytolytic mechanisms. Since nonspecific cytotoxicity does not involve antigen recognition, it thereby provided an effective system to delineate the interactions(s) in addition to that between antigen and its receptor, which was necessary for the expression of cytotoxicity. The cytotoxic activity of alloimmune CTL in antigen-specific (SCMC) and -nonspecific cytotoxicity-lectin[concanavalin A]-dependent cellular cytotoxicity (LDCC) and oxidation-dependent cellular cytotoxicity (ODCC)-was inhibited by the addition of Lyt 3 alloantiserum and monoclonal rat anti-mouse Lyt 2 antiserum but not by Lyt 1 antiserum as measured by a 3-h 51Cr-release assay. The specificity of blocking was established by the failure of Lyt alloantisera directed against the alternative allele of the Lyt 2,3 loci to inhibit cytotoxicity and by the ability of the monoclonal Lyt 2 antiserum to inhibit SCMC, LDCC and ODCC. The inhibitory activity of the Lyt 2 and Lyt 3 antisera was directed against membrane determinants of CTL, since comparable inhibition was observed when Lyt 2,3+ and Lyt 2,3- target cells were used. H-2 alloantiserum and monoclonal Thy-1 antiserum specific for surface determinants of effector lymphocytes did not inhibit SCMC LDCC or ODCC. Analysis of the mechanism of inhibition by Lyt 2,3 antisera indicated that the addition of Lyt 2,3 antisera, but not Lyt 1 antiserum before the formation of lymphocyte-target cell conjugates resulted in the reduction in the frequency of conjugates formed in antigen-specific and -nonspecific cytotoxic systems. Analysis by the single cell cytotoxicity assay in agarose revealed that the percent of bound target cells lysed was not affected by the presence of the Lyt antisera. When the antisera were added after conjugate formation, dissociation of preformed conjugates was not observed and the killing of bound target cells was not affected. Addition of Lyt antisera suspended in 10% dextran containing media at various times after the initiation of effector lymphocyte-target cell contact did not lead to the inhibition of antigen-specific or -nonspecific cytotoxicity. Lyt 2 and Lyt 3 antisera inhibit antigen-specific and -nonspecific cytotoxicity. Inhibition by Lyt antisera is attributed to the blocking of the formation of CTL-target cell conjugates and not the blocking of a post-binding step. Once effector lymphocyte-target cell binding has occurred. Lyt 2,3 antisera are unable to inhibit the expression of cytotoxicity. Lyt 2,3 alloantigens or closely linked determinants of CTL appear to be involved in the binding step of the cytolytic process. Evidence is provided that a common mechanism or molecular interaction(s) is involved in the triggering of the cytolytic process in antigen-specific and -nonspecific systems.