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 cell surface alloantigens of murine cytotoxic T lymphocytes (CTL) 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 were examined in conjunction with antigen-specific cytotoxicity in order to delineate possible similarities or differences in their cytolytic mechanisms. In addition, since nonspecific cytotoxicity does not involve antigen recognition, it thereby provided an effective system to delineate the interaction(s) in addition to that between antigen and its receptor, which was necessary for the expression of cytotoxicity. The present findings indicate that the cytotoxic activity of alloimmune CTL in antigen-specific (SCMC) and -nonspecific cytotoxicity—lectin-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-hr 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 utilized. Also, 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 1) 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 both antigen-specific and -nonspecific cytotoxic systems. 2) However, 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. 3) 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. 4) Furthermore, addition of Lyt antisera, suspended in 10% dextran containing media (DCM), at various times after the initiation of effector lymphocyte-target cell contact did not lead to the inhibition of either antigen-specific or -nonspecific cytotoxicity. These data demonstrate that Lyt 2 and Lyt 3 antisera inhibit both 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. Thus, Lyt 2,3 alloantigens or closely linked determinants of CTL appear to be involved in the binding step of the cytolytic process. Moreover, these results provide evidence that a common mechanism or molecular interaction(s) is involved in the triggering of the cytolytic process in both antigen-specific and -nonspecific system.