Recent observations have suggested striking similarities between complement-mediated and cell-mediated lysis. Both pathways share the terminal insertion of channels into target membranes, and unique esterases have been postulated to participate in the activation of cytolytic effector molecules. Since killer-specific esterases and channel-forming proteins can be demonstrated in in vitro cell lines, it is important to ascertain that the described esterase and channelforming proteins are also present in killer cells from in vivo sources. Results presented here show that killer cell-specific Na-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase is induced in vitro concomitant with the sensitization of cytotoxic effector cells. In contrast, in vivo-primed cytotoxic T cells or natural killer (NK) cells fail to express high levels of this enzyme. Assay of different cytotoxic effector cells reveals the presence of Na-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase in clones with T killer and NK activity, but enzyme levels do not correlate with cytolytic activity nor does inhibition of esterase activity interfere with granule-mediated cell lysis. A similar result is seen with granule-mediated cytolytic activity. Cloned NK and T killer cell lines possess granules that are able to lyse erythrocyte targets. However, T killer cells sensitized in mixed lymphocyte culture or in vivo have no detectable cytotoxic granules. Cytotoxic granules are also not detected in NK cells isolated from animals.

The channel-forming polyperforins P1 and P2 are thought to be formed from the contents of dense core vesicles of cytolytic effector cells. To test this hypothesis, granules from various cytotoxic effector cells were assayed for cytolytic activity on nucleated or unnucleated targets. The results show that in general, granules from cytolytic effector cells are cytolytic, whereas granules from noncytotoxic cells are not. Cytotoxicity of granules is not specific, but there appears to be a preference in that nucleated targets are lysed better than are erythrocytes by granules from T killer or natural killer cells. Granules from CTLL-2, however, preferentially lyse erythrocyte targets. This cell line has been in culture for a long period of time and has lost its cytotoxicity. We tested whether granules from CTLL-2 caused formation of transmembrane pores in erythrocyte target membranes. We found that granule- and complement-induced lesions have similar pore sizes. They are big enough to allow the total release of alpha-bungarotoxin, an 8000 Mr polypeptide with dimensions of 4 X 2.5 nm. Larger molecules are released partially or not at all. Under acidic conditions (pH 5.4) granules do not permeabilize target membranes. This may suggest a pH-dependent control mechanism in the formation, insertion, or function of polyperforin channels, in addition to a previously recognized Ca2+-dependent mechanism. Permeabilization of lipid vesicles by granules was studied to explore what the molecular requirements for channel insertion into membranes may be. Release of alpha-bungarotoxin induced by granules was observed in liposomes made of soybean lipid with or without cholesterol, suggesting that no membrane component other than lipid is required for the insertion of polyperforins, and that the action of polyperforins does not require other mechanisms in the target cell. When pure lecithin from soybean and egg, or synthetic phosphatidylcholines were used, slower release or no release of macromolecules was observed. We suggest that some kind of lipid specificity is required for perforin action. This may be related to the hydrophobic region of the lipid bilayer rather than to the polar portion, because different lecithins with varying fatty acid composition gave similar results.

Acute marrow graft rejection in allogeneic or semiallogeneic donor-recipient mouse combinations has been suggested to be caused by natural killer (NK) cells. The unique in vitro specificity of NK cells for tumor cells, however, does not explain the specific rejection of bone marrow grafts by NK cells. Recent experiments have implicated antibody in marrow graft recipients as the specificity-inducing component that guides NK cells in an antibody-dependent cytotoxic (ADCC) reaction to attack the marrow graft. On the basis of this hypothesis, one would postulate that nonresponder marrow graft recipients can be converted into responders by injection with antibody of appropriate specificity. Results presented in this report show that this is indeed possible. Specific monoclonal or polyclonal antibody of IgG isotype induces marrow graft rejection in nonresponder recipients. This can be demonstrated in allogeneic as well as in semi-allogeneic (hybrid resistance) donor-recipient strain combinations. Antibody-induced marrow graft rejection is independent of complement and dependent on the presence of NK cells. Surprisingly, graft rejection induced by antibody is quite efficient in allogeneic and semiallogeneic marrow donor-recipient combinations, whereas it is generally poor in syngeneic combinations. This result is not understood if NK cells lyse bone marrow cells solely in an ADCC-type reaction. Because NK cells can lyse targets in an antibody-dependent as well as independent reaction, it is proposed that the binding of NK cells to targets via their receptors plays an additional role in the rejection of bone marrow in vivo. Preliminary evidence for this possibility is that NK cells in the apparent absence of antibody may have a detectable suppressive effect on the growth of marrow grafts in F1 hybrid mice transplanted with parental marrow grafts.

Results of recent experiments have provided compelling evidence supporting the hypothesis that the acute rejection of bone marrow transplants by allogeneic and semiallogeneic recipients is principally due to the action of natural killer (NK) cells. The observed specificity of graft rejection is likely induced by target-specific antibody that guides the NK cells in an antibody-dependent cytolytic reaction resulting in the elimination of the graft. The sole involvement of NK cells in marrow graft rejection, however, is contradicted by several observations that point to the environment of specific T cells. Results presented in this paper demonstrate that in allogeneic marrow graft rejection models, T killer cells are capable of causing graft rejection provided a prior sensitization phase is allowed. Thus, mice not able to reject marrow grafts in a primary response via their NK cells will do so in a primed secondary response via their T cells. Rejection is specific in that only marrow grafts H-2 identical to the sensitizing marrow graft are rejected. Sensitization for NK cell independent marrow graft rejection can be accomplished by prior priming with allogeneic tumor cells or by injection of cloned T killer cells. In contrast to bone marrow allograft rejection, the hybrid resistance model in which F1 hybrid mice reject parental marrow grafts does not appear to induce T killer cells in vivo. Neither marrow grafts nor tumor cells prime F1 hybrids for a second-set parental graft rejection. Moreover, F1 hybrid antiparental T killer cells induced in vitro and adoptively transferred in vivo fail to transfer hybrid resistance. Therefore, there appear to be potent mechanisms acting in vivo that suppress the action or induction of F1 hybrid T killer cells specific to parental antigens.