Abstract Murine allogeneic cell-mediated lympholysis (CML) involves steps requiring cell surface sulfhydryl groups and separate steps that appear to require one or more cell surface proteases that become activated during the lytic process. Chloromethyl ketone derivatives of amino acids, which are specific irreversible inhibitors of isolated serine-dependent esterases, inhibit a variety of cellular functions including cell-mediated cytotoxicity. These reagents, such as the lysine derivative (TLCK), inhibited CML when the effectors were briefly treated and washed before the assay. However, the inhibition did not appear to result from inactivation of a serine-dependent esterase, because we found that TLCK inhibition was not reversed by including 10- to 30-fold molar excesses of protease substrates. Chloromethyl ketone derivatives are also alkylating agents that nonspecifically react with thiol groups. Iodoacetamide (IAcNH2), an alkylating agent with little reactivity for serine-dependent proteases, also inhibited CML. Furthermore, we showed that several other thiol-reactive compounds, including diamide, which penetrates cells, and oxidized glutathione and thiolyte monoquat, which do not penetrate cells, also inhibited CML. Therefore, a cell surface thiol group appeared involved in an obligatory step in CML. We obtained evidence in other experiments that proteases also appeared to participate in CML. CML was inhibited when low m.w. trypsin or chymotrypsin ester substrates or protein anti-proteases were included in the assay. Pretreatment of effector cells with serum α1AT, which irreversibly inactivates serine-dependent esterases, did not inhibit killing. Inhibition of CML by α1AT included in the assay increased as the assay time was lengthened. We interpreted these results to mean that CML involves one or more cell surface proteases that become activated as a consequence of effector-target interactions.

Redelman, D., & Hudig, D. (1980). The mechanism of cell-mediated cytotoxicity. I. Killing by murine cytotoxic T lymphocytes requires cell surface thiols and activated proteases. The Journal of Immunology, 124(2), 870–878.