The requirements for triggering of lysis by cytolytic T lymphocyte clones. II. Cyclosporin A inhibits TCR-mediated exocytosis by only selectively inhibits TCR-mediated lytic activity by cloned CTL.
TCR-mediated granule exocytosis, as measured by the release of serine esterase activity, has been implicated in the lytic process of Ag-specific CTL. Exocytosis appears to be the mechanism of release of other lysis-relevant molecules including cytotoxic lymphokines and proteins that have the capacity to induce membrane lesions as measured by the hemolysis of non-nucleated SRBC. In the studies presented here, we assessed the contribution of exocytosis and lymphokine production in CTL lysis of nucleated and non-nucleated target cells by using a panel of murine CTL clones. Ag-mediated activation of cytolysis, lymphokine production, and exocytosis could be mimicked by mAb against the TCR/CD3 complex, or by stimulation with the combination of PMA + calcium ionophore, which appear to bypass the TCR (neither PMA nor calcium ionophore alone induced these functions efficiently in our CD8+ CTL clones). Although lysis, IFN-gamma production and exocytosis of N-alpha-benzyloxycarbonyl-L-lysin esterase (BLTE) activity were induced by either stimulus, we were able to identify distinct activation requirements for each of these functions. We found that lymphokine production, exocytosis, and cytolysis could be selectively inhibited. Cycloheximide inhibited IFN-gamma production, but did not inhibit exocytosis of BLTE activity or cytolysis. In addition we showed that cyclosporine A (CsA) profoundly inhibited IFN-gamma production as well as exocytosis induced by stimulation through the Ag receptor or by PMA + calcium ionophore. In contrast, CsA had little or no effect on lysis of nucleated target cells that bear the relevant Ag. These findings indicate that our CTL clones can lyse target cells by a mechanism independent of exocytosis or (de novo) lymphokine production. To directly assess the capacity of our CTL clones to lyse target cells without inducing nuclear damage we developed a system of coating non-nucleated SRBC with anti-CD3 mAb for use as stimuli and as targets for lysis. We found that our cloned CTL were indeed activated to produce IFN-gamma by SRBC that were coated with anti-CD3 mAb, and, furthermore, they were able to lyse the SRBC in a short term cytolytic assay. Thus our CD8+ CTL are capable of lysing certain target cells by a mechanism independent of DNA degradation, presumably by inducing a membrane lesion. In addition, CsA did inhibit lysis of the non-nucleated SRBC targets as well as exocytosis of BLTE activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Lancki
D W
DW
Department of Pathology, University of Chicago, IL 60637.
Kaper
B P
BP
Fitch
F W
FW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology; D002999:Clone Cells / Q000145:classification / Q000187:drug effects / Q000201:enzymology; D003524:Cyclosporins / Q000494:pharmacology*; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D004338:Drug Combinations; D004987:Ethers / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D053804:Granzymes; D007371:Interferon-gamma / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015759:Ionomycin; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D010641:Phenotype; D011948:Receptors, Antigen, T-Cell / Q000187:drug effects* / Q000502:physiology; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors; D013602:T-Lymphocytes, Cytotoxic / Q000145:classification / Q000187:drug effects* / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D003524:Cyclosporins; D004338:Drug Combinations; D004987:Ethers; D011948:Receptors, Antigen, T-Cell; D015842:Serine Proteinase Inhibitors; D015759:Ionomycin; D007371:Interferon-gamma; D053804:Granzymes; D012697:Serine Endopeptidases; D013755:Tetradecanoylphorbol Acetate