Studies on the induction and expression of T cell-mediated immunity. XII. The concomitant loss and recovery of membrane-associated Lyt-2 antigens, lymphocyte-target cell binding, and the antigen-specific and -nonspecific cytotoxic activity of alloimmune T lymphocytes after treatment with trypsin
Fan, J.; Bonavida, B.
Journal of Immunology 127(5): 1856-1864
1981
ISSN/ISBN: 0022-1767 PMID: 6170675 Document Number: 174301
Treatment of alloimmune cytotoxic T lymphocytes (CTL) obtained from the peritoneal cavity of C57BL/6 mice 1-5 mg/ml trypsin for 30 min at 37.degree. C resulted in the complete removal of cell surface Lyt-2 antigens. Immunofluorescence studies using a fluorescence-activated cell sorter and fluorescent microscope demonstrated that trypsin treatment did not affect the percentage of effector lymphocytes that possessed H-2 and Thy-1 alloantigens and resulted in only the partial removal of Lyt-1 antigens. Treatment with trypsin resulted in 95% depletion of the antigen-specific cytotoxic activity of alloimmune CTL. This was also reflected by the inability of trypsinized CTL to form conjugates with the appropriate target cells. In the presence of 5-10 .mu.g/ml concanavalin A (Con A), trypsinized effector lymphocytes displayed significant levels of antigen-nonspecific cytotoxicity. With increasing amounts of Con A (20-30 .mu.g/ml), the antigen-nonspecific cytotoxic activity was 30-60% of the level manifested by the untreated cell population. Although there were substantial amounts of antigen-nonspecific cytotoxicity, as revealed by the 51Cr-release assay, effector lymphocyte-target cell conjugates were not observed. When the 51Cr-release assay was performed with the effector lymphocytes and target cells suspended in 10% dextran-containing media, thus preventing recycling, antigen-nonspecific cytotoxicity was not observed with trypsinized CTL. Thus, antigen-specific and -nonspecific cytotoxicity were similarly affected by trypsin treatment and the residual antigen-nonspecific cytotoxic activity found upon trypsin treatment of alloimmune CTL may be attributed to the regeneration of Lyt-2 antigens or other requisite cell surface proteins during the course of the 51Cr-release assay. This was substantiated by kinetics experiments that demonstrated that the effects of trypsinization on membrane-associated Lyt-2 antigens, cytotoxic activity and conjugate formation were reversible. Thus, when trypsin-treated CTL were incubated at 37.degree. C, the expression of Lyt-2 antigens was observed in 1.5-2 h. After 3 h incubation at 37.degree. C, 40-50% of the effector lymphocytes expressed Lyt-2 antigens. The cytotoxic activity of trypsinized CTL also increased with time of incubation at 37.degree. C and after 3 h the effector lymphocytes displayed 0-70% of the cytotoxic potential of the untreated population. Like the untreated population, the cytotoxic activity manifested by trypsinized CTL after incubation at 37.degree. C was inhibited by monoclonal Lyt-2 antibodies. The ability of trypsinized CTL to form effector lymphocyte-target cell conjugates also increased with time. As in the case of the recovery of Lyt-2 antigens and cytotoxic activity, there was a lag period of 1.5-2 h before significant amounts of lymphocyte-target cell binding was observed in the presence or absence of Con A. Demonstrations that both the antigen-specific and Con A-dependent, nonspecific cytotoxic activities of CTL are similarly affected by trypsin treatment suggest that these mechanisms share similar molecular interactions leading to lysis. Evidently, Lyt-2 alloantigens, cytotoxic activity and conjugate formation by effector lymphocytes are abrogated with trypsin treatment and these parameters display parallel courses of recovery at 37.degree. C. These findings provide additional evidence that Lyt-2 antigens and/or closely linked and trypsin-sensitive molecules are involved in T cell-mediated cytotoxicity.