A sensitive micromethod for generating and assaying allogeneically induced cytotoxic human lymphocytes

A sensitive micromethod for generating and assaying allogeneically induced cytotoxic human lymphocytes in vitro is described. Responding lymphocytes are cultured with mitomycin-C treated allogeneic stimulating cells in wells of replicate microtrays for both one-way mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML) assays. On day 5, MLC response is determined by measuring 3H-thymidine (3H-TdR) incorporation directly in wells of the MLC tray. On day 6 or 7 CML response is determined by measuring 51Cr released from labeled target cells added to replicate culture cells in the CML tray. It is thus possible to measure both MLC and CML responses of the 2.5 X 10(4) -U X 10(5) responding lymphocytes originally placed in replicate wells. 51Cr-labeled target cells can be added to wells containing dilutions of the stimulated cells and a log-linear relationship between the per cent specific 51Cr release and number of effector cells is observed. Significant levels of specific cytoxicity are detected at ratios as low as one effector cell per target cell; little cross-killing on third-party cells and no autokilling is observed. Lymphocytes purified from whole blood that is stored overnight at room temperature and purified lymphocytes stored overnight in the cold generate MLC and CML responses comparable to those of lymphocytes purified from fresh blood. Only 2 or 3 ml of whole blood are required to perform both MLC and CML assays, thus enabling the study of both proliferative and cytotoxic lymphocyte responses in young children and other individuals from whom only a few milliliters of blood can be obtained.