PRECLINICAL STUDIES USING IMMOBILIZED OKT3 TO ACTIVATE HUMAN T-CELLS FOR ADOPTIVE IMMUNOTHERAPY - OPTIMAL CONDITIONS FOR THE PROLIFERATION AND INDUCTION OF NON-MHC-RESTRICTED CYTOTOXICITY

In order to obtain large numbers of T cells for adoptive immunotherapy after bone marrow transplantation (BMT), we optimized conditions for long-term proliferation of T cells that exhibit non-MHC-restricted cyt otoxicity using immobilized anti-CD3 (OKT3) activation and culture in IL-2. Proliferation and cytotoxicity directed at Daudi, K562, and B ce ll lines were used to determine (1) the optimal concentration of IL-2 and the optimal time of exposure to immobilized OKT3 for maintaining g rowth and cytotoxicity, (2) the starting populations that can be used, (3) the T cell subsets that mediate cytotoxicity, and (4) the optimal medium and concentration of serum for maintaining growth and cytotoxi city. Peripheral blood lymphocytes (PBL) activated with OKT3 would pro liferate and mediate cytotoxicity at IL-2 doses as low as 30 IU/ml. In creasing the IL-2 concentrations beyond 600 IU/ml did not augment the proliferative or cytotoxic responses of PBL. A 24-hr incubation on OKT 3 was sufficient to activate PBL. Increasing the incubation time on OK T3 from 24 to 72 hr did not significantly enhance cytotoxicity. Compar isons between PBL and purified T cells (E-rosette) indicated that eith er cell population could be activated with OKT3 in the presence of IL- 2 to proliferate and mediate non-MHC-restricted cytotoxicity. Purified populations of CD4(+) or CD8(+) T cells demonstrated equivalent proli feration and cytotoxicity when activated using IL-2 and OKT3. With equ al concentrations of human or fetal bovine serum, RPMI 1640 and X-Vivo 10 were comparable for supporting proliferation and cytotoxicity. The se conditions are being used to activate and expand T cells for clinic al trials that involve infusing activated T cells into recipients afte r autologous BMT. (C) 1994 Academic Press, Inc.