[Cultivation of human tumor infiltrating lymphocytes for use in adoptive immunotherapy]

We have developed a method of in vitro stimulation of TIL and PBL using immobilized anti CD3 antibody instead of autologous tumors. After this treatment, proliferation of T cells was greatly enhanced. Separated TILs were cultured by OKT3 coated 96 well culture plate for about one week with 10% human serum and 1,000U/ml of rIL-2 supplemented RPMI-1640 medium, and then transferred to plain vessels. TIL increased in number by more than 5,000-fold after 3 weeks. PBLs obtained from 10 ml of peripheral blood were cultured in OKT3-coated culture flask (75 cm2) with 10 ml of medium for about one week, and then in plain flasks and finally expanded by culture in gas-permeable culture bags. PBL proliferated by more than 1,000-fold after 2 weeks. TIL or PBL cultured by this method contained over 90% activated T cells positive for the Leu4 marker, and HLA-DR. About 60% of these were CD8-positive and about 40% were CD4-positive. Only a small number of Leu 11-positive cells were detected in every case.