Cryopreservation of human peripheral blood lymphoid cells reactive to allogeneic antigens, virus-transformed autologous cells and autologous leukemia cells in in vitro sensitization.

Abstract

We asked if cryopreservation of human peripheral blood lymphoid cells (PBL) could maintain their function to generate cytotoxic activity against various antigens in vitro. Cells to be frozen were suspended in RPMI 1640 medium supplemented with 10% dimetylsulfoxide and 10% fetal calf serum at a concentration of I X 10' cells per mi. One and a halfml of this cell solution per polystyrene vial were frozen in an automated programmed freezer at a cooling rate of -1 0 C per minute. When used for assays, cells were recovered by rapidly thawing the cell solution in the vials. The ability of thus cryopreserved PBL to generate lymphocyte cytotoxicity was compared to that of fresh PBL from the same individuals. The micromethod of in vitro sensitization was used to generate and measure lymphocyte cytotoxicity in a single 96-well plate. Reactivities to alloantigens were examined in the PBL from 6 individuals and reactivities to Epstein-Barr (E-B) virus-transformed autologous lymphocytes were tested in 4 independent experiments using the PBL from two individuals. In addition, PBL reactivity to autologous leukemia cells was more precisely analysed in one patient with acute myelogenous leukemia. The cytotoxic activities of human PBL directed against alloantigens, E-B virus-transformed autologous lymphocytes, and autologous leukemia cells were satisfactorily recovered after cryopreservation. This encourages the idea to apply this procedure in further analysis of cell-mediated immune reactions in various disease conditions. It may also raise the possibility of producing immune lymphocytes cytotoxic to autologous leukemia cells that could be therapeutically usable for leukemia patients.