Limiting dilution analysis (LDA) of cells responding to recombinant interleukin-2 without previous stimulation: evidence that all responding cells are lymphokine-activated potent effectors.

The relationship between peripheral blood mononucleated cells spontaneously bearing the IL-2 receptor (IL-2R) and cell cytotoxicity for the natural killer (NK)-sensitive K562 target cell line was investigated. For this purpose, three types of experiments were performed. (i) Positive selection of cells spontaneously bearing the IL-2R was carried out by culturing peripheral blood lymphocytes (PBL) in the sole presence of recombinant IL-2 (rIL-2). Cytotoxicity was assessed at Day 6 of the culture in a 4 hr cytotoxic assay. (ii) Negative selection was performed by complement mediated lysis using the B1.49.9 monoclonal antibody which is specific for the IL-2R. (iii) Limiting dilution analysis of non-adherent PBL was carried out in the presence of rIL-2 alone. The colonies obtained were divided and daughter colonies assayed for anti-K562 cytotoxicity in a 6 hr cytotoxic assay and for proliferation. The results show that: (i) a 6-day culture of human non-adherent PBL in the presence of rIL-2 alone leads to a sharp increase in anti-K562 cytotoxicity; (ii) depletion of B1.49.9 positive PBL strongly decreases cytotoxicity against K562 targets; (iii) limiting dilution analysis indicates that all colonies grown without activation in the presence of autologous serum and rIL-2 can mediate cytotoxicity against K562 targets, which is not the case when the starting population is activated. Thus, our data taken together strongly suggest that lymphocytes spontaneously bearing the IL-2R are directly involved in K562 lysis by fresh PBL (classical NK activity). Moreover, we demonstrate that all colonies able to proliferate without any activation, in the sole presence of rIL-2, are potent K562 killers (in this case, these cells correspond to the so-called lymphokine activated killers, LAK).