RESTORATION OF LYMPHOCYTE PROLIFERATION AND CTL GENERATION BY MURINE rIL-2 AFTER TREATMENT OF ALLOGENEIC STIMULATOR CELLS BY ULTRAVIOLET B IRRADIATION, HEAT, OR PARAFORMALDEHYDE

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Abstract
Following a 5-day mixed lymphocyte culture (MLC), C3H/HeJ (H-2k) splenocytes stimulated with DBA/2 (H-2d) gamma-irradiated splenocytes (2000 rads) are specifically cytotoxic in a 4-hr 51Cr-release assay to P815 (H-2d) target cells (62±2% cytolysis) but not to third-party EL4 (H-2b). However, when the DBA/2 stimulator cells were treated with heat inactivation (45°C for 1 hr), fixed with 1% paraformaldehyde (15 min), or irradiated with ultraviolet-B light (104 J/M2), no cell proliferation or cytolytic activity developed in the MLCs. The levels of IL-1, IL-2, and IL-6 from the supernatants of MLC using stimulators undergoing either of the three treatments were markedly decreased compared with that from 7-irradiated stimulators. Both cell proliferation and specific cytolysis were restored in a dose-dependent fashion by the addition of murine rIL-2 to the MLCs. If the stimulator cells were first activated with 5 tig/ml pokeweed mitogen or lipopolysaccharide for 2 days, the subsequent treatment with heat, paraformaldehyde, or UV-B did not significantly affect the development of cytolysis (54–70% cytolysis). Suppressor cells were not detected when cells from the nonresponsive MLCs (2.5×108 cells) were added to an MLC freshly prepared with γ-irradiated stimulator cells, or were injected intraperitoneally (50×106 cells) into naive mice 2 days before recovery and in vitro sensitization of splenocytes. Therefore, modification of the stimulating alloantigen can prevent the release of cytokines that function as an essential second signal in the development of the proliferative response and subsequent cytolysis. The cytokine found to be essential for restoration of this response is IL-2.

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