Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes. © 1975.

The in vitro destruction of phytohemagglutinin (PHA) coated Beta L cells by non-immune human lymphocytes was resolved into two distinct phases--lymphocyte dependent and lymphocyte independent. The initial or lymphocyte-dependent phase occurred within the first 2 hr and proceeded equally well at 34 and 37 degrees C. The amount of lymphotoxin (LT) secreted by PHA-activated human lymphocytes in vitro to PHA stimulation was the same at 34 and 37 degrees C. Antiserum and complement inactivation of the aggressor lymphocytes at various intervals revealed that target cell lysis was lymphocyte independent. However, the latter phase was temperature dependent, i.e., proceeding at the permissive temperature of 37 degrees C, but inhibited at the restrictive temperature of 34 degrees C. Further experiments revealed that LT-induced destruction had the same temperature sensitivity as target cell cytolysis occurring during the lymphocyte-independent step. Trypsin treatment of target cells during an early period of the lymphocyte-independent phase protected the target cell from subsequent death, indicating the aggressor lymphocyte has deposited a cytotoxic effector material on its surface. These results suggest the lymphocyte-dependent stage involves the processes required for the induction of LT synthesis and secretion. The actual cytolysis occurring during the lymphocyte-independent stage may be caused by LT or LT-like material(s) deposited on the target cell surface by the mitogen-activated human lymphocyte.