In the absence of exogeneous stimulation, the murine macrophage cell line, P388D1, produced low but significant amounts of lymphocyte activating factor (LAF). When P388D1 cells were incubated with PHA-activated DBA/2 mouse or Hartley guinea pig T cells, LAF production was greatly enhanced. LAF activity was not detected in cultures of only PHA-activated T cells, or in cultures of T cells incubated with P388 cells, the parent nonmacrophage lymphoma from which the P388D1 cell line was obtained. P3888D1-derived LAF was also shown to be biologically distinct from the guinea pig lymphocyte mitogenic factor that, unlike LAF, is produced by PHA-activated T cells. These results, therefore, suggest that P388D1 cells are the cell source for most, if not all, the LAF produced in mixed cultures of P388D1 cells and T cells.

The stimulatory effect of activated T cells on LAF production was dependent on cell contact, since LAF production was not enhanced when P388D1 cells and T cells were separated by a semipermeable Nuclepore membrane in a Marbrook culture vessel. T cells did not have to proliferate in order to stimulate P388D1-LAF production, but T cell viability was essential. In addition, the level of LAF activity was dependent on at least three culture variables: the time of incubation of P388D1 cells and T lymphocytes, the ratio of P388D1 cells to T lymphocytes, and the fetal calf serum concentration.

Based on the results of these studies, it appears that P388D1 cells are an excellent source of LAF for both biologic and biochemical studies.

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