Effective supernatants (SUP), which potentiate mouse T-cell responses to phytohemagglutin (PHA), are obtained from cells of several species (human, rabbit, rat, mouse) and indeed from syngeneic spleen, thymus, or bone marrow cells. Unstimulated cells release some SUP activity but more is produced after stimulation. Lipopolysaccharide (LPS) produced very active SUP in all cultures tested. PHA was similarly active on human leukocytes only, whereas concanavalin A (Con A) gave highly efficient SUP only with mouse spleen cells. SUP production is not correlated with a mitotic response of the donor cells and is observed in cultures unable to respond mitotically to the stimulant. Adherent mouse spleen cell populations, consisting largely or entirely of macrophages, produce active SUP, while nonadherent cells do not. Similarly, purification of human peripheral leukocytes on nylon columns, with removal of macrophages and other adherent cells, destroys their ability to produce SUP. The importance of indirect effects in stimulating mitotic responses of T cells is emphasized by the fact that the mitotic response of mouse thymocytes to LPS and its ability to potentiate the response of these cells to PHA disappears with removal of adherent cells from the thymocyte population. Conversely the production of SUP from spleen cells stimulated by Con A requires the presence of T cells.

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