In vitro studies on the release of mitogenic factor by sensitized human lymphocytes

Abstract

In order to demonstrate mitogenic activity released by human lymphocytes, supernatants from “primary cultures” (sensitized cells together with antigen) were transferred to “secondary cultures” (autologous or homologous sensitized or non‐sensitized lymphocytes). It could be shown that sensitized human lymphocytes excrete a soluble mitogenic factor after exposure to antigen which induces DNA synthesis in secondary cultures. The production of this mitogenic factor was immunologically specific. Factor production could also be demonstrated in an autologous system; therefore the effect of mitogenic factor is independent of histocompatibility antigens. Maximal production of mitogenic activity was obtained after 24 h of primary culture. The amount of activity produced was dependent on the dose of antigen added. An effect of mitogenic factor on secondary cultures was already visible after 48–60 h; the maximal response, however, was seen after 120–136 h of incubation. The kinetics of DNA synthesis obtained were comparable to kinetics induced by specific antigen in short term lymphocyte cultures. DNA synthesis in secondary cultures was dependent on the amount of supernatant added. Since the addition of further antigen to different volumes of supernatants did not lead to an enhanced factor activity, the operation of mitogenic factor was not dependent on the amount of antigen present. Supernatants obtained after 3 days of primary culture, tested on autologous lymphocytes, exhibited a lower response in most instances when compared with the reconstituted controls. This effect may be due to an inhibitor produced by the cells.

Mitogenic Factor and Migration Inhibitory Factor in Supernatants of Serum-Free Human Lymphocyte Cultures Stimulated with Concanavalin A

“…The method for isolation and cultivation of human peri pheral blood lymphocytes has been described previously [Havemann and Bürger, 1971]. 15X10° lymphocytes (purity > 95°/o) were preincubatcd with Con A in amounts ranging from 5 to 1,000 «g per culture containing 6 ml of serum-free medium T C 199 (Difco Laboratories) and 100 U penicillin/ml and 100 ag of streptomycin/ml.…”