No abstract

This report describes a Ts costimulator assay and its use to analyze cofactors required for the expression of suppressor T cell function. Activation of primed MLR-Ts (alloantigen-activated suppressor T cells suppressive of mixed leukocyte reaction) to suppressor T cell factor (TsF) production typically fails in the presence of glutaraldehyde-fixed rather than irradiated allogeneic stimulator cells. However, MLR-TsF production was restored by the addition of 48-h primary MLR supernates; MLR-derived Ts costimulator neither activated primed MLR-Ts in the absence of fixed allogeneic stimulators nor directly suppressed assay MLR. Lack of antigen specificity or genetic restriction and failure to activate unprimed MLR-Ts precursors suggested that Ts costimulator activity differed from previously described Ts inducer functions and was more closely aligned with the lymphocyte- or monocyte-derived interleukins (IL). Three findings distinguished Ts costimulator from IL-2. Depletion of IL-2 activity from MLR supernates by HT2 adsorption failed to affect Ts costimulator function. In addition, MLR supernates prepared in the presence of cyclosporin A contained no IL-2 but expressed Ts costimulator activity. Finally, gel chromatography demonstrated Ts costimulator in peaks of 21,000 and 43,000 mol wt that were largely distinct from the IL-2-containing fractions. Ts costimulator activity was also identified in phorbol myristate acetate (PMA)-induced EL4 supernates and was retained in those supernates after IL-2 depletion by HT2 adsorption. In preliminary functional characterization, MLR supernate-derived Ts costimulator triggered MLR-TsF production from irradiated MLR-Ts in the absence of proliferation. Thus a differentiative rather than proliferative stimulus required for primed MLR-Ts function appears to be provided by this Ts costimulator and has been provisionally termed Ts differentiative factor ( TsDF ). This initial characterization may thus identify one of a possibly distinctive family of interleukins required in the alloantigen-driven activation of suppressor T cells to effector function.

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.