This application requests continuing support for examination of suppressor T cell (Ts) growth, differentiation and effector function. In previous studies, a Ts costimulator assay was used to identify a hormone-like lymphokine termed Ts differentiation factor (TsDF) that induces TsF production by primed Ts. A transformed T cell source of TsDF has been identified, its product characterized and its mRNA in vitro translated to a protein product with TsDF function. TsDF interaction with its target suppressor T cell is the consequence of TsDF binding to a transiently expressed alloantigen induced receptor (TsDFR). We now wish to further characterize the TsDF lymphokine, the activation of Ts to a TsDF responsive, TsDFR+ state, the consequences of TsDF-receptor interaction for Ts effector function and finally, the mechanisms whereby suppression is achieved. First, TsDF will be purified by standard biochemical technologies, sequenced and radiolabelled for use in TsDF receptor studies. TsDF cDNA will be identified by hybrid selection, in vitro translation and bioassay from enriched cDNA libraries created from mRNA of the TsDF producing thymoma BW5147. Second, the activation of primed Ts to TsDFR expression and TsDF responsiveness will be characterized. Constitutive and induced TsDFR expression on normal lymphocytes and on transformed cell lines will be established, and TsDFR+ lines will be used for anti-TsDFR antibody production. The roles of second messenger signaling pathways and of H-2 alloantigen recognition and cell subset presentation will be investigated. Regulatory lymphokines and cyclosporin A will be investigated for modulatory effects on TsDFR expression. Third, effects of TsDF interaction with TsDFR for TsF production and for Ts proliferation will be addressed. Second messenger pathways stimulated by TsDF binding will be characterized and related to TsF production and to TsDF enhanced IL2 dependent Ts proliferation. TsDF effect on IL2 receptor display on Ts will be addressed. Finally, mechanisms of TsF mediated suppression of IL2 induced T cell proliferation will be investigated regarding interference with IL2 induced second messengers and initiation of opposing second messenger pathways, effects on IL2 receptor and effects on cell cycle progression. TsF molecules suppressive of IL2 dependent T cell proliferation will be purified by standard biochemical approaches. These studies are anticipated to provide new and productive avenues to suppressor T cell isolation and analysis.
