Hierarchical costimulator thresholds for distinct immune responses: Application of a navel two-step Fc fusion protein transfer method

Activation of T cells is dependent upon coordinate engagement of Ag and cos timulator receptors on their surfaces. In the case of the Ag receptors (TCR s), activation thresholds have been defined, with the number of TCRs that m ust be triggered to stimulate cytokine secretion by individual activated T cells differing for the various cytokines. In the present study, we have de termined whether comparable activation thresholds exist for the costimulato r receptors on T cells. To facilitate this type of quantitative costimulato r analysis, we developed a novel two-step protein transfer approach that pe rmits delivery of graded amounts of proteins to APC surfaces. By adding a h uman B7-1.Fc gamma(1) (Fc domain of human IgG1) fusion protein to cells pre coated with palmitated protein A, fine titration of the B7-1 extracellular domain was achieved. The B7-1.Fc gamma 1, reincorporated into cell membrane s by this method retained costimulator function, as measured by an in vitro proliferation assay. The degree of proliferation was dependent on the surf ace density of B7-1.Fc gamma 1. Significantly, the threshold B7-1.Fc gamma( 1) density required for cytokine production differed between IFN-gamma and IL-2 and mirrored the hierarchy (IFN-gamma < IL-2) described previously for the TCR activation threshold. Hence, this: study invokes a novel protein t ransfer strategy to establish that the levels of surface costimulator on AP Cs can dictate both the magnitude and the quality of evoked T cell, respons es. The notion of costimulator receptor activation thresholds emerges.