AbstractIFN-γ exerts multiple biological activities in the modulation of immune responses by the induction of transcription factors. One transcriptional factor of the IFN regulatory factor family found to be critical in regulating IL-12-dependent IFN-γ production in vivo following infectious challenge has been designated IFN consensus sequence-binding protein (ICSBP). In this study, the role of ICSBP in regulating type 1 responses to T cell-specific stimulation in vitro was assessed. Total splenocytes from ICSBP−/− mice stimulated with soluble anti-CD3 were markedly impaired in the production of IFN-γ compared with similarly stimulated cells from ICSBP+/+ mice. Consistent with the decrease in IFN-γ production, splenocytes from ICSBP−/− mice stimulated with anti-CD3 in the presence or absence of IFN-γ or a soluble CD40 ligand agonist failed to produce IL-12 p40 and IL-12 p70 protein; however, the deficient production of IFN-γ from ICSBP−/− mice could be restored by the addition of anti-CD28 Ab in an IL-12-independent manner. In contrast to the previous data, production of IFN-γ from naive CD4+/LECAM-1high cells of ICSBP−/− mice that had been primed in vitro with anti-CD3 was similar to or greater than that of ICSBP+/+ controls. In addition, the presence of IFN-γ in priming cultures enhanced both priming for IFN-γ and IL-12 responsiveness from ICSBP−/− CD4+ T cells. Overall, these results provide evidence that ICSBP is differentially required for the ability of IFN-γ to regulate type 1 cytokine responses from APCs and CD4+ T cells.