IL-6 and GITR signal is essential for elicitation of NY-ESO-1–specific CD4⁺ Th1 cells. (A) Whole CD4⁺ T cells were isolated from PBMCs of NC235 or NW681 and cultured with APCs infected with S typhimurium–NY-ESO-1 or S typhimurium control strain without or with 10 μg/mL of indicated antibodies. At 15 to 20 days later, responses were analyzed by specific IFN-γ secretion for recognition of T-APCs pulsed with NY-ESO-1 (NC235, NY-ESO-1 157-170; NW681, NY-ESO-1 143-154) or control HIV peptide. (B) Whole CD4⁺ T cells were isolated from PBMCs of NC235 or NW681 and cultured with APCs infected with S typhimurium–NY-ESO-1 or pulsed with indicated NY-ESO-1 peptides with as well as recombinant IL-6 (10 ng/mL), GITRL-Fc (5 μg/mL), or both. At 15 to 20 days later, responses were analyzed by specific IFN-γ secretion for recognition of T-APCs pulsed with NY-ESO-1 (NC235, NY-ESO-1 157-170; NW681, NY-ESO-1 143-154) or control HIV peptide. (C) APCs used for presensitization were prepared from PBMCs of NC235 or NW681 and infected with S typhimurium–NY-ESO-1. The kinetics of GITRL expression after S typhimurium infection was analyzed. These experiments were performed independently at least twice with similar results. Data in panels A and B are expressed as means plus or minus SD. *P < .05; **P < .01.