NY-ESO-1–specific CD4⁺ T cells are elicited by S typhimurium–NY-ESO-1 without the need for CD4⁺CD25⁺ T-cell depletion. Presence of NY-ESO-1 peptide-specific CD4⁺ Th1 cells in whole CD4⁺ T cells and CD4⁺CD25⁻ T cells was analyzed by ELISPOT assays following a 15- to 20-day culture with APCs pulsed with indicated NY-ESO-1 peptides (except NC155; NY-ESO-1 157-170) or infected with S typhimurium–NY-ESO-1 (S typh–NY-ESO-1) or S typhimurium control strain (S typh–control strain). Responses were analyzed by specific IFN-γ secretion for recognition of autologous T-APCs pulsed with indicated peptide (except NC155; NY-ESO-1 157-170 [▩], or HIV peptide [□]), in 4 healthy donors and in 4 patients with NY-ESO-1–expressing tumors but without NY-ESO-1 antibody. These experiments were performed independently at least twice with similar results. Data are expressed as means plus or minus SD.