S typhimurium–induced NY-ESO-1–specific CD4⁺ Th1 cells are able to recognize naturally processed NY-ESO-1 protein. (A) Whole CD4⁺ T cells or CD4⁺CD25⁻ T cells of NC235 or NW681 were isolated from PBMCs and cultured with APCs infected with S typhimurium–NY-ESO-1 or pulsed with indicated NY-ESO-1 peptide, respectively. At 15 to 20 days later, the capacity of elicited NY-ESO-1–specific Th1 cells to recognize naturally processed NY-ESO-1 protein was analyzed by ELISPOT assay using NY-ESO-1 (NC235, NY-ESO-1 157–170; NW681, NY-ESO-1 143–154) or control HIV peptide-pulsed or NY-ESO-1 or SSX-2 protein-pulsed DCs as APCs. (B) Avidity of induced NY-ESO-1–specific Th1 cells was analyzed by ELISPOT assay using T-APCs pulsed with serial dilutions of peptides. These experiments were performed independently at least twice with similar results. Data are expressed as means plus or minus SD.