S typhimurium–NY-ESO-1 activates high-avidity NY-ESO-1–specific CD4⁺ T-cell precursors from a naive T-cell population (A) CD4⁺CD25⁻ T cells isolated from PBMCs of NC235 or NW681 were further separated into CD45RA⁺ or CD45RO⁺ cells using magnetic beads as described in “Methods.” These T cells were cultured with APCs pulsed with a NY-ESO-1 peptide or infected with S typhimurium–NY-ESO-1. At 15 to 20 days later, specific T-cell elicitation and 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.