CD4 help is required for the appropriate development of a secondary H60-specific CD8 T-cell response. (A) Mice that had been previously immunized with male H60 cells were boosted with male or female H60 cells. PBLs from the twice-challenged mice (6 mice per group) were periodically checked for the binding of H60-tetramer, and the proportion of H60-tetramer⁺ CD11a⁺ cells among the CD8⁺ cells was plotted. Representative data from more than 10 experiments conducted on day 7 after secondary immunization are shown (gated for CD8⁺ cells). (B,C) CD8 effector cells from MLCs of splenocytes from the boosted mice on day 7 after secondary immunization were tested for specific cytotoxicity (B) and IFN-γ production (C) after stimulation with H60-peptide and VSV (control)–peptide. (D) The mice that had been previously immunized with male H60 cells were depleted of CD4 T cells by treatment with GK1.5 before boosting with male H60 cells. The FACS data showing the peak days for the primary (day 10 after primary immunization) and secondary (day 7 after secondary immunization) responses are compared. FACS data obtained from nondepleted mice (no treat) are shown as a control. The FACS data were gated for the CD8 T cells and are representative of 3 independent experiments.