Primary H60-specific CD8 T cell response in the presence or absence of CD4 help. (A) Female B6 mice were immunized with male or female H60 cells and pooled PBLs from the immunized mice (12 mice per group) were stained with H60-tetramer-PE, anti–CD8-APC, and anti–CD11a-FITC. The proportion of H60-tetramer⁺ CD11a⁺ cells among the CD8⁺ cells is plotted. Representative flow cytometric data (gated for CD8⁺ cells) showing the peak primary response (day 10 after immunization) from more than 10 independent experiments are shown. (B) Splenocytes from mice immunized with male or female H60 cells were prepared on day 10 after immunization and tested ex vivo for IFN-γ production after stimulation with H60-peptide and VSV-peptide (control peptide). The cells were surface-stained with anti–CD8-FITC and anti–CD4-PE, and intracellular staining was performed with APC-conjugated anti–IFN-γ mAb. Representative flow cytometric data from 3 independent experiments are shown. The values in the upper right quadrants indicate IFN-γ–producing cells as a percentage of CD8 T cells. (C) Splenocytes from mice immunized with male or female H60 cells were cultured with irradiated male H60 splenocytes (mixed lymphocyte culture, MLC) for 7 days, and the CD8 effector T cells from each MLC were tested for IFN-γ. The cells were surface stained with H60-tetramer and anti–CD8-APC, and were stained for intracellular IFN-γ with FITC-conjugated mAb. Representative flow cytometric data from 3 independent experiments are shown (gated for CD8⁺ cells).