H4/H60-specific T-cell depletion reduces H4/H60-specific immune responses but does not alter total anti-BALB.B responses. Splenocytes from female B6 mice were either depleted with K^b-H4-and K^b-H60 tetramers or mock depleted. Subsequently, 25 × 10⁶ splenocytes plus 5 × 10⁶ T-cell–depleted BM cells were transferred into lethally irradiated male BALB.B recipients (n = 3 in both groups). Blood was sampled at day 6 after HSCT, and 1 day later, spleens and livers were harvested for T-cell isolation. Antigen-specific CD8⁺ T-cell responses were determined ex vivo by intracellular IFNγ staining for each individual mouse upon incubation with 0.1 μg/mL of the indicated peptide for 5 hours. (A) Dot plots show H60-specific CD8⁺ T-cell responses in peripheral blood (PBL), spleen, and liver samples of 1 representative mouse. (B) Graph shows the mean percentage of IFNγ-producing CD8⁺ T cells of 3 mice ± SD. (C-E) Splenocytes from mice analyzed in panels A and B were restimulated in vitro for 14 days with CD8⁺ T-cell–depleted irradiated male BALB.B target splenocytes. The restimulated cells were tested for reactivity toward the various MiHAgs (C), and for anti-BALB.B reactivity (D) by intracellular IFNγ staining. (E) Graph shows the mean percentage of IFNγ-producing CD8⁺ T cells of 3 mice ± SD. Numbers in upper right quadrants reflect the percentage of IFNγ-producing CD8⁺ T cells of total CD8⁺ T cells (panels A, C, and D).