Naive, effector, and nonresponsive H2^d-specific CD8⁺ T cells distinguished by differential expression of cytokines and activation markers. Splenocytes from naive C57BL/6 mice, from C57BL/6 mice that were injected with BALB/c splenocytes (BALB/c DST) 7 days previously, or from C57BL/6 mice that were tolerized with anti-CD154 and BALB/c DST were stimulated in vitro with either BALB/c (H2^d) splenocytes (A) or CBA (H2^k) splenocytes (B), as described in “Materials and methods.” Samples were then stained for cell surface markers and for intracellular cytokines. Samples were gated on CD8⁺ cells, and the values shown represent the percentage of CD8⁺ T cells staining positive for TNF-α. (C) H2^d-specific CD8⁺ T cells producing TNF-α were evaluated for IFN-γ production and the values represent the percentage of TNF-α–producing CD8⁺ T cells that were positive for IFN-γ. The total number of CD8⁺ T cells producing TNF-α following stimulation with BALB/c (H2^d) splenocytes is shown in panel D (n = 4). One-way ANOVA was used to compare naive C57BL/6 mice with mice treated with DST or with mice treated with DST and MR1 (*P < .05). The data are representative of 3 experiments. Error bars indicate SD.