Rapid production of TNF-α by naive alloreactive CD8⁺ T cells in C57BL/6 mice. Splenocytes from naive C57BL/6 mice were stimulated with syngeneic (C57BL/6, H2^b) or allogeneic (BALB/c, H2^d or CBA, H2^k) splenocytes, as described in “Materials and methods.” Splenocytes were then stained for cell surface markers and for intracellular (A) TNF-α or (B) IFN-γ. For analysis, samples were gated on CD8⁺ cells, and the values shown represent the percentage of CD8⁺ T cells staining positive for either cytokine. The total number of splenic alloreactive CD8⁺ T cells producing TNF-α in response to the indicated allo-stimulation is shown in panel C. The data are representative of 5 experiments. Error bars indicate SD.