Naive T cells rapidly produce TNF-α but not IFN-γ after TCR engagement. (A-B) Splenocytes from naive C57BL/6 mice were stimulated for 5 hours with anti-CD3ϵ mAb and stained for cell surface markers and for intracellular TNF-α and IFN-γ, as described in “Materials and methods.” Samples were gated on CD8⁺ (A) or CD4⁺ (B) T cells and examined for expression of TNF-α or IFN-γ and for expression of CD11a and CD44. The values represent the percentage of T cells producing cytokine or the percentage of T cells that were either low or high for CD11a or CD44 that produced TNF-α. The data are representative of 5 experiments. (C-D) Splenocytes from (C) naive KB5 TCR CD8⁺ alloreactive transgenic mice or (D) naive D10 TCR CD4⁺ alloreactive transgenic mice were stimulated for 5 hours with either syngeneic (CBA, H2^k) or allogeneic (C57BL/6, H2^b) splenocytes and stained as described. For analysis, samples were gated on (C) DES⁺CD8⁺ cells or (D) 3D3⁺CD4⁺ cells. The values represent the percentage of TCR transgenic T cells positive for TNF-α or IFN-γ. The data are representative of 3 experiments.