Figure 3.

TCRxgag mice do not proliferate or produce IL-2 but retain IFN-γ and TNF-α production after antigen stimulation. (A) To measure antigen-specific proliferation, 0.1 × 10⁶ CD4-depleted splenocytes from TCR-TG plus 0.9 × 10⁶ B6 splenocytes and 10⁶ splenocytes from TCRxgag mice were stimulated with 2 × 10⁴ irradiated FBL for 3 d, and then pulsed with ³[H]-thymidine for 8 h. For analysis of IL-2 production (B), 0.1 × 10⁶ CD4-depleted splenocytes from TCR-TG plus 0.9 × 10⁶ B6 and 10⁶ CD4-depleted TCRxgag were stimulated with 2 × 10⁴ irradiated FBL in a total volume of 200 μl in a 96-well round bottomed plate. After 72 h, 50 μl of culture supernatant was harvested and murine IL-2 measured by ELISA. (C) The antigen-specific response of TCRxgag in the presence of IL-2 was measured as in A, but with indicated amounts of IL-2 added to the wells. Intracellular staining for IFN-γ and TNF-α production (D) was performed after stimulation of TCR-TG or TCRxgag splenocytes with irradiated FBL. After 48 h, secretion of protein was prevented by addition of Golgi-Block reagent for 6 h and cells permeabilized by Cytofix/Cytoperm. The cells were stained with anti-CD8 and anti–IFN-γ or anti–TNF-α. Plots were gated on CD8⁺ cells.