FIGURE 3.

Transgenic expression of Bcl-x_L in PKC-θ^−/− T cells restored cardiac allograft rejection. a, Bcl-x_L^Tg prevented the apoptosis of the PKC-θ^−/− T cells. T cells purified from WT, Bcl-x_L^Tg, PKC-θ^−/−, and PKC-θ^−/− /Bcl-x_L^Tg mice were left in medium (□) or subjected to stimulation with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (2 μg/ml) Abs (CD3/28) for 36 h. Apoptotic cells were then detected by flow cytometric analysis of annexin V-staining cells. Percentage of annexin V-positive cells was calculated. b, T cells obtained from WT, Bcl-x_L^Tg, PKC-θ^−/−, and PKC-θ^−/− /Bcl-x_L^Tg mice were either left alone (□) in medium or stimulated with BALB/c splenocytes (MLR, ■). Proliferation of the activated T cells was monitored by [³H]thymidine incorporation. c, Same treatment as described in b, and the IL-2 concentration in the supernatants was measured by ELISA. d, The same treatment as described in b, and the IFN-γ concentrations in the supernatant were measured by ELISA. e, Rag1^−/− mice reconstituted with PKC-θ^−/− /Bcl-x_L^Tg T cells reject cardiac allografts. T cells purified from WT (n = 6), PKC-θ^−/− (n = 6), and PKC-θ^−/− /Bcl-x_L^Tg (n = 5) mice were adoptively transferred into syngeneic Rag1^−/− mice that received transplanted hearts from BALB/c donors. Survival of the cardiac allografts was assessed over time. f–i, Immunohistochemical analyses of the tissue sections of the cardiac allografts removed from Rag1^−/− mice reconstituted with T cells obtained from WT (Rag1⁻/PKCθ⁺), PKC-θ^−/− (Rag1^-/PKCθ⁺), and PKC-θ^−/− /Bcl-x_L^Tg (Rag1^-/PKCθ^-/Bcl-x_L^Tg) mice. Cryosections were prepared from cardiac allografts removed from recipients. Immunohistochemical analyses of the sections were performed with H&E staining (f), anti-CD4 Ab (g), anti-CD8 Ab (h), and isotype control Ab (i). The sections were examined using regular light microscope (×400) (*, p < 0.05; **, p < 0.001; ***, p < 0.0001).