FIG. 4.

Cbp deficiency does not control T-cell sensitivity to TCR-mediated signals in vitro. (A) Cbp deficiency does not alter TCR-induced calcium flux. Calcium flux of CD4 SP, CD8 SP, and DP thymocytes and of splenic CD4 T cells is depicted. Wild-type (gray area) or Cbp-deficient (black line) cells were stimulated with titrated amounts of anti-CD3 antibody (in micrograms), and an overlay of the histograms of their calcium fluxes is shown. Arrowheads indicate the addition of anti-CD3 and cross-linking antibodies. (B) Unaltered regulation of activation markers in the absence of Cbp. CD4 T cells isolated from spleen and lymph nodes were incubated for 12 h either in medium alone or with plate-bound anti-CD3 (black lines) and analyzed by flow cytometry for the expression of CD25, CD44, CD62L, and CD69. Gray areas represent uninduced controls, and black lines represent stimulated cells. (C) Unaltered TCR-induced proliferation in Cbp^−/− T cells. CD4 T cells isolated from spleen and lymph nodes were incubated for 3 days either in medium alone or with plate-bound anti-CD3 (in micrograms) with or without plate-bound anti-CD28 (in micrograms). Proliferation was assessed by carboxyfluorescein succinimidyl ester (CFSE) dilution and analyzed by flow cytometry. Gray areas represent uninduced controls, and black lines represent stimulated cells. Stimulation with phorbol myristate acetate (P+I; 2.5 ng/ml) and the ionophore A23187 (100 ng/ml) was used to control for the proliferative potentials of wild-type and Cbp^−/− cells. (D) Unaltered kinetics of TCR-induced proliferation in Cbp^−/− T cells. CD4 T cells were purified from spleen and lymph nodes from two mice per genotype, wild type (open symbols) and Cbp^−/− (closed symbols). The cells were incubated for 12 h, 24 h, or 36 h on titrated amounts of anti-CD3 antibody, transferred into uncoated 96-well plates, and further incubated for a total of 72 h. Proliferation was scored by [³H]thymidine incorporation for the last 8 h of the culture. (E) Normal Th1/Th2 differentiation in the absence of Cbp. CD4 T cells were differentiated in vitro under Th1-polarizing and Th2-polarizing conditions. Numbers show percentages of live gated cells.