Patient T cells expressing mutant CARD11 are hyporesponsive to polyclonal stimulation. (A) Expression of CARD11 and p65 visualized by immunofluorescence and confocal microscopy in fixed resting T cells, quantified as described for Fig. 4 A. Insets in bottom row show individual cells from the same field. Bars: (top) 1 µm; (bottom) 10 µm. (B) Representative immunoblots of total T cell lysates prepared from normal donor controls (C1, C2, and C3) and patients for the indicated proteins. A separate matched control (C3) was compared with P4 for subsequent experiments. (C) Representative flow cytometric analysis of CD69 and CD25 measured ± stimulation with either soluble anti-CD3ε + anti-CD28 mAbs (blue lines) or bead-coupled anti-CD2/anti-CD3/anti-CD28 mAbs (red lines). MFI values are plotted at right in the corresponding color. (D) Calcium flux in purified resting T cells from controls (C1 and C2) and patients (P3 and P4) as measured by flow cytometry after stimulation with anti-CD3/anti-CD28 + 1 µg/ml protein A. Addition of ionomycin served as a positive control (bottom). (E) Western blot analysis of ERK and JNK phosphorylation relative to total ERK/JNK levels, after anti-CD3/anti-CD28 Ab stimulation of T cells cycling in IL-2. (F) Proliferation of resting T cells purified from patients and controls was measured by [³H]thymidine incorporation in counts per minute to stimuli described in C. Patient responses to soluble Ab stimulation were significantly lower than controls (P = 3.97 × 10⁻⁴). (G) IL-2 accumulation measured by ELISA in cell supernatants 4 d after stimulation as described in C. (H) Proliferation assay for purified control and P3 T cells stimulated with anti-CD3/CD28 Abs ± 100 U/ml of exogenous recombinant IL-2. (A and D–G) Data are representative of two (A, D, and E), three (F and G), or four (C) independent experiments. (A and F–H) Data show mean ± standard deviation of triplicate wells.