T-cell activation in PDE4 LMWI–treated wild-type and PKCθ^–/– T cells. (A) Titration curve of the PDE4 LMWI with normalized percent of CD3/CD28-induced IL-2 production of CD3⁺ wild-type and PKCθ-deficient T cells (DMSO control was set as 100% in each genotype). (B-C) Proliferative response and (D-E) IL-2 production of purified mature CD3⁺ T cells in the presence or absence of PDE4 LMWI inhibitor as specified. Cells were left unstimulated or were stimulated with anti-CD3 (precoated at a concentration of 10 μg mL^–1) plus soluble anti-CD28 (1 μg mL^–1) or PDBu/ionomycin, as indicated, and analysis was done by using standard procedures. Results shown are the mean ± SD of at least 3 independent experiments. (C, inset) Western blot of CD3⁺ T-cell lysates immunostained for the endogenous PKCθ isotype and fyn, as indicated. Note the complete breakdown of IL-2 production in the PKCθ^–/– T cells when stimulated with anti-CD3 plus anti-CD28. The suboptimal low concentration of 0.4 nM PDE4 LMWI reduces neither the proliferation nor the IL-2 production in the wild-type significantly, but does so in the PKCθ^–/– T cells (P = .008 in the proliferation assay; P = .002 in the IL-2 assay; t test).