Functional consequences of aberrant IL-21R signaling in B and T cells from P2 of family A. (A) Representative FACS analysis showing proliferation of IL-21R–deficient naive B cells that were co-cultured with 3T3 CD40L-expressing cells and stimulated with anti-IgM beads (50 ng/ml), CpG (50 nM), and rh IL-2 (20 U/ml) in the presence or absence of rh IL-21 (30 ng/ml) for 6 d. Histograms illustrate proliferation of CFSE-labeled B cells isolated from healthy donors (HD) and patient (P2, A.II-5). Gray shaded, unstimulated B cells; gray line, CD40L-stimulated B cells; red line, CD40L-/IL-21–stimulated B cells. (B) FACS analysis of immunoglobulin class-switch in naive IL-21R–deficient B cells upon stimulation with rh IL-21 for 6 d. (C) Expression of AICDA and PRDM1 mRNA was determined in CD40L-/IL-21–stimulated B cells after 6 d using Real-time PCR analysis. Values represent the expression of respective genes in relation to RPS9 (ribosomal protein S9). (D) Analysis of NK cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) using ⁵¹Cr-release assay. (E) T cell proliferative response to stimulation with antigens was determined by [³H]-thymidine incorporation assay. PBMCs isolated from healthy donors (n = 4), father, and P2 were stimulated with C. albicans (Cand), DTT, and TT. Depicted data represent average of duplicates and are representative of two independent experiments. (F) Secretion of multiple cytokines in IL-21R–deficient T cells measured by protein array. Data shown are compiled from two independent experiments. The bars render logarithms (to the base of 2) of fold changes between unstimulated cells and cells upon stimulation with anti-CD3/anti-CD28 for 18 h. For each bar, the 95% confidence interval is given. Asterisks indicate cytokines for which the 95% (*), 99% (**), and 99.9% (***) confidence intervals do not overlap.