rAd35 and CD46 ligation inhibit CD3/CD28-induced IL-2 in CD4+ T cells. (A–F) Sorted total or naive CD4+ T cells were activated with CD3/CD28 with or without immobilized rAd5, rAd35 (100 ip/cell), anti-CD46 mAb, or anti-TfR mAb for 5 h. Intracellular IL-2 and IFN-γ production was measured by flow cytometry. Flow plots depict one representative donor of five for sorted total (A) or naive (B) CD4+ T cells. Graphs summarize the mean from five donors for frequencies of single-positive IL-2–, double-positive IL-2/IFN-γ–, and IFN-γ–producing total (C) and naive (D) CD4+ T cells. Wilcoxon signed-rank test: *P = 0.031. (E) Flow cytometry plots show naive CD4+ T cells stimulated with anti-CD3/CD28 mAb with or without rAd5 (100 ip/cell), rAd35 (10 ip/cell), rAd35.5knob (10 ip/cell), rAd35.5fiber (100 ip/cell), or anti-CD46 mAb. (F) Proliferation of anti-CD3/CD28–activated naive CD4+ T cells was measured on day 5 with 3H-thymidine. Cells were stimulated with or without rAd (200 ip/cell) or anti-CD46 mAb in the presence of increasing concentrations of exogenous IL-2. The graph is a summary (mean ± SEM) of data from two donors.