rAd35 vectors infect and activate DCs. (A) MDCs, PDCs, or MDDCs were exposed to rAd5-GFP (●) or rAd35-GFP (■) at doses from 0.1 to 1,000 ip/cell for 24 h, at which time the frequency of GFP⁺ cells was measured by flow cytometry. Graphs depict mean ± SD for rates of infection assessed on DCs from at least six donors. (B) MDCs or MDDCs were exposed to rAd5 (100 ip/cell), rAd35 (10 ip/cell), or TLR ligands for 24 h. CD86 expression of all DCs was measured by flow cytometry. Histograms depict mock (gray-filled) or stimulated (black line) DCs for one representative donor of three independent experiments.

rAd35-exposed DCs or monocytes suppress allogeneic naive CD4⁺ T-cell proliferation. (A) Purity of naive CD4⁺ T cells was assessed by flow cytometry from a representative donor. Immature MDCs (B), PDCs (C), MDDCs (D), or monocytes (E) were exposed to rAd5 or rAd35 for 24 h and then transferred to sorted allogeneic naive CD4⁺ T cells at a ratio of 1 to 10. Proliferation was measured on day 5 by ³H-thymidine incorporation. Graphs depict mean ± SD for MDCs (n = 4), PDCs (n = 3), MDDCs (n = 16), and monocytes (n = 4). Wilcoxon signed-rank test: *P < 0.05; **P < 0.005; ***P < 0.0005. (F) DCs or monocytes were exposed to either rAd5 (100 ip/cell) or rAd35 (10 ip/cell) for 24 h and then transferred to sorted allogeneic CFSE-labeled naive CD4⁺ T cells. CFSE dilution was evaluated on day 5 by flow cytometry. Flow plots are from one representative donor of three independent experiments. (G) Sorted donor-matched naive and memory CD4⁺ T-cell proliferation induced by mock- or rAd-infected MDDCs. Proliferation was measured on day 5 with ³H-thymidine. Graphs depict mean ± SD (n = 4). Paired t test: *P < 0.05, **P < 0.005. (H) MDDCs were exposed to either rAd5 or rAd35 for 24 h, and the supernatants were harvested and transferred to untreated DCs cocultured with sorted allogeneic CD4⁺ T cells. Proliferation was measured on day 5 with ³H-thymidine. One representative donor is shown.

rAd35 binding leads to lower CD46 expression on naive CD4⁺ T cells. Naive CD4⁺ T cells were mock-treated or exposed to immobilized rAd5 or rAd35 (100 ip/cell), anti-CD46 mAb, or anti-TfR mAb. Surface CD46 expression on live cells (Annexin-V⁻/7-AAD⁻) was evaluated by flow cytometry after 1–5 d as indicated. Histograms depict CD46 expression on mock and treated naive CD4⁺ T cells of one representative donor from three independent experiments. Graphs are a summary (mean ± SD) of CD46 and CD3 mean fluorescence intensity data of three donors. Paired t test: **P < 0.01.

rAd35 and CD46 engagement blocks proliferation of CD3/CD28-activated naive CD4⁺ T cells. Naive CD4⁺ T cells were activated with immobilized anti-CD3/CD28 mAb with or without immobilized (A) or soluble (B) rAd5, rAd35 (200 ip/cell), or anti-CD46 mAb. A chimeric rAd35 vector with rAd5 knobs (rAd35.5knob) was also used. Graphs are a summary (mean ± SEM) of data from three donors. Paired t test: *P < 0.05. (C) Memory CD4⁺ T cells were activated with anti-CD3/CD28 with or without rAd (200 ip/cell) or anti-CD46 mAb. The graph is a summary (mean ± SEM) of data from two donors. (D) Naive CD8⁺ T cells were activated with anti-CD3/CD28 with or without rAd (200 ip/cell) or anti-CD46 mAb. The graph is a summary (mean ± SEM) of data from four donors. Proliferation was evaluated in triplicate on day 5 with ³H-thymidine.

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.

CD46 engagement suppresses NF-κB activation in CD4⁺ T cells. SDS/PAGE and Western blot analysis for p65, IκBα, and β-tubulin in lysed C and N fractions of sorted CD4⁺ T cells after 4 h of no stimulation (mock) or stimulation by immobilized anti-CD3/CD28 mAb with or without anti-CD46 mAb (13/42). C, cytoplasmic; N, nuclear. Densitometry measurements are summarized (mean ± SD) for three donors. Paired t test: *P < 0.05; **P < 0.005; or not significant (n.s.) for P > 0.05.