Adenovirus type-35 vectors block human CD4+ T-cell activation via CD46 ligation

Abstract

Recombinant adenoviruses (rAds) based on types 5 (rAd5) and 35 (rAd35) have emerged as important vaccine delivery vectors in clinical testing for a variety of pathogens. A major difference between these vectors is their binding to cellular receptors used for infection. Whereas rAd5 binds coxsackie-adenovirus receptor (CAR), rAd35 binds the complement regulatory protein CD46. Although rAd35 infected and phenotypically matured human blood dendritic cells (DCs) more efficiently than rAd5, we show here that rAd35 markedly suppressed DC-induced activation of naive CD4(+) T cells. rAd35 specifically blocked both DCs and anti-CD3/CD28 mAb-induced naive T-cell proliferation and IL-2 production. This effect was also observed in CD4(+) memory T cells but to a lesser extent. The suppression occurred by rAd35 binding to CD46 on T cells and was independent of infection. CD46 engagement with mAb mimicked the effects of rAd35 and also led to deficient NF-κB nuclear translocation. In contrast, rAd5 and rAd35 vectors with ablated CD46 binding did not inhibit T-cell activation. Our findings provide insights into the basic biology of adenoviruses and indicate that CD46 binding may have an impact on the generation of primary CD4(+) T-cell responses by Ad35.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.

Fig. 6.

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.