Respiratory syncytial virus impairs T cell activation by preventing synapse assembly with dendritic cells

Abstract

Respiratory syncytial virus (RSV) infection is one of the leading causes of infant hospitalization and a major health and economic burden worldwide. Infection with this virus induces an exacerbated innate proinflammatory immune response characterized by abundant immune cell infiltration into the airways and lung tissue damage. RSV also impairs the induction of an adequate adaptive T cell immune response, which favors virus pathogenesis. Unfortunately, to date there are no efficient vaccines against this virus. Recent in vitro and in vivo studies suggest that RSV infection can prevent T cell activation, a phenomenon attributed in part to cytokines and chemokines secreted by RSV-infected cells. Efficient immunity against viruses is promoted by dendritic cells (DCs), professional antigen-presenting cells, that prime antigen-specific helper and cytotoxic T cells. Therefore, it would be to the advantage of RSV to impair DC function and prevent the induction of T cell immunity. Here, we show that, although RSV infection induces maturation of murine DCs, these cells are rendered unable to activate antigen-specific T cells. Inhibition of T cell activation by RSV was observed independently of the type of TCR ligand on the DC surface and applied to cognate-, allo-, and superantigen stimulation. As a result of exposure to RSV-infected DCs, T cells became unresponsive to subsequent TCR engagement. RSV-mediated impairment in T cell activation required DC-T cell contact and involved inhibition of immunological synapse assembly among these cells. Our data suggest that impairment of immunological synapse could contribute to RSV pathogenesis by evading adaptive immunity and reducing T cell-mediated virus clearance.

Fig. 1.

RSV infects murine DCs. DCs were incubated overnight with RSV at a moi equal to 1 and analyzed 48 h later for RSV infection. (A) DC surface expression of RSV F protein. Left shows a representative flow cytometry histogram for F protein expression on the surface of RSV- and UV-RSV-pulsed DCs. As a control, uninfected DCs were included. Right shows the mean fluorescence intensity for RSV F protein staining in DCs. Data are means ± SEM of three independent experiments, **, P < 0.01. (B) Real-time PCR detection of RSV nucleocapsid transcripts in DCs. Data are means ± SEM of three independent experiments.

Fig. 2.

RSV induces DC maturation and cytokine secretion. RSV-induced DC maturation requires viable virus. (A) Uninfected, LPS-, RSV-, or UV-RSV-pulsed DCs at a moi equal to 1 were analyzed 48 h after pulse for surface expression of maturation markers CD40, CD80, and CD86 and MHC-I (H-2K^b) and MHC-II (I-A^b) by flow cytometry. Bar graphs show fold increases for mean fluorescence intensities (MFI) relative to uninfected DCs. Data are means ± of six independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. (B) DC secretion of IL-6, IL10, IL-12p70, and TGF-β cytokines was assessed 48 h after pulse by ELISA in the supernatants of treated cells. Data are means ± SEM of six independent experiments. **, P < 0.01; ***, P < 0.0001.

Fig. 3.

RSV-infected DCs show an impaired capacity to prime T cells. (A and B) IL-2 secretion, (C and D) CD4 expression and proliferation, and (E and F) surface expression of activation markers CD69 and CD25 was determined for T cells stimulated with allogenic DCs (A, C, and E) or for OT-II T cells stimulated with pOVA-pulsed DCs (B, D, and F), by using uninfected, RSV- or UV-RSV-pulsed DCs. Proliferation was determined by CFSE dilution within the CD4^high T cell population. Data are means ± SEM of four to five independent experiments, *, P < 0.05; **, P < 0.01; ***, P < 0.0001 compared with uninfected treatment in A and B.

Fig. 4.

T cell activation impairment by RSV-infected DC is not mediated by IL-10. (A) IL-2 release by T cells stimulated with allogenic DCs or (B) OT-II T cells stimulated with pOVA-pulsed DCs in the presence or absence of 1 μg/ml of a neutralizing anti-IL-10 antibody. Data are means ± SEM of at least three independent experiments. *, P < 0.05; **, P < 0.01.

Fig. 5.

T cell activation impairment by RSV-infected DC is not predominantly mediated by soluble factors secreted by DCs. (A) IL-2 release by polyclonal T cells or (B) OT-II T cells in response to plate-bound anti-CD3ε, in the presence of supernatants obtained from uninfected, RSV-, or UV-RSV-pulsed DCs. Data are means ± SEM of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

Fig. 6.

Interaction with RSV-infected DCs renders T cells unresponsive to subsequent stimulation with anti-CD3ε. T cells recovered from 48-h cocultures with (A) allogenic DCs or (B) pOVA-pulsed DCs were seeded over plate-bound anti-CD3ε. IL-2 secretion derived from T cells was measured 24 h later in the supernatants. Data are means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01.

Fig. 7.

Immunological synapse assembly between DCs and T cells is impaired by RSV. Uninfected, RSV-, or UV-RSV-pulsed DCs (red fluorescence) were cocultured with transgenic OT-II T cells (green fluorescence). Polarization of the Golgi apparatus of T cells was determined by laser confocal microscopy. (A) One representative microphotograph per treatment is shown (Scale bar, 5 μm). Arrowheads show Golgi apparatus (green fluorescence) polarization from T cells toward a DC (red fluorescence). (B) Quantification of T cells with Golgi apparatus polarization toward DCs within total DC-T cell conjugates. Data are means ± SEM of at least 800 visualized DC-T cell conjugates obtained from 50 fields per treatment, captured in three independent experiments. ***, P < 0.0001.