Respiratory syncytial virus decreases the capacity of myeloid dendritic cells to induce interferon-gamma in naïve T cells

Abstract

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants under 6 months of age. Since an RSV infection does not necessarily prevent a reinfection, we asked whether RSV might subvert an effective immune response by interfering with the function of dendritic cells (DCs). Immature DCs cultured from cord blood stem cells and infected with RSV reduced the rate of interferon-gamma (IFN-gamma) production in co-cultured autologous naïve T cells stimulated with the superantigen TSST-1. Maturation of DCs in response to poly(IC) but not to CD40 ligand did overcome the inhibitory effect of RSV. Further experiments demonstrated that induction of apoptosis, a selective increase in CD86 expression and lack of release of pro-inflammatory cytokines were associated with inhibition of IFN-gamma generation. In addition, RSV replication seemed to be essential for modulation of IFN-gamma production because a virus preparation inactivated by UV irradiation had no effect. Hence, one reason for multiple reinfections by RSV might be the subversion of antiviral immune responses by interference of RSV with DC function.

Figure 1

Infection of DCs with RSV. Human cord-blood-derived DCs were infected with increasing MOI of RSV. After 24 hr of infection at 37°, cells were fixed, permeabilized, and stained for RSV antigen. The percentage of infected cells was determined by flow cytometry. (a) The rate of infection depending on multiplicity of infection (MOI) and pretreatment of the DCs. The results are expressed as mean ± SD of four independent experiments. ▾, CD40L-matured DCs; □, immature DCs; ▴, poly(IC)-matured DCs. (b–d) Histograms obtained with (b) CD40L-matured DCs, (c) immature DCs and (d) poly(IC)-matured DCs at a MOI of 10. The solid lines delineate controls with FITC-streptavidin, the shaded histograms controls with FITC-streptavidin and an irrelevant biotinylated antibody and the bold lines delineate measurements with anti-RSV antibody.

Figure 2

Immature DCs infected with RSV die by apoptosis. Immature and mature DCs were uninfected or infected with RSV (MOI 10). The extent of apoptosis and necrosis was determined by staining the cells with FITC–annexin V and propidium iodide (PI) after 24 hr. Results shown are representative of four experiments.

Figure 3

Surface phenotype of DCs exposed to RSV. Immature DCs were left untreated or were stimulated for 24 hr with CD40L or poly(IC) and subsequently treated with RSV (MOI 10) for further 24 hr. The histograms show fluorescence values on gated large cells. The open histograms represent the controls of DCs stained with isotype-matched irrelevant mAb. The figures indicate the percentage of highly positive (HLA-DR, CD86) or positive (CD83) cells for each sample. The data are from one representative experiment out of six performed.

Figure 4

Modulation of cytokine production by RSV. DCs were treated with RSV (MOI 10) and supernatants were collected after 24 hr and analysed with specific ELISA. The results represent the means ± SD of six independent experiments. Measurements with and without RSV were compared by paired t-test. n.s., denotes not significant, *P < 0·05, **P < 0·01 and ***P < 0·001.

Figure 5

Stimulation of naive CD4⁺ CD45RA⁺ T cells. Immature DCs were left untreated or were stimulated for 24 hr with the indicated stimuli. An mixed lymphocyte reaction assay was then set up where mitomycin-C-treated DC were cultured at different cell numbers with 5 × 10⁴ purified autologous CD4⁺ CD45RA⁺ T cells and incubated with the superantigen TSST-1. The proliferative response was measured after 5 days induced by RSV-infected DCs (MOI 10) is compared with (a) immature DCs; (b) CD40L-matured DCs and (c) poly(IC)-matured DCs;. The data from six experiments are presented as mean ± SD. Measurements with and without RSV were compared by paired t-test. *P < 0·05; **P < 0·01.

Figure 6

Analysis of intracellular cytokine production by T-cells. Cytokine production by TCR-Vβ2⁺ T cells stimulated in the presence of autologous DCs pretreated with the indicated stimuli and incubated with the superantigen TSST-1. The T-cells expressing TCR-Vβ2 chain, which are activated by TSST-1, were 10% of the purified CD4⁺ CD45RA⁺ T cells. (a) The data are from one representative experiment. (b) shows the comparison of all six cultures treated with medium alone or with RSV. Horizontal bars represent the mean. The results were compared by paired t-test.