Human metapneumovirus keeps dendritic cells from priming antigen-specific naive T cells

Abstract

Human metapneumovirus (hMPV) is the second most common cause of acute lower respiratory tract infections in children, causing a significant public health burden worldwide. Given that hMPV can repeatedly infect the host without major antigenic changes, it has been suggested that hMPV may have evolved molecular mechanisms to impair host adaptive immunity and, more specifically, T-cell memory. Recent studies have shown that hMPV can interfere with superantigen-induced T-cell activation by infecting conventional dendritic cells (DCs). Here, we show that hMPV infects mouse DCs in a restricted manner and induces moderate maturation. Nonetheless, hMPV-infected DCs are rendered inefficient at activating naive antigen-specific CD4(+) T cells (OT-II), which not only display reduced proliferation, but also show a marked reduction in surface activation markers and interleukin-2 secretion. Decreased T-cell activation was not mediated by interference with DC-T-cell immunological synapse formation as recently described for the human respiratory syncytial virus (hRSV), but rather by soluble factors secreted by hMPV-infected DCs. These data suggest that although hMPV infection is restricted within DCs, it is sufficient to interfere with their capacity to activate naive T cells. Altogether, by interfering with DC function and productive priming of antigen-inexperienced T cells, hMPV could impair the generation of long-term immunity.

Figure 1

Dendritic cells (DCs) are infected by human metapneumovirus (hMPV). (a) Fluorescence micrographs (100 × magnification) of DCs 24 hr post-inoculation with mock, viable or UV-inactivated hMPV CZ0107 and NL/1/00 strains (Green-Fluorescence: anti-hMPV Nucleoprotein/Fusion protein monoclonal antibodies, Red: Evan's Blue counterstain). One representative experiment out of four independent assays is shown. (b) Flow cytometry detection of hMPV infection in DCs (gate on CD11c⁺ cells) inoculated either with mock, UV-inactivated [multiplicity of infection (MOI 20)] or increasing amounts of hMPV (MOIs 1– 20). (c) Flow cytometry analysis of DCs inoculated with the green fluorescent protein (GFP) -expressing hMPV_NL/1/00 strain (MOI 10), at different time-points post-infection (2–72 hr). Gated CD11c⁺ cells were assessed for virus-derived GFP expression. Mature (treated with 5 μg/ml LPS) and immature (untreated) DCs were inoculated with mock or the GFP-expressing hMPV_NL/1/00 strain at MOI 10. Bars represent flow cytometry data as the percentage of CD11c⁺ cells expressing GFP. Maturation of lipopolysaccharide (LPS) -treated DCs was confirmed by flow cytometry by measuring MHC class-II, CD40, CD80 and CD86, which were up-regulated as compared to untreated cells (data not shown). (d) Flow cytometry analysis of LLC-MK2 cells inoculated with the GFP-expressing hMPV_NL/1/00 strain (MOI 10), at different time-points post-infection (2–72 hr). Untreated or LPS-treated cells were assessed for virus-derived GFP expression. (e) Titration of hMPV in the supernatants of DCs inoculated either with mock or hMPV (strains CZ0107 and NL/1/00). Supernatants were harvested 24 hr post-inoculation and applied over LLC-MK2 monolayers. Supernatants from hMPV infected-LLC-MK2 were used as positive controls for virus-detection. Bars in (b–e) are means of at least three independent experiments ± SEM. One-way analysis of variance and the Bonferroni post-test were used for (b), (d) and (e). Two-way analysis of variance and the Bonferroni post-test were used to compare each time-point against its paired mock in (c). ****P < 0·0001; ***P < 0·001; **P < 0·01; *P < 0·05; n.s.: non-significant.

Figure 2

Secretion of cytokines and up-regulation of maturation markers after human metapneumovirus (hMPV) inoculation. (a) Supernatants from hMPV-inoculated and control dendritic cells (DCs) were assessed by ELISA for the presence of interleukin-6 (IL-6). (b) Expression of MHC class II (I-A^b) was measured by flow cytometry at 24 hr post-inoculation (geometric mean fluorescence intensity; GMFI). The hMPV-infected [CD11c⁺/green fluorescent protein (GFP)⁺] and non-infected (CD11c⁺/GFP⁻) DC populations (within the hMPV_GFP-inoculated DC population) were analysed separately and compared with mock- and lipopolysaccharide (LPS) -inoculated DCs. Bars are means of at least four independent experiments ± SEM. Statistical analysis was performed by one-way analysis of variance and the Bonferroni post-test comparing all groups against mock-inoculated DCs. ***P < 0·001; **P < 0·01; *P < 0·05; n.s.: non-significant.

Figure 3

Human metapneumovirus (hMPV)-inoculated dendritic cells (DCs) display a reduced capacity to activate naive CD4⁺ T cells. Increasing amounts of either uninfected, mock-inoculated, UV-inactivated-, or viable hMPV-inoculated DCs were used to stimulate antigen-specific T cells in the presence of exogenously added antigenic peptide (pOVA_323–339). T cells were assessed for (a) interleukin-2 (IL-2) secretion in the supernatant by ELISA (left panel: hMPV clinical isolate CZ0107, right panel: hMPV reference strain NL/1/00) and surface expression of (b) CD4, (c) CD25, (d), CD69 and (e) CD71, 24 hr after co-culture (gating on CD4⁺ T cells). Representative histograms are shown for each marker assessed. (f) The percentage of proliferating T cells after co-culture with antigen-pulsed DCs was determined using a CFSE dilution assay. (g) Flow cytometry density plots depict proliferating subsets of CD4⁺ T cells at 24 hr of co-culture (low-CFSE fluorescent populations). Data are means ± SEM of at least three independent experiments. Negative (non-antigenic peptide/unpulsed) control is not shown in (a). Positive (Type IV concanavalin A) controls are not shown in (a–g). One-way analysis of variance and the Bonferroni post-test were used to compare all groups against T cells co-cultured with mock-inoculated DCs. ***P < 0·001; **P < 0·01; *P < 0·05; n.s.: non-significant.

Figure 4

Human metapneumovirus (hMPV) does not prevent T-cell polarization towards dendritic cells (DCs) during immunological synapse (IS) formation. DC–T-cell IS assembly was assessed for uninfected, hMPV- and UV-hMPV-inoculated DCs (red fluorescence) co-cultured with antigen-specific T cells (green fluorescence). Golgi apparatus polarization within T cells was measured as a read-out of synapse formation by laser confocal microscopy. (a) A representative micrograph is shown per treatment. Arrowheads show polarization of the Golgi apparatus (green fluorescence) at the interface formed between T cells and contacting DCs (red fluorescence). (b) IS assembly was quantified by determining the percentage of T cells with their Golgi apparatus polarized towards DCs within the total number of DC–T-cell conjugates. Data are means ± SEM of at least 400 visualized DC–T-cell conjugates captured in three independent experiments. One-way analysis of variance and the Bonferroni post-test were used to compare groups. **P < 0·01; n.s.: non-significant.

Figure 5

Reduced T-cell activation by human metapneumovirus (hMPV) -inoculated dendritic cells (DCs) is mediated by soluble factors. (a) Interleukin-2 (IL-2) secretion (ELISA) by T cells incubated in the presence of supernatants from hMPV-inoculated DCs and plate-bound anti-CD3ε plus anti-CD28 antibodies (50 ng/well each). Increasing multiplicities of infection (MOIs) were used (range 1–20) to assess the effects of virus doses over the inhibitory capacity of DC supernatants from both hMPV_CZ0107- and hMPV_NL/1/00-inoculated DCs. Supernatants from DCs treated with 25 μg/ml of poly (I : C) or inoculated with mock or UV-hMPV (MOI 20) were used as controls (white bars, right side of each graph). Data are means ± SEM of at least three independent experiments. (b) FACS analyses assessing the expression of CD25 and CD69 in CD4⁺ T cells used for the experiments shown in (a). Data represent means ± SEM of at least four independent experiments performed in duplicate. One-way analysis of variance and the Bonferroni post-test were used to compare all groups against T cells treated with supernatants from mock-inoculated DC. ***P < 0·001; **P < 0·01; n.s.: non-significant.

Figure 6

Neutralization of type I interferons (IFNs) does not restore T-cell activation, (a) IFN-α and (b) IFN-β secretion by dendritic cells (DCs) was measured by ELISA in supernatants collected 24 hr post-inoculation (hr p.i.) with either mock or different multiplicities of infection (MOIs; varying from 1 to 20) of the human metapneumovirus (hMPV) NL/1/00 and CZ0107 strains. Bars represent means of at least three independent experiments performed in duplicate ± SEM. (c, d) To assess potential inhibitory roles of IFN-α/β in the supernatants, a cocktail of antibodies were used to block the type I IFN receptor as well as soluble IFN-α/β during DC–T-cell co-cultures. Blockade of the IFN-α/β receptor was corroborated by an IFN-α/β ELISA that showed an increase in soluble IFNs in the supernatants of DC–T-cell co-cultures (data not shown). (c) Interleukin-2 (IL-2) ELISA of co-culture supernatants recovered at 36 hr of culture. (d) Flow cytometry analyses were performed over cells harvested from (c). The percentage of CD4^High+/CD69⁺/CD25⁺ activated T cells observed for each condition is shown. Black bars: untreated co-cultures, white bars: co-cultures treated with the antibody cocktail. Bars represent means of at least three (a, b) and four (c, d) independent experiments ± SEM. For IFN-α/β ELISA statistical analyses were performed by one-way analysis of variance and the Bonferroni post-test. For IFN-blockage assays, statistical analyses were performed by two-way analysis of variance and the Bonferroni post-test compared with mock both, for the effects of either virus infection or IFN-blockade over T-cell activation. ***P < 0·001; **P < 0·01; n.s.: non-significant.