FIG 3 .

Role of the lung microenvironment on the inhibition of iNKT cell activation in the context of bacterial superinfection. (A) Pulmonary iNKT cells from mock-infected or IAV-infected mice were stimulated in an APC-free system with plate-coated CD1d/α-GalCer. The production of IFN-γ was quantified 48 h later. Results from a pool of two experiments are shown. (B) RNAs were extracted from the lungs of mock-infected and IAV-infected mice. IL-10 mRNA copy numbers were measured by quantitative RT-PCR. Data are expressed as fold increase ± SD over average gene expression in mock-treated mice (n = 5). (C) IAV-infected mice (6 dpi) were injected with the anti-IL-10 receptor (IL-10R) or the isotype control MAb (1 mg) 24 h before i.n. inoculation of α-GalCer. For each group, the percentages ± SD of IFN-γ- and IL-17A-producing iNKT cells (16 h after α-GalCer) are shown (n = 6 to 8, two pooled experiments). (D) The proportions ± SD of conventional DCs (cDCs), monocyte-derived DCs (MoDCs), and inflammatory monocytes (IMs) among CD45⁺ cells were determined in mock-infected and IAV-infected (7 dpi) mice (n = 8 to 10, three pooled experiments). (E, left panel) A representative histogram depicts the expression of CD1d on conventional DCs (naive mice), monocyte-derived DCs (7 dpi), and inflammatory monocytes (7 dpi). White peak, isotype control; gray peak, CD1d. (Right panel) Conventional DCs, monocyte-derived DCs, and inflammatory monocytes were sorted from mock-infected (cDCs) or IAV-infected (7 dpi) mice, incubated with α-GalCer for 1 h, and cocultured with naive iNKT cells. The concentration of IFN-γ present in the supernatant was determined after 48 h. Shown are the mean concentrations ± SD from two (cDCs) or three (MoDCs and IMs) experiments. ns, not significant. **, P < 0.01; ***, P < 0.001 (panels B and C, one-way ANOVA Kruskal-Wallis test).