PD-L1 and PD-L2 expression on dendritic cells regulates the iNKT cell cytokine secretions after IAV infection.

<p>Pulmonary DCs (10³) were isolated from lungs of influenza A infected PD-L1^−/−, PD-L2^−/− and wild type BALB/c mice and loaded with 1 µg of α-galactosylceramide for 3 hours and then cultured with negatively enriched iNKT cells (1×10⁴) from the spleen of naïve wild type mice. Supernatants were collected after 48 h and IL-4 and IFN- γ levels were measured by ELISA. Data presented as mean ± SEM. *** P>0.001 as calculated by a one-way ANOVA with Tukey post hoc test and is representative of four independent experiments with n of 5 per group.</p

iNKT cell subpopulations are modulated in PD-L1^−/− and PD-L2^−/− animals after IAV infection.

<p>(<b>a</b>) Wild type BALB/c, PD-L1^−/− and PD-L2^−/− mice (n = 3) were infected with IAV intranasally. Lungs were collected at day 1, 5 and 10 post infection and the frequency of CD4⁺ and DN iNKT cells determined by flow cytometry and compared (DN: black asterisk, CD4⁺: gray asterisk). (<b>b</b>) CD4⁺ and DN iNKT cells subsets were sorted from IAV infected lungs on day 5 from PD-L1^−/−, PD-L2^−/− and wild type mice BALB/c and IL-4 and IFN- γ were analysed by quantitative RT-PCR normalized to β-actin levels (n = 8). Data presented as mean ± SEM. * P>0.05, ** P>0.01 and *** P>0.001 as calculated by a one-way ANOVA with Tukey post hoc test and is representative of five independent experiments.</p

PD-L1 and PD-L2 affect the cytokines produced by lung iNKT cells after IAV infection.

<p>iNKT cells were positively selected from influenza A infected lungs on day 5 from PD-L1^−/−, PD-L2^−/− and wild type BALB/c mice and different cytokines were analysed by quantitative RT-PCR normalized to β-actin levels. Data presented as mean ± SEM. * P>0.05, ** P>0.01 and *** P>0.001 as calculated by a one-way ANOVA with Tukey post hoc test and is representative of five independent experiments with n of 5 per group.</p

Lack of PD-L1 expression on iNKT cells reduces sensitivity to IAV infection.

<p>iNKT cells (1×10⁶) were positively isolated from spleen of BALB/c, PD-L1^−/− and PD-L2^−/− mice and adoptively transferred into iNKT cell deficient Jα18^−/− mice. After 24 hours Jα18^−/− mice received 3000 PFU influenza A intranasally. Weight loss was monitored at different time points after the infection with n = 5 per group and is representative of three independent experiments.</p

iNKT cells reduce the sensitivity of BALB/c mice to IAV infection.

<p>Jα18^−/− mice and BALB/c mice (n = 10) were intranasally infected with a lethal dose (3000 PFU) of IAV. (<b>A</b>) Increased frequency of iNKT cells after IAV infection. Representative FACS plots of iNKT cell frequencies in the lung of BALB/c mice stained with CD1d: PBS57 loaded tetramer and TCR-β antibody, gated on viable lymphocytes with percentage of cells in the iNKT cell gate indicated next to the gate. (<b>B</b>) Total iNKT cell numbers in the lung were determined by flow cytometry at different days post infection (DPI). Data is presented as mean ± standard error of the mean (SEM), n = 3. * P>0.05 (<b>C</b>) The weight loss of Jα18^−/− mice and BALB/c mice has been monitored daily after IAV infection. Jα18^−/− mice show augmented weight loss compared to wild type mice. Data are representative of three independent experiments.</p

PD-L1 and PD-L2 affect the sensitivity and the degree of lung inflammation after infection with IAV.

<p>PD-L2^−/−, PD-L1^−/− and BALB/c (n = 10) mice received a lethal dose of IAV (3000 PFU) intranasally. (<b>A</b>) Weight loss has been monitored by measuring body weight daily post infection (DPI) (<b>B</b>) BAL was performed in mice (n = 5) five days post IAV infection and analyzed for differential cellular infiltration. Bar graph shows total cell number (Total) and absolute number of eosinophils (EOS), monocytes/macrophages (MAC), lymphocytes (LYM) and neutrophils (PMN). ND: not detectable. (<b>C</b>) Representative image of lung sections taken 5 days post infection with 3000 PFU IAV stained with H&E (original magnification ×40, insert magnification ×100, scale bar 50 μm) from BALB/c, PD-L1^−/− and PD-L2^−/− mice. (<b>D</b>) Quantification of lung sections as shown by measurement of thickness of epithelium, left panel and number of inflammatory cells per 250 µm², right panel. (<b>E</b>) Virus titration in the lungs shown as plaque forming units (PFU) per gram of lung tissue. Data are presented as mean ± SEM, * P>0.05, ** P>0.01 and *** P>0.001 as calculated by a one-way ANOVA with Tukey post hoc test and is representative of four independent experiments.</p

Differential expansion of iNKT cell subsets to IAV infection.

<p>Expression of PD-1, PD-L1 and PD-L2 on iNKT cells isolated from the lungs of control or 5 day post IAV infection in BALB/c mice. Dot plots are gated on iNKT cells that were identified as CD1d: PBS57 loaded tetramer⁺ TCR-β^intermediate and show CD4 expression on the iNKT cells (Dot plots are representative of 5).</p