EAE-induced lung CD11b⁺ cells effectively suppress CD8⁺ T cell activation by iNOS. (A–F) CD11b⁺ cells were isolated from lungs obtained from each of the groups of mice (EAE only [10 d after EAE], influenza only [3 d after flu], and comorbid [10 d after EAE/3 d after flu]) and added to cultures of CFSE-labeled CD8⁺ T cells, which were activated with plate-bound α-CD3 and soluble α-CD28 at a CD11b⁺ myeloid cell/CD8⁺ T cell ratio of 10:1. Then, cells were analyzed by flow cytometry. (A) CD8⁺ T cell proliferation is shown by representative flow histograms of CFSE dilution and compiled percentages of undivided CD8⁺ T cells gated on live cells from three independent experiments. (B) IFN-γ production from CD8⁺ T cell activated under the same conditions is shown by representative flow cytometric plots and compiled percentages of two independent experiments. After 72 h, supernatants were harvested and assayed for IL-10 secretion (C) and nitrite production (E). IL-10 or NO-producing iNOS was blocked by addition of anti–IL-10 neutralizing antibody or l-NMMA, respectively, at the start of culture. After 72 h, cells were analyzed by flow cytometry. Shown are representative flow cytometric plots and bar graphs of compiled experiments of CFSE dilution indicating division of CD8⁺ T cells and IFN-γ production with IL-10 blockade (D) and l-NMMA–mediated iNOS blockade (F). Data are combined from two independent experiments. (G) Cytokine production by influenza-specific CD8⁺ T cell function in comorbid mice given no treatment or 60 mg/kg/d sildenafil citrate was analyzed by conducting intracellular cytokine staining on lung cell suspensions 9 d after influenza infection. Shown are representative flow cytometric plots of live cells gated on CD8⁺ T cells, which were harvested from comorbid mice given no treatment or sildenafil citrate, with bar graphs of compiled cell percentages from two independent experiments, with four to five mice per group. Un-act., unactivated. Student’s t tests were conducted for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The mean and standard error of means are represented.