Comorbid mice fail to resolve influenza infection and exhibit deficient lung antiviral CD8⁺ T cell function. (A–G) Mice were immunized and inoculated with influenza for analysis of viral replication, immunity, and pathology 11–16 d after influenza infection. (A) Left lungs were perfused, harvested, and sectioned for immune infiltration evaluation via hematoxylin and eosin tissue staining 14 d after influenza infection. Bar, 100 µm. (B) Lung influenza viral titer was determined in influenza and comorbid mice by qPCR analysis of influenza matrix protein, normalized to β-actin. Data are compiled from two independent experiments per time point with n = 8–10 mice per group. (C–F) Lung cell suspensions were prepared from influenza and comorbid mice at the specified time points, and flow cytometry was used to identify lung cell type. Shown are kinetic analyses of lung NK cells (C), CD8⁺ T cells (D), inflammatory monocytes (E), and MDSC abundance (F). Each time point contains 8–10 mice per group. (G) Cytokine production by influenza-specific CD8⁺ T cell function in influenza-infected and comorbid mice was analyzed by conducting intracellular cytokine staining on lung cell suspensions 11–16 d after influenza infection. Shown are representative flow cytometric plots of IFN-γ production from influenza NP tetramer⁺ CD8⁺ T cells harvested from either singly influenza–infected or comorbid mice, with bar graphs of compiled cell percentages and numbers of two independent experiments, with 9–10 mice per group. Student’s t tests were conducted for statistical analysis. *, P < 0.05. The mean and standard error of means are represented.