Figure 8. LRRC8C deficiency enhances T cell mediated antiviral immunity to influenza.

(a) Experimental design of influenza A virus (IAV) infection of wild-type and Lrrc8c^−/− mice using the PR8 strain (H1N1, left), and body weight change (right) relative to the initial weight before infection. i.n., intranasal; LD, lethal dose. (b) Viral titers detected in the lung of IAV infected mice by measuring PR8 M2/M1 mRNA expression using RT-qPCR. TCID₅₀, 50% tissue culture infective dose. TCID₅₀ values where transform to TCID₅₀+1 to plot titers on log10 scale. (c) anti-PR8 IgG antibodies in the sera of wild-type and Lrrc8c^−/− mice detected by ELISA. (d) Representative flow cytometry plots and bar graphs showing the frequencies of T follicular helper (T_FH) cells in the mediastinal lymph nodes (medLNs, left) and absolute number of T_FH cells in spleen and medLNs (right). (e) Representative contour plots and bar graphs showing the frequencies of germinal center (GC) B cells in the medLNs (left) and absolute numbers of GC B cells in the spleen, medLNs and lungs (right). (f) Expression of IL-2 and TNF-α in CD4⁺ T cells isolated from IAV infected mice. Representative contour plots from lung-infiltrating T cells (left) and frequencies (right) of cytokine-producing T cells isolated from the spleen, medLNs and lungs after PMA+Iono re-stimulation for 6h. (g) T cell proliferation measured by CFSE dilution in wild-type and Lrrc8c^−/− CD4⁺ T cells isolated from the lung of mice infected with IAV and re-stimulated with anti-CD3+CD28. (h) Apoptosis of CD4⁺ and CD8⁺ T cells from wild-type and Lrrc8c^−/− mice shown in (g) measured by annexin V staining 4 days after stimulation with anti-CD3+CD28. (i) CD80 surface expression by CD4⁺ and CD8⁺ T cells in the lungs of wild-type and Lrrc8c^−/− mice infected with IAV. Representative histogram plots (left) and quantification (right) of CD80 expression (MFI). (j) Correlation of CD80 expression by T cells in the lungs of IAV infected mice vs. body weight (% original BW of mice 15 days after IAV infection). Data in (a-j) are pooled from 2 independent experiments using 8 wild-type and 7 Lrrc8c^−/− mice. Data in (g,h) are pooled from 2 independent experiments using 5 mice/genotype. All data are mean ± s.e.m. and were analyzed by 2-way ANOVA with Sidak’s multiple comparison test (a), two-tailed Mann Whitney test (b), and two-tailed, unpaired Student’s t test (b-f, h,i), *P < 0.05, **P < 0.01, ***P < 0.001.