Figure 3. Lung T RM are predominant... | Rockefeller University Press

Figure 3.

$Lung TRMare predominant producers of IL-10 in the secondary response. Naïve and memory IL-10-GFP reporter mice were challenged with PR8 influenza virus, and lungs were analyzed 2–3 dpi. Data are representative of three independent experiments (n = 10–15 mice per time point and condition). (A) IL-10(GFP) production by total CD45+ lung immune cells in the primary response (red) or secondary response (blue) at 0 dpi (uninfected baseline) and 3 dpi shown in representative flow cytometry plots (left) and graphs showing compiled mean frequency ± SD (top right) and mean absolute numbers ± SD (lower right) from three to six mice per time point and condition. Statistical significance was determined by two-way ANOVA. (B) Immune composition of CD45+IL10(GFP)+ cells in the secondary response at 2–3 dpi expressed as the mean ± SD from four to six mice per time point. Statistical significance was determined by one-way ANOVA. (C) IL-10(GFP) production by lung CD4+ (top, green) and CD8+ (bottom, orange) at baseline in memory mice (0 dpi) and 3 dpi shown in representative flow cytometry plots (left) and as the number of IL-10+ cells (mean ± SD) (upper right) and fold change at 3 dpi relative to baseline (lower right) from three to six mice per group. Statistical significance was determined by Student’s paired t test. (D) IL-10(GFP) expression by circulating and tissue-resident T cells. Fluorescently labeled anti-CD45 antibody was administered IV prior to euthanasia (see Materials and methods) to differentiate circulating (CD45(IV)+) and tissue-localized (CD45(IV)−) cells. IL-10(GFP) expression by lung CD4+ (top) and CD8+ (bottom) T cells in primary infection (red) and secondary infection (blue) at 0 and 3 dpi shown in representative flow cytometry plots. (E) Frequency (left) and absolute numbers (right) of IL-10(GFP)+ cells within the CD45(IV)+ and CD45(IV)− fractions of lung CD4+ (top) and CD8+ (bottom) T cells determined as in D. Statistical significance was determined by two-way ANOVA. (F) Expression of TRM and Treg markers by IL-10+ and IL-10− CD4+ (green) and CD8+ (orange) T cells in the lungs. Columns represent mean values, and error bars indicate standard deviation. ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05. Refer to the image caption for details.$

Lung T _RM are predominant producers of IL-10 in the secondary response. Naïve and memory IL-10-GFP reporter mice were challenged with PR8 influenza virus, and lungs were analyzed 2–3 dpi. Data are representative of three independent experiments (n = 10–15 mice per time point and condition). (A) IL-10(GFP) production by total CD45⁺ lung immune cells in the primary response (red) or secondary response (blue) at 0 dpi (uninfected baseline) and 3 dpi shown in representative flow cytometry plots (left) and graphs showing compiled mean frequency ± SD (top right) and mean absolute numbers ± SD (lower right) from three to six mice per time point and condition. Statistical significance was determined by two-way ANOVA. (B) Immune composition of CD45⁺IL10(GFP)⁺ cells in the secondary response at 2–3 dpi expressed as the mean ± SD from four to six mice per time point. Statistical significance was determined by one-way ANOVA. (C) IL-10(GFP) production by lung CD4⁺ (top, green) and CD8⁺ (bottom, orange) at baseline in memory mice (0 dpi) and 3 dpi shown in representative flow cytometry plots (left) and as the number of IL-10⁺ cells (mean ± SD) (upper right) and fold change at 3 dpi relative to baseline (lower right) from three to six mice per group. Statistical significance was determined by Student’s paired t test. (D) IL-10(GFP) expression by circulating and tissue-resident T cells. Fluorescently labeled anti-CD45 antibody was administered IV prior to euthanasia (see Materials and methods) to differentiate circulating (CD45(IV)⁺) and tissue-localized (CD45(IV)⁻) cells. IL-10(GFP) expression by lung CD4⁺ (top) and CD8⁺ (bottom) T cells in primary infection (red) and secondary infection (blue) at 0 and 3 dpi shown in representative flow cytometry plots. (E) Frequency (left) and absolute numbers (right) of IL-10(GFP)⁺ cells within the CD45(IV)⁺ and CD45(IV)⁻ fractions of lung CD4⁺ (top) and CD8⁺ (bottom) T cells determined as in D. Statistical significance was determined by two-way ANOVA. (F) Expression of T_RM and Treg markers by IL-10⁺ and IL-10⁻ CD4⁺ (green) and CD8⁺ (orange) T cells in the lungs. Columns represent mean values, and error bars indicate standard deviation. ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.