Fig. 6

 H&E images (left column) showing exact sections (black boxes) that were double stained against PD-1 and CD4. PD-1 and CD4 positivity is demarcated with green and red respectively, while DAPI which was used to stain the nuclei is demarcated with blue. Double stain images indicated higher PD-1 and CD4 expression upon bleomycin administration. Right column shows 2X zoomed images. White arrows are pointing representative double positive cells. (error bars: 50 μm) (Panel A). Quantification analysis showed that bleomycin treated mice presented with higher PD-1 compared to saline treated mice (p = 0.04), while mice treated with bleomycin and low dose pembrolizumab presented with lower PD-1 compared to mice treated with bleomycin only (p = 0.03, Kruskal-Wallis test) (Panel B). Pearson’s Rr quantification measurement of the colocalization of PD-1 and CD4. Treatment with pembrolizumab and/or bleomycin did not result to a significant change in the percentage of PD-1/CD4 colocalization (Panel C). Median PD-1 mRNA expression was significantly higher in lung tissues isolated from mice treated with bleomycin compared to littermates treated with saline at day 14 (Kruskal–Wallis test; saline vs. bleomycin: p = 0.008). Pembrolizumab treatment did not lead to statistically significant change in PD-1 mRNA expression (Panel D)