Figure 3. CS-induced TRM cells, especially TRM-Tregs, depended on replenishment from circulating T cells.
(A) The scheme indicated the time points of FTY720 treatment (20 μg in vehicle).
FTY720 treatment from 0W blocked circulating T cell recruitment. FTY720 treatment from 4W, in which circumstance, lymphocytes could be recruited into the lung at the inflammatory stage but blocked at the fibrogenesis stage. (B) H&E staining to the lung section of different treated mice. Scale Bar = 500 μm and 200 μm. (C) The inflammation scores were assessed in the lung sections (n = 5). (D) FC analysis of CD4+TRM cells in the lungs of distinct FTY720-challenge. The bar graph showed percentages and counts of CD4+ TRM cells (n = 5). (E) Typical flow histogram indicated Ki-67 expression in CD4+TRM cells. (F) FC analysis of CD4+TRM cells for CD69 and CD103 expressions. The bar graph displayed the ratios of CD103 to CD103+ in the CD69+ TRM cells (n = 5). (G) FC analysis of Tregs (FOXP3+) in the CD4+TRM cells. The bar graph showed the percentages of TRM-Tregs (n = 5). (H) Schematic overview of parabiosis experiment. CD45.1/1 and CD45.1/2 mice were approximated with sutures. With 14 days’ recovery, CD45.1/1 mice were treated by CS particle instillation and analyzed 7 days later. (I) Typical FC plot of circulating blood leukocytes of the parabiont. (J) Flow plot analysis of the CD4+ TRM cells and circulating TEM cells of the CS-treated conjoined mice. CD45.2+ TRM cells indicated the recruited cells from circulating. (K) FC plots demonstrated the CD45.2 expression in TRM-Treg or TRM-Teff cells. (L) FC plots demonstrated CD103 and CD45.2 expressions in TRM-Treg or TRM-Teff cells. N number also indicated independent experimental replicates. Individual mice are plotted on the graphs. Values are reported as the mean ± SD. Pvalue was determined by one-way ANOVA followed by Tukey’s test.