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.