Fig. 3: CS-induced T_RM cells derived from circulating lymphocyte recruitment and in situ proliferation.

a The scheme indicated the time points of FTY720 treatment (20 μg in vehicle per time). FTY720 treatment from 4 weeks, 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 (upper) and 200 μm (lower). c The inflammation scores were assessed in the lung sections. d FC analysis of CD4⁺ T_RM cells in the lungs of distinct FTY720-challenge. The bar graph shows percentages and counts of CD4⁺ T_RM cells. e Flow histogram indicated Ki-67 expression in CD4⁺ T_RM cells. f FC analysis of CD4⁺ T_RM cells for CD69 and CD103 expressions. The bar graph displayed the ratios of CD103^– to CD103⁺ in the CD69⁺ T_RM cells. g FC analysis of Tregs (FOXP3⁺) in the CD4⁺ T_RM cells. The bar graph shows the percentages of T_RM-Tregs. 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⁺ T_RM cells and circulating T_EM cells of the CS-treated conjoined mice. CD45.2⁺ T_RM cells indicated the recruited cells from circulating. k Typical flow plots showed CD69 and CD103 expressing patterns on T_RM-Teff cells or T_RM-Tregs. Flow plot analysis of CD45.2⁺ cells in CD69⁺CD103⁺ Tregs, CD69⁺CD103⁺ Teffs, CD69⁺CD103^- Tregs, and CD69⁺CD103^- Teffs in CD4⁺ T_RM cells. For panels a–g, n = 5 biologically independent animals, n number also indicated independent experimental replicates. Individual mice are plotted on the graphs. Values are reported as the mean ± SD. P value was determined using one-way ANOVA followed by Tukey’s test. For panels j and k, n = 4 biologically independent animals, n number also indicated independent experimental replicates. CD45.1/1 and CD45.1/2 mice approximated with sutures were used in these experiments. Values are reported as the mean ± SD. P value was determined by paired two-tailed Student’s t-test.