Figure 2. Pulmonary CD4+ TRMcells mediated severe lung inflammatory response to CS particles.
(A) The reconstitution sketch of Rag1–/– mice with different cell origins. CD4+ T cells were sorted from the lung or spleen of CS-treated mice, or from the spleen of saline-treated mice by magnetic-activated cell sorting (MACS) method. The MACS-purified CD4+ T cells and PBS werei.v. transferred into Rag1–/– mice 2 weeks before CS instillation. One week after the CS instillation, recipient mice were analyzed. (B) H&E staining to the lung sections of distinct reconstituted Rag1–/– mice. Scale Bar = 500 μm and 200 μm. (C) The inflammation scores were assessed in the lung sections (n = 4 to 5). (D) Relative RNA levels of Ifng , Tnfa , Il17a ,Tbx21 , and Il6 in each group (n = 4 to 5). (E) FC analysis of pulmonary resident CD4+ T cells in transferred Rag1–/– mice. The percentages and counts of the resident CD4+ T cells were shown (n = 4 to 5). (F) Flow histogram indicated Ki-67 intensity in lung-resident CD4+ T cells. The graph compared the MFI of Ki-67 (n = 4). (G) FC analysis of CD69 and CD103 expressions within CD4+ TRM cells. The graph compared the proportions of CD103 to CD103+ in the CD69+ CD4+ TRMcells (n = 4 to 5). (H) FC analysis of TRM-Tregs (FOXP3+). The percentages of TRM-Tregs were displayed (n = 4). (I) FC plots for KLRG1 and CD103 in the TRM-Tregs. The graph compared the ratios of CD103+KLRG1+ in the TRM-Tregs (n = 4). (J) Flow histogram compared CD25 and ICOS expressions in the TRM-Tregs. The graph showed the MFI (n = 4). The graphs showed the combined results of 4-5 independent experiments. The recipient Rag1–/– mice were littermates. Individual mice are plotted on the graphs. Values are reported as mean ± SD. P value was determined by one-way ANOVA followed by Tukey’s test.