3.1 CS particles stimulated CD4+TRM cell emergence and expansion along with silicosis progression.
First, we utilized the in vivo labeling method distinguishing tissue-resident cells commonly used in multi-vascular tissues (Fig 1A)[26]. We observed a remarkable appearance of CD4+ TRM cells (CD44+CD45 i.v.) in the silicotic lung (Fig 1B), whereas few CD4+ TRMcells were found in naive mice. Unlike the circulating CD4+ TEM cells (CD44+CD45 i.v.+), CD4+TRM cells surged continuously with the progression of silicosis (Fig 1B and Fig S1B), suggesting a causal relation between CD4+ TRM cells and silicosis progression. Furthermore, the elevated expressions of cell retention marker, CD69, CD103, and CXCR6 further confirmed their lung retention ability (Fig 1C). With the expansion of CD4+TRM cells, there was an increasing number of CD69+CD103+ and CD69+CD103subsets (Fig 1D), implying their pathogenic role in silicosis. Comparatively, the phenotype of circulating CD4+TEM cells was analogic in saline and CS-treated mice, which differed from TRM cells (Fig 1E). Notably, CD69 and CD103 expressions on splenic CD4+TEM cells were not affected by CS injury in the lung (Fig 1F), indicating CS led to a tissue-local response. Collectively, these data demonstrated CS stimulated the emergence and expansion of pulmonary CD4+ TRM cells that were tightly related to silicosis progression.