3.2 Pulmonary CD4+ TRM cells mediated severe lung inflammatory response to CS particles.
In view of the immunologic memory role of TRM cells, we next sought to explore the response of CD4+TRM cells to CS particles by T-cell transfer studies. CD4+ T cells were sorted from the CS-treated lung containing certain TRM cells, the spleen under CS treatment only involving TEM cells, or the spleen of saline-treated mice, including naïve T cells, respectively. The sorted cells were adoptively transferred into Rag1–/– mice that lack T cells and then treated with CS (Fig 2A). H&E staining revealed that CS-induced the severest inflammation in the mice lung transferred CD4+ T cells sorted from CS-treated lung. In contrast, the mice who transferred CD4+ cells from the saline-treated spleen exerted relatively mild responses (Fig 2B and C). These phenotypes were further confirmed by the transcripts of cytokine and transcription factors associated with adaptive immunity, including Ifng , Tnfa ,Il17a , and Tbx21 , but not Il6 , related to innate immunity (Fig 2D). We further adopted flow analysis to the lungs. Significantly, more pulmonary resident T cells were observed in the mice transferred with CS-treated pulmonary CD4+ T cells (CD4+TRM) than those of naive counterparts (saline-treated splenic T cell) (Fig 2E), highlighting a rapid reaction and expansion of the CD4+ TRM cells to CS particles. In supporting the notion, a higher Ki67, cell proliferating marker, was observed in the TRM cells from the mice transferred CD4+ TRM (Fig 2F). While lung resident T cells in the mice transferred CD4+TEM cells (CS-treated splenic T cell) resembled those transferred with certain CD4+ TRMcells, implying a potential of TEM cells converting into TRM cells (Fig 2E). Besides, we discovered the ratio of CD103 to CD103+ in CD69+TRMwas affected by the distinct cellular sources that more CD69+CD103 subsets resided in the CD4+ TRM transferred mice (Fig 2G), implying the CD69+CD103TRM cells mediated proinflammatory effects to the invaded CS particles. Unexpectedly, there were fewer TRM-Tregs in the mice transferred with CS-treated pulmonary CD4+ T than those transferred with saline-treated splenic CD4+ T cells (Fig 2H). Further characterization indicated the TRM-Tregs from lung-transferred CS-treated pulmonary CD4+ T cells exerted a decreased ratio of KLRG1+CD103+ subsets (Fig 2I), suggesting an impaired Treg immunosuppressive function [27]. Moreover, these Tregs expressed lower CD25 and ICOS (Fig 2J), confirming the deficiency of suppressive functions [28,29]. Collectively, these results demonstrated CS particles stimulating TRM cell expansion mediated severe inflammatory responses in the lung, which was related to TRM’s rapid reaction to invaded CS particles, as well as the impaired Treg immunosuppressive function.
3.3 CS-induced TRM cells, especially TRM-Treg, depended on replenishment from circulating T cells.
We next sought to study the origin of CD4+TRM cells in silicotic lung. By applying FTY720, we blocked leukocyte egress from the peripheral lymphoid tissue, minimizing the recruitment of circulating leucocytes [22]. Particularly, the treatments at different time points within silicosis progression were employed to elucidate the origin and maintenance of CD4+ TRM cells (Fig 3A). H&E staining demonstrated that FTY720 treatment exerted protective effects on silicosis. However, though half-time intervention (4W-8W) reduced inflammatory cell recruitment, we could not get an equal attenuated phenotype compared to full-time blockage (0W-8W) (Fig 3B and C), suggesting that the TRM cells are derived from peripheral circulating cells. Specifically, full-time FTY720 treatment resulted in a significant vanish of CD4+TRM cells in number, albeit with surged percentages, while half-time FTY720 intervening reduced TRM cells but not as much as those of full-time treatment (Fig 3D), implying that CS-induced pulmonary TRM cells actively expanded in situ . In supporting the notion, high levels of Ki-67 were observed in the TRM cells but not impaired by FTY720 treatments (Fig 3E). Notably, we noticed affected ratios of CD103 to CD103+ in CD69+TRM by the FTY720 intervention. Full-time FTY720 treatment resulted in a high portion of CD103CD69+ TRMsubsets (Fig 3F), which was analog to the phenotypes of previous transfer experiments. We further tested the FTY720’s effects on the emergence of Tregs and found that full-time treatment decreased the portion of FOXP3+ Tregs in TRM cells (Fig 3G), reminding us that TRM-Tregs were prone to be replenished by circulation.
To further confirm the hypothesis, we aimed to perform a parabiosis model to illuminate the cellular source of the TRM cells in the silicotic lung. To do this, naïve CD45.1/2 mice were cojoined with naive congenic mice (CD45.1/1) by parabiosis surgery (Fig 3H). The blood circulation between parabionts was established 14 days later, indicated by an equal portion of CD45.1 and CD45.2 lymphocytes in the blood (Fig 3I), after which the CD45.1/1 congenic mice were exposed to CS particles. Another seven days later, we checked the component of circulating TEM cells and the pulmonary TRM in the CS-exposed mice. We found that the composition of CD45.2 lymphocytes in circulating effector cells was equal to the proportion in the blood (Fig 3J). Notably, we found that there were CD45.2+ T cells in the CD4+ TRM cells of CS-treated CD45.1/1 mice lung (Fig 3J), suggesting circulating T cells contributed to TRM cells formation. Significantly, when scrutinizing the origin of TRM subsets, we found that CD45.2+ cells accounted for a higher proportion in Tregs than those of Teff cells, further proving our hypothesis that Tregs depended more on circulating T cell replenishment (Fig 3K). Additionally, we observed that TRM-Tregs expressed higher CD103 than the Teff cells, while within TRM-Tregs, the newly recruited Tregs (CD45.2) expressed high adhesive molecule CD103, but not the TRM-Teff cells (Fig 3L). These results remind us that the pattern of CD103 and CD69 seems related to the constitutions of CD4+TRM subsets in the lung. Collectively, these data proved that CS-induced pulmonary CD4+ TRM cells came in two ways: recruited from circulation and proliferating in situ . While the TRM-Tregs were prone to be replenished by the circulation.