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+CD103–TRM 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
CD103–CD69+ 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.