IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish
Fig 7
Tilapia requires mTORC1 signaling for Th1 cell development during E. piscicida infection.
(A, B) Spleen leukocytes from rapamycin treated or untreated tilapia were stimulated with CD3ε/CD28 mAbs for 12 h. (A) Western blot assay showed protein or phosphorylation levels of S6 and 4EBP1 in spleen leukocytes. (B) Overlaid histograms showed the phosphorylation levels of S6 in CD4-1+ T cells. (C-K) Tilapia individuals that infected with E. piscicida were treated with rapamycin or PBS. (C) Flow cytometry showed the spleen leukocytes stained with CD4-1 and CD3ε mAbs on 7 DPI. (D) Scatter plot figures showed the percentage and absolute numbers of T cells, n = 4. (E) Scatter plot figures showed the percentage and absolute numbers of CD4-1+ T cells, n = 4. (F, G) Spleen leukocytes harvested from the tilapia on 7 DPI were stimulated with P+I or not with the presence of Golgiplug in vitro for 4 h, and flow cytometry (F) and Scatter plot figures (G) showed the percentages of IFN-γ+CD4+ T cells, n = 4. (H, I, K) The mRNA levels of indicated molecules were examined by qPCR on 4 DPI, n = 6. (J) Tilapia individuals that infected with E. piscicida were treated with rapamycin or PBS in the presence or absence of recombinant IL-2. Tilapia was i.p. injected with BrdU 1 day before sacrifice, and flow cytometry showed the BrdU staining on gated CD4-1+ T cells on 5 DPI. These experiments were repeated for at least two independent times. *: p<0.05, **: p<0.01, ***: p<0.001, determined by a two-tailed Student’s t-test.