Fig. 2

TNF-α increases the survival and proliferation of Th cells in vitro. (a) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing conditions with or without the addition of TNF-α for 6 h. The experiments were repeated three times. Cell samples (S1–3) were analyzed by RNA-seq. Pink-blue heatmap shows the log₂-fold change of the differentially regulated expression of genes related to cell death pathways. Pink, higher expression; blue, lower expression. (b) Mouse naïve CD4⁺ T cells were cultured under Th0 or Th9 polarizing conditions in the presence or absence of TNF-α for 3 days. Flow cytometry analyzed Annexin V⁺ T cells. Numbers in the histograms represent the percentages of Annexin V⁺ T cells. Right, summarized results of three independent experiments obtained as the left. (c) Naïve CD4⁺ T cells were labeled with CFSE and cultured under Th0 or Th9 polarizing conditions in the presence or absence of TNF-α for 3 days. Flow cytometry analyzed CFSE-stained T cells. Numbers in the histograms represent the fluorescence intensity (FI) of CFSE-stained T cells. Right, summarized results of three independent experiments obtained as the left. MFI, mean fluorescence intensity. (d, e) Naïve CD4⁺ T cells were cultured as shown in (b). (d) qPCR assessed the expression of Pdcd1 in cultured T cells. (e) Flow cytometry analyzed the expression of CD44 by T cells. Numbers in the histograms represent the percentages of CD44⁺ T cells. Right, summarized results of three independent experiments obtained as the left. Data are representative of three (b, c, e) independent experiments or presented as mean ± SD of three (b-e) independent experiments. *P < 0.05