Figure 2

Effect of NTG-A-009 on CD4⁺ T cell proliferation, viability and apoptosis. (a) Naïve CD4⁺ T cells isolated from spleen and lymph nodes were cultured with or without NTG-A-009 (1 μM) under Th1 condition for 72 hour and percentage of live cells were detected by Annexin-V and PI staining and analyzed by flow cytometry. (b) Apoptotic cells percentage was analyzed by treating with the different doses of NTG-A-009. (c) Naïve CD4⁺ T cells labelled with CFSE were activated with anti-CD3 and anti-CD28 under Th1 and Th17 conditions with the different doses of NTG-A-009. Cell proliferation was assessed by CFSE dilution by flow cytometry. (d) CD4⁺ T cell proliferation detection by the analysis of Ki-67 protein expression under Th1 condition with or without NTG-A-009. Bar diagram represent the percentage of Ki-67 positive CD4⁺ T cells. (e) Naïve CD4⁺ T cells and APCs were isolated from spleen and lymph node and stimulated under Th1 condition in the presence of BrdU (10 μM) with or without NTG-A-009. (f) Splenic lymphocytes were isolated from normal C57BL/6 mice and incubated with the different concentrations of NTG-A-009, Tofacitinib and Triamcinolone and Cell viability was measured by MTS assay. (g) CD4⁺ T cells isolated from spleen and lymph nodes were stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 24 hours in the presence of NTG-A-009 (1 μM), tofacitinib (1 μM) or triamcinolone (1 µM). The expression of CD25 was determined by flow cytometry. Data are the representative of three independent experiments. ***P < 0.001.