Figure 4

Effects of LANCL2 pathway in CD4+ T cells and Th17 cells. CD4+ T cells and naïve T cells from WT and LANCL2^−/− spleens were isolated and cultured for 48 h. Naïve T cells were differentiated to Th17 cells. In CD4+ T cells, proportion of Th17 cells (A) were measured using flow cytometry analysis, IL17 (B) secretion was quantified through cytokine bead array, and proliferation (C) through CFSE staining. In Th17 differentiated cells, percentage of IL17+ cells (D) and Th17 proliferation (E) were assessed through flow cytometry analysis. Metabolic assessment was also conducted using Agilent Seahorse (F,I–K) and commercial metabolic kits (G,H,L–N). Extracellular Acidification Rate (F), ATP production from glycolysis (K) and LDH activity (L) were assessed in Th17-differntiated cells. Lactate production (G), hexokinase enzymatic activity (H), percentage of utilization of complete glycolytic pathway (I) and XF ATP Rate Index (mitoATP/GlycoATP, J) were measured in CD4+ T cells. CD4+ T cells were treated with the adenylate cyclase activator Forskolin and lactate production (M) and hexokinase enzymatic activity (N) were measured using the metabolic kits. CD4+ T cells were also treated with sodium oxamate, FX11, and gossypol (three inhibitors of LDH activity) and proliferative index (O) was assessed by CFSE staining and flow cytometry analysis. *P ≤ 0.05.