α-KG or glutamine restores Treg-phenotype caused by aPC. (A) Bar graph with dot plot showing FOXP3 expression in human CD4⁺CD25⁻ cells stimulated in an MLR (96 hours) (A, n = 4). Control CD4⁺CD25⁻ cells (T) compared with CD4⁺CD25⁻ cells preincubated with aPC (T + aPC) (1 hour, 20 nM) or with supplementation of α-KG (48 hours, 3.5 mM, T + aPC + α-KG) or glutamine (48 hours, 4 mM, T + aPC + Q). (B-C) Bar graphs with dot plots summarizing the expression of CD4⁺T-bet⁺ (B, n = 4) and CD4⁺IFNγ⁺ (C, n = 4) in CD4⁺CD25⁻ cells in the MLR as estimated by flow cytometry and described in panel A. (D) Representative flow cytometry histograms (top) and bar graph with dot plot (bottom) reflecting the percentage of proliferating human CD4⁺CD25⁻ cells in the MLR as described in panel A and quantified by CellTrace Violet cell proliferation dye (n = 5). (E) Representative flow cytometry plots and bar graph with dot plots of CD4⁺Ki-67⁺ cells after stimulation of human CD4⁺CD25⁻ cells with plate-bound αCD3 and αCD28 for 96 hours and treated as described in panel A. The data are shown as the mean ± SEM, and statistical significance was determined by 1-way ANOVA for panels A-E: ∗P < .05, ∗∗P < .01, and ∗∗∗P < .005.