T-cell metabolism is homeostatically regulated by aPC, which induces Treg frequency in vivo. (A) Bar graph with dot plot showing the percentages of CD4⁺FOXP3⁺ cells in the spleen and thymus in WT and APC^high mice (flow cytometry) (n = 4). (B) Seahorse analysis showing the OCRs (line graph summarizing the results) in CD4⁺CD25⁻ cells isolated from WT and APC^high mice and stimulated ex vivo with plate-bound αCD3 and αCD28 for 48 hours, n = 5. (C) Seahorse analysis showing the OCR (line graph summarizing the results) in CD4⁺CD25⁻ isolated from WT and APC^high mice and stimulated ex vivo with plate-bound αCD3 and αCD28 without (APC^high) or with α-KG supplementation (48 hours, 3.5 mM, WT + α-KG; APC^high + α-KG). (D) Representative bar graph with dot plot summarizing CD4⁺FOXP3⁺ expression in CD4⁺CD25⁻ cells isolated from WT and APC^high mice and stimulated with plate-bound αCD3 and αCD28 and experimental conditions as described in panel C (n = 5). (E) Schematic illustration of the experimental timeline and analysis. WT mice were injected once (1×) and 3 times (3×) with aPC (1 mg/kg). Control mice denoted later as WT were injected with PBS. (F) Seahorse analysis depicting the OCR (line graph summarizing results) in CD4⁺CD25⁻ cells isolated from experimental mice as described in panel E, and stimulated ex vivo with plate-bound αCD3 and αCD28 for 48 hours. (G) Representative flow cytometry plots and bar graph with dot plot summarizing CD4⁺FOXP3⁺ expression in splenocytes⁻ isolated from WT mice injected with PBS or aPC once (1×) or thrice (3×) (n = 5). The data are shown as the mean ± SEM; statistical significance was determined by 2-tailed Student t test for panel A; 2-way ANOVA for panels B-C,F; 1-way ANOVA for panels D,G. Significance is represented in panel B: ∗P < .05 (WT vs APC^high); panel C: ∗P < .05 (WT vs APC^high), ^#P < .05 (WT vs WT + α-KG), ^$P < .05 (APC^high vs APC^high + α-KG); panel F: ∗P < .05 (WT vs aPC 1×), ^#P < .05 (WT vs aPC 3×); and panels A,D,G: ∗P < .05, ∗∗P < .01, and ∗∗∗P <.005.