Fig. 3: HMB, a BCAT1-mediated leucine metabolite, is involved in the regulation of Th17 responses.

a Public RNA-seq data (GSE140244) were analyzed to examine the expression of BCAA catabolic enzymes at the indicated times in TCR-stimulated human CD4⁺ memory T cells. Heatmap analysis illustrating the time course of changes in the expression of major BCAA catabolic enzymes. b The expression of the HPD, HPDL, and BCKDK in human CD4⁺ T cells was validated by RT‒qPCR at 24 h poststimulation (n = 4). c Scheme of cytosolic leucine metabolism. d CD4⁺ memory T cells were pretreated with Bi2 (10 μM), a statin (10 μM), or HMB (0.4 mM) for 1 h and stimulated with anti-CD3/CD28-coated microbeads for 3 days. The accumulation of cholesterol in cell lysates was measured (n = 3). e The amount of IL-17A in the culture supernatant from TCR-stimulated CD4⁺ memory T cells was measured on Day 3 by ELISA (n = 4). f TCR-stimulated CD4⁺ memory T cells were cultured for 3 days with Bi2 and the indicated concentration of acetate. The amount of IL-17A was measured by ELISA (n = 5). g The amount of IL-17A in the culture supernatant of CD4⁺ memory T cells supplemented with HMB was measured by ELISA (n = 5). h, i Freshly isolated human CD4⁺ memory T cells were activated and infected with GFP lentivirus containing HPD or HPDL shRNA for 24 h. shRNA⁺ cells expressing GFP were sorted and cultured for another 3 days with TCR stimulation. Expression of the indicated genes in sorted shRNA⁺ cells (n = 3) (h). The mRNA (left) and protein (right) levels of IL-17 and IFN-γ in the culture supernatant were analyzed by RT‒qPCR (n = 3) and ELISA (n = 3) (i). The graphs show the means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 according to the Mann‒Whitney U test (b, e), one-way ANOVA with the Kruskal‒Wallis test (d, f, g), or two-tailed unpaired t test (h, i), respectively.