Fig. 2: SLC7A5-mediated leucine influx regulates Th17 responses.

a mRNA expression of SLC7A5, BCAT1, and BCAT2 was analyzed by RT‒qPCR at 24 h poststimulation in human naive and memory CD4⁺ T cells (n = 5). b CFSE-labeled naive and memory CD4⁺ T cells were stimulated with anti-CD3/CD28-coated microbeads for 4 days with or without BCAAs, Bi2 (10 μM), or JPH203 (10 μM) (n = 5). The proportion of proliferating cells was measured by CFSE dilution assay. c, d The amount of cytokines in the culture supernatant of TCR-stimulated CD4⁺ naive T cells (c) and CD4⁺ memory T cells (d) stimulated with anti-CD3/CD28-coated microbeads for 3 days under the indicated conditions (n = 5). e The amount of IL-17A (left) and IFN-γ (right) in culture supernatant from TCR-stimulated CD4⁺ memory T cells supplemented with leucine and Bi2 (n = 7–8). f, g Freshly isolated human CD4⁺ memory T cells were activated and infected with GFP lentivirus containing BCAT1 shRNA for 24 h. shRNA⁺ cells expressing GFP were sorted and cultured for another 3 days with TCR stimulation. BCAT1 expression in sorted shRNA⁺ cells (n = 5) (f). The mRNA (left) and protein (right) levels of IL-17 and IFN-γ in the culture supernatant were analyzed by RT‒qPCR (n = 5) and ELISA (n = 5) (g). The graphs show the means ± SEMs. *p < 0.05 and **p < 0.01 according to the Mann‒Whitney U test.