a–c, ChIP analysis of H3K27ac, p300 and H3K4 mono-methylation (H3K4me1) on the Foxp3 gene locus. Chromatin obtained from DMSO- and isoalloLCA-treated wild-type cells were immunoprecipitated with IgG, anti-H3K27ac, anti-p300 or anti-H3K4me1 antibodies, followed by real-time PCR analysis (n = 3 biologically independent samples per group). Primers targeting Foxp3 promoter (Pro), CNS1, CNS2 and CNS3 region and Hsp90ab1 promoter were used for qPCR quantification. Relative enrichment was calculated as fold change relative to the ChIP signal at the Foxp3 promoter of the DMSO-treated control. d, e, Flow cytometry and quantification of CD4+ T cells stained intracellularly for FOXP3. Naive CD4+ T cells isolated from wild-type mice (n = 2 biologically independent samples per group) were cultured with anti-CD3/28, IL-2 and TGFβ (0.05 ng ml−1) in the presence of DMSO or isoalloLCA (20 μM) in the presence or absence of iBET. f, ChIP analysis of H3K27ac on the Foxp3 promoter region. Naive CD4+ T cells isolated from wild-type or CNS3-knockout mice (n = 3 biologically independent samples per group) were treated with DMSO or isoalloLCA (20 μM). g, Seahorse analysis of oxygen consumption rate (OCR) with naive CD4+ T cells isolated from wild-type or CNS3-knockout mice cultured with anti-CD3/28 and IL-2 for 48 h, in the presence of DMSO or isoalloLCA (20 μM). Measurements from six wells from two mice for each genotype. h–k, T cells were cultured with DMSO, LCA, isoLCA, alloLCA, isoalloLCA or 3-oxoLCA at 20 μM for 48 h. Their mitochondrial and cytoplasmic ROS were measured by mitoSOX (h) and 2′,7′-dichlorofluorescein diacetate (DCFDA) (i), respectively. Total mitochondria mass was measured by MitoTracker (j) and the mitochondrial membrane potential measured by JC-1 dye (k). Mean fluorescence intensities of different treatments were normalized as fold changes of those of the DMSO control (n = 3 biologically independent samples per group). l, MitoROS production measured by mitoSOX with T cells cultured with DMSO, isoalloLCA (20 μM), retinoic acid (1 nM), or isoalloLCA (20 μM) + mitoQ (0.5 μM) for 48 h. m, ChIP analysis (n = 3 biologically independent samples per group) of H3K27ac on the Foxp3 promoter of T cells, treated with DMSO, isoalloLCA, isoalloLCA + mitoQ or isoalloLCA + anti-TGFβ for 72 h. n–q, MitoROS production measured by mitoSOX with T cells cultured with different concentrations of anti-CD3 and treated with DMSO, isoalloLCA (20 μM), TGFβ (0.05 ng ml−1) or isoalloLCA plus TGFβ (n = 2 biologically independent samples per group) (n); or with T cells treated with DMSO or isoalloLCA (20 μM) plus an isotype control or anti-TGFβ antibody (n = 4 biologically independent samples per group) (o); or with T cells cultured under T H 1, T H 2, T H 17 or T reg cell conditions (n = 3 biologically independent samples per group) (p); or with naive CD4+ T cells isolated from wild-type or CNS3-knockout mice and cultured with anti-CD3/28 and IL-2 (n = 3 biologically independent samples per group) (q). r, MitoROS production measured by mitoSOX with T cells cultured with DMSO or mitoPQ (5 μM) for 48 h. s, Dose-dependent effects of mitoPQ on T reg cell differentiation (n = 3 biologically independent samples per group). t, Quantification of T reg cell differentiation in vitro on naive CD4+ T cells cultured in the presence of DMSO or mitoPQ (5 μM) and treated with isotype control or anti-TGFβ antibody (n = 3 biologically independent samples per group). u, A model showing the mechanism of isoalloLCA enhancement of T reg cell differentiation. Data are representative of two independent experiments (l, r) and shown as mean ± s.d., by unpaired t-test with two-tailed P value. Source Data