Fig 2: HIF1a and AhR mediate butyrate induction of IL-22 in CD4+ T cells.a WT CD4+ T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions ± butyrate (0.5 mM) for 2 days (n = 3 biologically independent samples per group). RNA sequencing was performed. Hif1a, Ahr, and Prdm1 were shown in heatmap. b–f CD4+ T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) under Th1 conditions (n = 3/group). Hif1a (b) and Ahr (c) were analyzed by qRT-PCR. HIF1a (d) and AhR (e) protein was analyzed by western blot on day 2. HIF1a activity was measured using HIF1a Transcription Factor Assay Kit (f). g Raw 264.7 cells were transduced with XRE/AhR Luciferase Reporter Gene Lentivirus, and treated ± butyrate (0.5 mM) 3 days post transduction. AhR activity was assessed by luciferase. h–j Cbir1 Tg CD4+ T cells were activated with APCs and Cbir1 peptide under Th1 conditions with butyrate (0.5 mM) ± YC-1 (5 µM) or/and CH-223191 (3 µM) for 60 h (n = 3/group). IL-22 mRNA (h) and protein (i) were measured by qRT-PCR and ELISA. j IL-22 was measured by flow cytometry on day 5. k WT and HIF1a-/- CD4+ T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 5 days (n = 3/group). IL-22 was assessed by flow cytometry. l, m CD4+ T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM), AR420626 (5 µM), or TSA (10 nM) for 60 h (n = 3/group). Hif1a (l) and Ahr (m) were measured by qRT-PCR. One representative of three independent experiments was shown (b–m). Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t-test (b–g) or two-tailed one-way ANOVA (h–m). b **p = 0.0033 (24 h), ***p = 0.0002 (36 h), **p = 0.0032 (48 h), *p = 0.0310 (60 h); c *p = 0.0338 (24 h), **p = 0.0054 (36 h), ***p = 0.0003 (48 h), ***p = 0.0007 (60 h); d *p = 0.0178; e *p = 0.0325; f *p = 0.0273; g *p = 0.0435; h ****p < 0.0001; i ****p < 0.0001, ***p = 0.0002 (butyrate + YC-1 vs butyrate), ***p = 0.0006; j ****p < 0.0001; k **p = 0.0015, *p = 0.0325; l **p = 0.0014 (butyrate vs control) and 0.0036 (AR429626 vs control); m ****p < 0.0001, ***p = 0.0009.

Fig 3: Butyrate promotes HIF1a binding to Il22 promoter in CD4+ T cells.a Schematic diagram of HIF1a binding to Il22 promoter. b WT CD4+ T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions for 2 days (n = 3/group). HIF1a binding to Il22 promoter was analyzed by CHIP assay. c–e WT CD4+ T cells were cultured under Th1 conditions with or without butyrate (0.5 mM) for 2 days (n = 3/group). HIF1a binding to Il22 promoter was analyzed by CHIP assay (c). The H3K9 acetylation (d) and trimethylation (e) levels in HIF1a-binding site on Il22 promoter were assessed by CHIP assay. One representative of three independent experiments (b, c), or two independent experiments (d, e) was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t-test. b **p = 0.0047; c *p = 0.0278; d *p = 0.0105; e **p = 0.0094.

Fig 4: Compound M351-0056 further enhanced the expression of human CD4+FoxP3+ in PBMCs. PBMCs (1 × 105) were stimulated with anti-CD3 co-coated with human VISTA-ECD (2.5-µg·ml-1 ) in the presence of IL-2 (10 ng·ml-1) and anti-CD28 (250-ng·ml-1 ) in the culture media, and compound M351-0056 was added as indicated. Staining of PBMCs with FITC-anti-human CD4 followed by intracellular staining with PE-anti-human Foxp3 using the Foxp3 staining buffers on Day 5

Fig 5: Stat3 and mTOR regulate IL-22 production by CD4+ T cells.a–d WT CD4+ T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) (n = 3/group). Phosphorylated Stat3 (6 h) (a, b) and phosphorylated mTOR (24 h) (c, d) were assessed by western blot and flow cytometry. Phosphorylated S6K was analyzed by flow cytometry (e). f–i CBir1 Tg CD4+ T cells were activated with APCs and CBir1 peptide under Th1 conditions with butyrate (0.5 mM) ± rapamycin (1 µM) or HJC0152 (1 µM). IL-22 mRNA (f) and protein (g) were assessed by qRT-PCR and ELISA at 60 h (n = 3/group). Expression of Hif1a (h) and Ahr (i) was analyzed at 48 h by qRT-PCR. j WT and Stat3-/- CD4+ T cells were treated with or without butyrate (0.5 mM) for 5 days (n = 3/group). IL-22 production was measured by flow cytometry. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t-test (a–e, j) or two-tailed one-way ANOVA (f–i). a *p = 0.0134; b ***p = 0.0002; c **p = 0.0059; d ***p = 0.0002; e **p = 0.0010; f, ****p < 0.0001; g **p = 0.0019 (butyrate vs control) and 0.0069 (butyrate + rapamycin vs butyrate), *p = 0.0141; h ***p = 0.0004, **p = 0.0012; i, ***p = 0.0004 (butyrate vs control) and 0.009 (butyrate + HJC0152 vs butyrate), **p = 0.0030; j **p = 0.0017, *p = 0.0338.