Fig 2: Sirt6 deletion affects the cell number and function of thymic tuft cells.a Downregulated genes or upregulated genes in Sirt6-deficient mTECs are enriched in KEGG pathways, and the top five pathways are ordered by p value. All pathways were selected under the standard of p < 0.05. b Scatter plot shows the difference in TPM values of taste transduction genes between WT and Sirt6-deficient mTECs. c Differential expression of thymic tuft cell-related genes between mTECs sorted from WT and Sirt6 cKO mice. The color scale indicates the level of gene expression. d Representative images of frozen thymic sections from WT and Sirt6 cKO mice (n = 4 per group). Green, KRT5. Red, DCLK1. Scale bars: 100 µm. e Representative flow cytometric profiles and frequency of thymic tuft cells (CD45–EpCAM+MHCIIlowDCLK1high) obtained from 4-week-old Sirt6 cKO mice and littermates (n = 4 per group). f Representative flow cytometric profiles and frequency of L1CAM+ thymic tuft cells from 4-week-old Sirt6 cKO mice and littermates (n = 4 per group). g Representative flow cytometric plots and frequency of TCRßintCD1d+ thymic iNKT cells in 4-week-old Sirt6 cKO mice and littermates (n = 6 per group). h Representative flow cytometric plots and frequencies of NKT1 (PLZF–ROR?t–), NKT2 (PLZF+ROR?t–), and NKT17 (PLZF–ROR?t+) in 4-week-old Sirt6 cKO mice and littermates (n = 6 per group). i Representative flow cytometric plots and frequency of EOMES+TCRß+CD8 single-positive thymocytes in 4-week-old WT (n = 4) and Sirt6 cKO (n = 5) mice. j Representative flow cytometric plots of Lin–TCR–CD127+ thymic ILCs (upper) and ILC subsets (lower) in Sirt6 cKO mice and littermates. k, l Frequencies of Lin–TCR–CD127+ thymic ILCs (k) and Lin–TCR–CD127+Gata3+Ror?t– ILC2s (l) of WT (n = 5) and Sirt6 cKO (n = 6) mice. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the identical WT control mice (Student’s t-test). The error bar represents one standard deviation.

Fig 3: GATA3 suppresses CD39 expression.(A) T cells were transduced with GFP-expressing lentivirus, carrying the pCDH or pCDH-GATA3 construct. After 5 days following activation, CD39 expression was evaluated on GFP+ T cells by flow cytometry. Representative contour plots and summary data are shown. (B) 2 GATA3 binding sites are predicted in the ENTPD1 promoter region (–1468 to +211 from TSS) by MatInspector. Purified total T cells were activated for 3 days and collected for ChIP using anti-GATA3 Abs. Real-time qPCR was done on 2 predicted sites. The GATA3-binding site in the IL-4 gene was detected as a positive control. Data are shown as percentage of input. (C) HEK293 T cells were transfected with pcDNA3.1-GATA3 and pGL3-ENTPD1 promoter plasmids. pcDNA3.1 and pGL3-basic were used as control vectors. Renilla luciferase reporter plasmid was taken as a control for transfection efficiency. After 48 hours, luciferase activities were determined. Results are pooled from 3 experiments. Data are shown as mean ± SEM and compared by 2-tailed paired or unpaired Student’s t test in A and C. **P < 0.005.

Fig 4: GATA3 prevents RUNX3 binding to the ENTPD1 promoter.(A and B) T cells were transduced with GFP-expressing lentivirus, expressing pCDH-RUNX3 or pCDH (control vector), RUNX3-shRNA, or TR30021 (control vector) to overexpress or knockdown RUNX3 during T cell activation. After 5 days, CD39 expression on GFP+ T cells was determined by flow cytometry. Representative contour plots and summary data are shown. (C) HEK293 T cells were transfected with pcDNA3.1-RUNX3-FLAG, pcDNA3.1-GATA3-HA, or both. pcDNA3.1 was used as a control vector. After 48 hours, cells were collected for CoIP assay using anti-FLAG magnetic beads to pulldown RUNX3. Western blots of total lysates (upper panel) and anti-FLAG pull downs (lower panel) are shown. (D) Purified total T cells were activated for 4 days by anti-CD3/CD28 Dynabeads in the presence or absence of IL-4. Cells were collected for RUNX3 ChIP, followed by qPCR of 2 RUNX3 binding sites in the ENTPD1 promoter. Data are shown as a percentage of input. (E) Purified total T cells were activated by anti-CD3/CD28 Dynabeads under indicated conditions and collected on day 3 for Western blotting of RUNX3. (F) Splenocytes were isolated from WT and Cbfb–/– B6 mice and cultured on plates precoated with anti-CD3 and CD28 Abs (both 8 µg/mL) in the presence or absence of IL-4 (20 ng/mL). After 4 days, CD39 expression was compared by flow cytometry. Results are representative of 2 experiments. Data in D are shown as mean ± SEM and compared by 2-tailed paired or unpaired Student’s t test in A, B, and D.