Fig 2: STAT6 inhibition in a T cell response upregulates CD39 expression.(A) Purified total T cells were activated for 5 days by anti-CD3/CD28 Dynabeads. Each day, cells were examined for the expression of pSTAT6, GATA3, and CD39 in CD4 or CD8 T cells by flow cytometry. In addition, the level of pSTAT6 was also compared in T cells with or without IL-4 receptor blocking Abs. (B) Purified total T cells were activated for 3 days by anti-CD3/CD28 Dynabeads in the presence or absence of IL-4 (20, 50, or 100 ng/mL), IL-4 receptor blocking Abs (1 or 2 µg/mL), or STAT6 inhibitor AS1517499 (100 nM) as indicated. GATA3 expression was assessed by Western blotting. (C) Purified total T cells were activated in the presence or absence of the STAT6 inhibitor AS1517499 (100 nM). After 4 days, CD39 expression was evaluated by flow cytometry. (D) Total T cells were activated for 2 days before nucleofection with STAT6-specific siRNA smartpool. After 2 additional days, cells were collected for ENTPD1 and STAT6 mRNA quantification by real-time qPCR. (E) CD45.1+ naive SMARTA cells were isolated and injected into CD45.2 B6 WT mice via tail vein, followed by LCMV Armstrong infection and daily i.p. injection of the STAT6 inhibitor AS1517499 or DMSO solvent control. After 6 days, SMARTA cells were isolated from spleen and examined for CD39 expression using flow cytometry. Representative contour plots (upper panel) and summary data (lower panel). Data in D are shown as mean ± SEM and compared by 2-tailed paired or unpaired Student’s t test in C and E.

Fig 3: 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.

Fig 4: 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.