IL-4 prevents adenosine-mediated immunoregulation by inhibiting CD39 expression
Fengqin Fang, Wenqiang Cao, Yunmei Mu, Hirohisa Okuyama, Lingjie Li, Jingtao Qiu, Cornelia M. Weyand, Jörg J. Goronzy
Fengqin Fang, Wenqiang Cao, Yunmei Mu, Hirohisa Okuyama, Lingjie Li, Jingtao Qiu, Cornelia M. Weyand, Jörg J. Goronzy
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Research Article Aging

IL-4 prevents adenosine-mediated immunoregulation by inhibiting CD39 expression

Abstract

The ectonucleotidase CD39 functions as a checkpoint in purinergic signaling on effector T cells. By depleting eATP and initiating the generation of adenosine, it impairs memory cell development and contributes to T cell exhaustion, thereby causing defective tumor immunity and deficient T cell responses in older adults who have increased CD39 expression. Tuning enzymatic activity of CD39 and targeting the transcriptional regulation of ENTPD1 can be used to modulate purinergic signaling. Here, we describe that STAT6 phosphorylation downstream of IL-4 signaling represses CD39 expression on activated T cells by inducing a transcription factor network including GATA3, GFI1, and YY1. GATA3 suppresses ENTPD1 transcription through prevention of RUNX3 recruitment to the ENTPD1 promoter. Conversely, pharmacological STAT6 inhibition decreases T cell effector functions via increased CD39 expression, resulting in the defective signaling of P2X receptors by ATP and stimulation of A2A receptors by adenosine. Our studies suggest that inhibiting the STAT6 pathway to increase CD39 expression has the potential to treat autoimmune disease while stimulation of the pathway could improve T cell immunity.

Authors

Fengqin Fang, Wenqiang Cao, Yunmei Mu, Hirohisa Okuyama, Lingjie Li, Jingtao Qiu, Cornelia M. Weyand, Jörg J. Goronzy

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Figure 4

GATA3 prevents RUNX3 binding to the ENTPD1 promoter.

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GATA3 prevents RUNX3 binding to the ENTPD1 promoter. (A and B) T cells w...
(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.