EGFR-HIF1α signaling positively regulates the differentiation of IL-9 producing T helper cells

Interleukin 9 (IL-9)-producing helper T (Th9) cells are essential for inducing anti-tumor immunity and inflammation in allergic and autoimmune diseases. Although transcription factors that are essential for Th9 cell differentiation have been identified, other signaling pathways that are required for their generation and functions are yet to be explored. Here, we identify that Epidermal Growth Factor Receptor (EGFR) is essential for IL-9 induction in helper T (Th) cells. Moreover, amphiregulin (Areg), an EGFR ligand, is critical for the amplification of Th9 cells induced by TGF-β1 and IL-4. Furthermore, our data show that Areg-EGFR signaling induces HIF1α, which binds and transactivates IL-9 and NOS2 promoters in Th9 cells. Loss of EGFR or HIF1α abrogates Th9 cell differentiation and suppresses their anti-tumor functions. Moreover, in line with its reliance on HIF1α expression, metabolomics profiling of Th9 cells revealed that Succinate, a TCA cycle metabolite, promotes Th9 cell differentiation and Th9 cell-mediated tumor regression.


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All studies must disclose on these points even when the disclosure is negative. Sequence data that support the findings of this study have been deposited in GEO with the primary accession code, GSE163056. Publicly available data with accession code, GSE100634, were reanalyzed. The authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files.
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Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research All antibodies were validated by the supplier (BioLegend, BioXcell, abcam) and were checked in the lab by comparing to the manufacturer's or inhouse results. Statement from BioLegend: BioLegend antibodies undergo an extensive series of testing to ensure quality at every step in the manufacturing process, as well as maintaining quality after the sale. Statement from Bio X Cell: Our InVivoPlus™antibodies feature all the great qualities of our InVivoMab™ antibodies. The InVivoPlus™ versions of our products are structurally and functionally identical to the InVivoMab™ versions with the added benefit of additional QC measures.
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C57BL/6 (#00064), OT-II TCR (#004194) and Nos2-/-(#002596), Egfrflox/floxXCd4cre mice were provided by D.M.W. Zaiss, Egfrflox/ floxXCd4cre mice were performed at the University of Edinburgh, Areg-/-mice were provided by Fiona Powrie and Phd2kd and Hif1! kd mice were provided by Chris W. Pugh respectively. All animals used in the study were 6-12 weeks old and mixed gender. Laboratory animals were housed at institutional animal house facility maintained between 19 to 26°C ambient temperature with 30-70% humidity and 14 h light and 10 h dark cycle. All animals procedures were performed in laminar flow hoods.
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The experiments on Egfrflox/floxXCd4cre mice were performed at the University of Edinburgh in accordance with university ethical guidelines. The experiments on Areg-/-, Phd2kd and Hif1!kd were performed at Kennedy Institute of Rheumatology, University of Oxford, United Kingdom in accordance to the institutional ethical guidelines. The samples were further shipped on dry ice to THSTI,

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6-12 weeks old WT mice were euthanized and spleen and lymph nodes were collected aseptically. Single cell suspensions from spleen and lymph nodes were prepared after lysing red blood cells using ACK lysis buffer. Cells were then stained with the cell surface antibodies-anti-mouse CD4 PerCP ( In Vitro human T helper cells differentiation 10 ml of peripheral blood was collected from healthy human volunteers after written informed consent in accordance with the approval of the institutional human ethics committee. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using ficoll-paque based density gradient centrifugation and were then stained with the following cell surface fluorochrome-labelled antibodies: