15 deoxy δ12 14 pgj2

Isolated CD8⁺ T cells from OT-I mice were maintained in the naive status after CRISPR-Cas9 gene targeting by resting them overnight in complete medium added with IL-7 (Peprotech) at 10 ng/ml. Isolated T cells were otherwise activated using plate-bound anti-CD3 (5 μg/ml, inVIVOMAb Cat# BE0002) in complete medium added with 100 U/ml IL-2 (Peprotech) and soluble αCD28 (0.5 μg/ml, inVIVOMAb Cat# BE0015) under 5% CO₂, atmospheric oxygen, at 37 °C in a humidified incubator. At day 2 post activation, and then daily up to day 6, media were refreshed and cells counted and replated at a density of 10⁶ cells/ml [for (IL-2)-T cells] or 1.5 × 10⁶ cells/ml [for (IL-15/TGF-β)-T cells]. (IL-2)-T cells were maintained in 100 U/ml IL-2 throughout the culture; to generate (IL-15/TGF-β)-T cells, cells were cultured in 10 ng/ml IL-15 and 10 ng/ml TGFβ starting from day 3 post-activation. Where indicated cells were treated with vehicle control (0.1% ethanol), PGE₂ (Sigma) at the indicated concentrations and for the indicated time, PGE₁ (Sigma), PGF_2α (Sigma), PGI₂ sodium salt (Sigma) and 15-deoxy-Δ12-14 PGJ₂ (Sigma) at 100 nM, bafilomycin A1 (Sigma) at 100 nM, Wortmannin (PI3K inhibitor, Selleckchem) at 10 μM and SBI-0206965 (ULK1 inhibitor, Sigma) at 2 μM.

Carbaprostacyclin (cPGI₂), AL 8810, baicalein, and 17-octadecynoic acid (17-ODYA) were purchased from Cayman Chemical. 15-Deoxy-Δ^12,14 PGJ₂, PGE₂, PGF_2α, SC-560, SC-236, and indomethacin were all purchased from Sigma-Aldrich. PD98059, U0126, and GF109203X were obtained from Enzo Life Sciences and BAPTA-AM from Tocris Bioscience.