Journal: Cell reports
Article Title: AHR prevents human IL-1R1hi ILC3 differentiation to natural killer cells
Figure Lengend Snippet: Human IL-1R1 hi ILC3s express AHR and respond to AHR ligands in vitro ( A, B ) Real-time RT-PCR was used to assess: ( A ) TBX21 (TBET), EOMES, RORC and AHR mRNA expression ex vivo in human IL-1R1 hi ILC3s (Lin - CD34 - CD117 + CD94 - IL-1R1 hi ) and stage 4 (Lin - CD34 - CD117 +/- CD94 + ) mature CD56 bright NK cells, FACS-purified from SLT; or ( B ) IL-22, CYP1A1, RORC, EOMES , and TBX21 (TBET) mRNA expression after culture of IL-1R1 hi ILC3s for 14 d in the presence of IL-15 (1 nM; ) or IL-15 + IL-1β (10 ng/ml; ■), plus either carrier alone (DMSO, 1 μl/ml), or carrier containing AHR agonist (FICZ, 300 nM) or AHR antagonist (CH-223191, 3 μM). For ( A, B ), gene expression levels were normalized to 18S mRNA, and relative quantification was performed using the ΔΔCt method. Y axis depicts fold difference in mRNA expression, quantified relative to the condition in which expression was lowest (arbitrarily normalized to 1). ( C ) Absolute number of viable cells generated from culture of IL-1R1 hi ILC3s for 14 d with IL-15 plus DMSO, IL-15 plus AHR agonist FICZ, or IL-15 plus AHR antagonist CH-223191. Data in A, B and C presented as mean ± SEM (for A and B , n ≥ 4; for C , n = 7; * for P
Article Snippet: FACS-purified IL-1R1hi ILC3s were cultured in a round-bottom 96-well plate (Costar) at a starting density of 25,000 cells/ml in α-MEM medium containing 10% FBS, penicillin G (100 μg/ml), and streptomycin (100 μg/ml) (Invitrogen), supplemented with recombinant human IL-15 (1 nM; Amgen) with or without IL-1β (10 ng/ml; Peprotech), and either carrier alone (DMSO, 1 μl/ml; Invitrogen) or carrier containing AHR agonist (FICZ, 300 nM; Enzo Life Sciences) or AHR antagonist (CH-223191, 3 μM; Calbiochem) at concentrations previously reported to modulate TH 17 differentiation ( ).
Techniques: In Vitro, Quantitative RT-PCR, Expressing, Ex Vivo, FACS, Purification, Generated