Journal: Journal for immunotherapy of cancer
Article Title: Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes.
doi: 10.1136/jitc-2023-006728
Figure Lengend Snippet: Figure 4 AHR activation by kynurenine and its derivatives is enhanced in the absence of tryptophan. (A)HEK293-E cells were treated for 72 hours with kynurenine (80 µM), AHR inhibitor (CH223191, 10 µM) or both in tryptophan-free medium supplemented or not with 80 µM tryptophan in the presence of 10% FBS. CYP1A1 expression was measured by RT-qPCR analysis. (B)HEK293-E cells were treated for 24 hours with different concentrations of kynurenine in tryptophan-free medium supplemented or not with 80 µM tryptophan in medium without FBS. CYP1A1 expression was measured by RT-qPCR analysis. (C)Flow cytometric evaluation of kynurenine uptake in HEK293-E cells. Kynurenine fluorescence in HEK293-E cells treated with 80 µM kynurenine, in the presence or absence of the System L inhibitor, BCH (10 mM) in PBS at 37°C or 4°C. (D.)Kynurenine fluorescence in HEK293-E cells treated with 80 µM kynurenine and different concentrations of tryptophan in PBS for 4 min. (E, F)RT-qPCR analysis (E)and western blot analysis (F) of SLC7A5 expression in wildtype or GCN2-KO HEK293-E cells cultured for 24 hours in tryptophan medium or tryptophan-free medium. The quantification of LAT1 protein expression is shown in the lower panel, each point representing a technical replicate. (G)Scramble or GCN2-KO HEK293-E cells were cultured overnight in tryptophan-free medium supplemented or not with 80 µM tryptophan without FBS. Cells were then collected and incubated with or without 80 µM kynurenine during 4 min in PBS without tryptophan. Kynurenine uptake was measured by flow cytometry analysis of kynurenine fluorescence. (H)RT-qPCR analysis of CYP1A1 expression in wild-type (upper panel) or GCN2-KO (lower panel) HEK293-E cells treated for 24 hours with kynurenine (80 µM) or FICZ (1 µM) in tryptophan-free medium supplemented or not with 80 µM tryptophan in the absence of FBS. (I)HEK293-E cells were treated for 72 hours with 80 µM kynurenine metabolites in tryptophan-free medium supplemented or not with 80 µM tryptophan in the presence of 10% FBS. CYP1A1 expression was measured by RT-qPCR analysis. (J)HEK293-E cells were treated for 24 hours with different concentrations of kynurenic acid in medium with or without tryptophan and without FBS. CYP1A1 expression was measured by RT-qPCR analysis. (K) RT- qPCR analysis of AADAT expression in wildtype or ATF4-KO HEK293-E cells cultured for 72 hours in tryptophan-free medium supplemented or not with 80 µM tryptophan in the presence of 10% FBS. The mRNA levels of different genes were measured by quantitative RT-qPCR and normalized to GAPDH. Mean±SD of technical triplicates from one representative experiment out of three independent experiments, except for panel F and K, which are Mean±SD of technical replicates from one representative experiment out of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. AHR, aryl hydrocarbon receptor; FBS, fetal bovine serum; ns, not significant; PBS, phosphate-buffered saline.
Article Snippet: AHR (clone D5S6H) rabbit monoclonal antibody, ATF4 (clone D4B8) rabbit monoclonal antibody, GCN2 (#3302S) rabbit polyclonal antibody, LAT1 (#5347S) rabbit polyclonal antibody was purchased from Cell Signaling and diluted at 1/1000.
Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Fluorescence, Western Blot, Cell Culture, Incubation, Flow Cytometry, Saline
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