Fig 1: Selective AHR inhibition reverses IDO/TDO-mediated tumor progression and improves the efficacy of PD-1 blockade.a Top left: scheme figure with experimental approach of targeting multiple steps of Trp-catabolic pathway. Top right: therapy regimen (PO, QD). Bottom: tumor progression of orthotopically injected B16WT, B16IDO and B16TDO tumors in mice treated with vehicle or inhibitors (Epacadostat/IDO inhibitor 300 mg/kg/d, 680C91/TDO inhibitor 20 mg/kg/d, CH-223191/AHR inhibitor 50 mg/kg/d). (n = 5 mice per group for two independent experiments). b FACS analysis of MHCII expression (n = 4 per group, n = 5 B16IDO vehicle). c Quantification of frequency and representative plots of intratumor Tregs (CD4+FoxP3+CD25+/% of CD45+) and d, quantification of frequency and representative plots of Ki67+PD-1+ CD8+ T cells from B16WT and B16IDO-bearing mice treated with vehicle or AHR inhibitor (CH-223191) (n = 4 per group). e Top: therapy regimen. bottom: mean tumor size and overall survival of B16IDO and B16TDO tumor-bearing mice treated with AHR inhibitor (CH-223191), anti-PD-1 alone or in combination with AHR inhibitor (combo) (n = 5 B16TDO and n = 10 B16IDO). f Mean tumor size of B16IDO and CT26 and g, overall survival of B16IDO tumor-bearing mice treated with the optimized AHR inhibitor (KYN-101), anti-PD-1 alone or in combination with AHR inhibitor (combo) (n = 10 mice per group). Results are representative of two independent experiments. Data represented as mean values ± SEM. Two-tailed Student’s t test was used when only two groups were compared and log-rank (Mantel–Cox) test was used for survival comparison. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 2: EVs facilitated CD8+ T cell exhaustion.A The infiltrating CD8+ T cells sorted from HCC mice were divided into three groups: normal CD8+ T cells (PD1-TIM3-), partially exhausted CD8+ T cells (PD1int TIM3+), and completely exhausted CD8+ T cells (PD1hi TIM3+). PKH67-labeled EVs were co-cultured with CD8+ T cells. B The absorption of EVs by CD8+ T cells was detected using immunofluorescence. C The expressions of inhibitory receptors in CD8+ T cell surface (PD1 and TIM3) were detected using flow cytometry. D The expressions of effector cytokines (IFN-?, IL-2, and TNF-a) were detected using flow cytometry. E The proliferation ability of CD8+ cells was measured using flow cytometry. F The ability of CD8+ cells to kill Hep1-6 cells was measured using flow cytometry. The cell experiment was repeated 3 times. Data are presented as mean ± standard deviation and analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test, **p < 0.01.
Fig 3: Overexpression of YOD1 reversed the effect of EVs on CD8+ T cell exhaustion. pcDNA-YOD1-transfected CD8+ T cells were treated with EVs.A YOD1 mRNA expression was detected using RT-qPCR. B The expressions of PD1 and TIM3 were detected using flow cytometry. C The expressions of IFN-?, IL-2, and TNF-a were detected using flow cytometry. D The proliferation ability of PD1-TIM3- and PD1int TIM3+ cells was measured using flow cytometry. E The ability of PD1-TIM3- and PD1int TIM3+ cells to kill Hep1-6 cells was measured using flow cytometry. The cell experiment was repeated 3 times. Data are presented as mean ± standard deviation and analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test, **p < 0.01.
Fig 4: Combination therapy-mediated MB49 tumour rejection is dependent on CD4+ and CD8+ T cells.Isolated TILs from 13–15 days tumours, 1–3 days after the last treatment with a-CTLA-4, a-PD-1, combination of both (Combo) were analyzed by flow cytometry for (a) Total CD4+ T cells and CD8+ T cells. Representative flow panels are shown on the left and pooled results from five mice are depicted in bar graphs on the right. (b) Foxp3+ Treg in CD4+ T cells. Pooled results from five mice are shown in the right bar graph. (c) Ratios of Foxp3- to Foxp3+ in CD4+ T cells. (d) Ratios of CD8+ T cells to Foxp3+ CD4+ T cells. Mice depleted of CD4+ (GK1.5), CD8+ (2.43) or both (GK1.5+2.43) were treated with the combo and mouse survivals are presented (e). Data in the bar graphs are means±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by one-way ANOVA with Bonferroni's post hoc test. Data are representative of three independent experiments.
Fig 5: An active IDO/TDO-Kyn-AHR pathway associates with immune suppressive features in human cancers.a Top: heat-map representing the gene-expression analysis of Kyn-degrading enzymes (KMO, KYNY, HAAO) and KD-Score (grayscale) in responsive (complete response/CR or partial response/PR) highlighted in black (n = 8), or non responsive (stable disease/SD or progressive disease/PD) highlighted in gray (n = 31), melanoma patients after PD-1 blockade. Bottom: bar graph with quantification of KD-Score. b ELISA quantification of L-Kynurenine in blood serum of melanoma patients (left) categorized as IDOhigh or IDOlow (n = 4 IDOhigh and n = 4 IDOlow) based on intracellular IDO staining by FACS of melanoma cell suspensions (right) (n = 8 IDOhigh and n = 10 IDOlow). c mRNA of immunoregulatory markers by qRT-PCR analysis in IDOhigh and IDOlow melanoma cell suspensions. d mRNA of AHR-target genes CYP1A1 and CYP1B1 by qRT-PCR analysis in IDOhigh and IDOlow melanoma cell suspensions after treatment with selective AHR inhibitor KYN-101 for 24 h (n = 6 IDOhigh and n = 6–7 IDOlow). e Distribution of log-transformed expression levels of AHR-related genes (TDO2, AHR, and CYP1B1) across six immune subtypes of cancer (C1: wound-healing, C2: IFN-? dominant, C3: inflammatory, C4: lymphocyte-depleted, C5: immunologically quiet, C6: TGF-ß dominant). Data plotted as box and whiskers with the median and limits within the 10–90% percentile. f Correlation analysis between Treg marker (FOXP3), myeloid-cell marker (MRC1/CD206), inhibitory checkpoint (PDCD1/PD-1), and AHR-related genes IDO1, TDO2, and CYP1B1 in TCGA RNAseq data of skin melanoma (SKCM), squamous lung (LUSC) and pancreatic adenocarcinoma (PDAC) analyzed by Spearman rank correlation. Data shown are represented as mean values ± SEM with two-tailed unpaired Student’s t test in (a–d), one-way ANOVA test with Tukey correction in (a) and Kruskal–Wallis with Dunn correction in (e). P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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