Supplementary Figure 4: Kyn regulates TAM polarization in vivo via AHR.

(a) Nanostring analysis of peripheral infiltrated macrophages (Lin^NegCD11b⁺CD45^Hi) in GBM from WT and AHR^LysM mice 15 days after GL261 cells implantation (pool of 4 mice per group). Ingenuity pathway analysis of macrophage polarization genes is shown. (b) Tumor size in WT mice 7 days after implantation of GL261-control and GL261-TDO/IDO cells (n = 3 independent mice). Representative images and quantification (left and right, respectively). Data are representative of two independent experiments with similar results. (c,e) Ido1 and Tdo2 expression in tumor tissue from WT mice injected with GL261- control and GL261-TDO/IDO (n = 3 independent mice) (c), and in CT2A cells (n = 3 biologically independent samples) (e). Representative of two independent experiments with similar results. (d) Arg1 expression in sorted TAMs from WT mice injected with GL261-control and GL261-TDO/IDO cells (n = 3 independent mice). Representative of two independent experiments with similar results. (f) Schematic of AHR binding sites (XREs) in the Klf4 promoter. The arrows indicate primers designed to study AHR (sites 1-6) recruitment. (g,h) Klf4 gene was silenced by siRNA in bone marrow derived macrophages. The cells were stimulated with TCM from GL261 cells for 24 hours and gene expression of M1- and M2-like genes was analyzed by qPCR (n = 3 technical replicates). Data are representative of two independent experiments with similar results. (i) Ingenuity Pathway Analysis of NF-κB signaling using data from Nanostring analysis of peripheral infiltrated macrophages (Lin^NegCD11b⁺CD45^Hi) in GBM from WT and AHR^LysM mice 15 days after GL261 cells implantation (pool of 4 mice per group). Colors indicate up- and down-regulation of individual pathway components in red and green, respectively. (j,k) Socs2 expression (j) and TRAF6 protein levels (k) in TCM-stimulated BMDMs from WT and AHR^LysM mice. Data in j were repeated two times with similar results, with three technical replicates. The experiment in k was repeated two times with similar results; images were cropped and the full scans are shown in Supplementary Fig. 2d. All data are presented as mean ± s.e.m, Unpaired two-tailed t test was used to compare two groups (b-e, h and j) and one-way ANOVA was used to compare three or more groups (g).