Extended Data Fig. 3: EPO-EPOR downstream signaling is activated in cDC1s following TLI/ATS.
From: Erythropoietin receptor on cDC1s dictates immune tolerance
a-c, Gene Set Enrichment Analysis (GSEA) of transcriptional profiles using the Hallmark gene set of MSigDB. NES, normalized enrichment score; FDR, false discovery rate. Red: upregulated; Blue: downregulated. TLI/ATS vs. UNT. b, Upregulated gene sets. c, Downregulated gene sets. d-e, Intracellular phospho-flow cytometric analysis of EPO-EPOR downstream signaling in live-dead blue−Lin−SiglecH−PDCA-1−CD11chighMHCIIhigh. Spleens were harvested on the next day following the last dose of TLI or TLI/ATS. UNT (n = 4) vs. TLI (n = 4) vs. TLI/ATS (n = 4). d, XCR1+CD8α+ cDC1s and e, XCR1−CD8α− cDC2s. f,g, Histograms and MFI of the indicated EPO-EPOR downstream signaling molecules with fluorescence minus one (FMO) as controls by intracellular phospho-flow staining on the next day following the last dose of TLI/ATS treatment. Eporflox/flox (n = 4) vs. EporΔXcr1 (n = 5). cDC1s (f) and cDC2s (g). h, Ex vivo analysis of EPO-EPOR downstream signaling in splenic cDC1s. Splenic cDCs were MACS-purified with a pan-DC isolation kit and cultured at 5 × 106 cells/ml, then rested overnight. Cells were isolated from UNT or TLI/ATS-treated Eporflox/flox (n = 4; n = 4) and EporΔXcr1 (n = 4; n = 4) mice. cDCs from TLI/ATS-treated mice were stimulated ex vivo with EPO (10 IU/200 μl) or PBS (control) overnight. Phosphorylation of downstream signaling molecules was assessed by flow cytometry, after gating on XCR1+SIRPα− splenic cDC1s. Data are shown from one experiment, representative of at least two independent experiments with similar results (d-h). Statistical analysis was performed using unpaired two-tailed Student’s t-test (f,g), or one-way ANOVA Tukey’s multiple-comparison test (d, e and h left), or paired two-tailed Student’s t-test (h right). Data are mean ± s.e.m. (d-h).
