Fig 2: ETV3 and ETV6 are essential for mo-DC differentiation.a, Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484). a.u., arbitrary units. b–g, Monocytes were cultured with M-CSF, IL-4 and TNF. b, At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. GP96 was used as a loading control. Representative results are shown (n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t-test). c–f, ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c,e, Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown (n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t-test). d,f, Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown (n = 8) and the median (n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g, ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor (n = 6 in three independent experiments). h, Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor (n = 5 in two independent experiments; paired one-way ANOVA). For all panels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.Source data

Fig 3: Type I interferon increases mo-Mac differentiation and ETV3 and ETV6 repress interferon-stimulated genes.(a-c) Monocytes were cultured for 5 days with M-CSF, IL-4 and TNFα, in absence or presence of 100 or 1000 U/mL IFNα or IFNβ. (a) Monocyte differentiation after 5 days. DN = double negative. One representative donor is shown (n = 6). Percentage of mo-Mac, mo-DC or DN cells. Median is shown (n = 6 in 2 independent experiments). Paired-one way Anova. (b) Percentage of live cells. Each symbol represents an individual donor (n = 6). Median is shown. Paired-one way Anova. (c) Percentage of CD163+ or CD1b+ cells among DN cells. Median is shown (n = 6 in 2 independent experiments). Paired-one way Anova. (d-f) Monocytes were cultured with M-CSF, IL-4 and TNFα for 3 days. ChIP-seq analysis was performed for ETV3 or ETV6. (d-e) Overlap of differentially expressed ISG found in RNA-seq and genes identified in ChIP-seq for ETV3 (d) and ETV6 (e) (Wald test). (f) Gene tracks from ChIP-seq analysis for the genomic regions of ISG15, MX1 and MX2. IP = immuno-precipitation. All statistical tests were two-sided.

Fig 4: ETV3 and ETV6 repress mo-Mac transcriptional program and differentiation.a–d,f,g, Monocytes were cultured with M-CSF, IL-4 and TNF for 3 d. ChIP–seq analysis was performed for ETV3 or ETV6. a, Motif enrichment analysis obtained with HOMER. b, Most enriched motifs. c, Overlap of identified genes for ETV3 or ETV6 immunoprecipitation (IP). Peaks in all gene regions or only in the transcription start site (TSS) region are shown. d, Overlap of DEGs found in RNA-seq and genes identified in ChIP–seq. e,f, Data from RNA-seq analysis. e, GSEA of monocyte, mo-Mac and mo-DC signatures in control (red) versus silenced (blue) samples. NES, normalized enrichment score. f, Heatmap of DEGs belonging to the monocyte, mo-Mac or mo-DC signatures. Samples were ordered by condition and genes were ordered manually. Genes detected in ChIP–seq analysis are emboldened. g, Gene tracks from ChIP–seq analysis for the genomic region of MAFB. h–j, Monocytes were cultured with M-CSF for 5 d. ETV6 was overexpressed using a lentivirus containing an expression plasmid. h, Protein quantification by immunblotting. GP96 was used as a loading control. Representative results are shown (n = 5 in two independent experiments). Paired samples derive from the same experiment and were processed in parallel. i, Mo-Mac differentiation. One representative donor is shown (n = 11). j, Percentage of mo-Macs after 5 d (n = 11 in four independent experiments; paired Student’s t-test: ***P < 0.001). All statistical tests were two sided.Source data

Fig 5: ETV3 and ETV6 control monocyte differentiation independently of their effect on ISGs.a,b, Monocytes cultured for 5 d with M-CSF, IL-4 and TNF, in the absence or presence of 100 or 1,000 U ml−1 of IFN-α. a, Monocyte differentiation. DN, double negative. One representative donor is shown (n = 8). Percentage of mo-Mac, mo-DC or DN cells at day 5. The median is shown (n = 8 in three independent experiments; paired one-way ANOVA). b, Expression of CD163 and CD1b in the DN cells. Histograms of one representative donor are shown (n = 8). Percentage of CD163+ or CD1b+ cells among DN cells. The median is shown (n = 8 in three independent experiments; paired one-way ANOVA). c, Monocytes were cultured with medium only or combinations of M-CSF, IL-4 and TNF, in the presence or absence of 1,000 U ml−1 of IFN-α. MX1 and MAFB expression is measured by RT–qPCR after 3 h and PPARG and ZNF366 after 6 h. The median is shown (n = 5 in two independent experiments; paired two-way ANOVA). The asterisks represent tests against the same condition without IFN-α. Ctrl, Control. d–h, Monocytes cultured with M-CSF, IL-4 and TNF for 5 d, in the presence or absence of recombinant B18R. ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. d, Monocyte differentiation. One representative donor is shown (n = 9). e,f, Percentage of mo-Macs, mo-DCs or DN cells at day 5 after ETV3 (e) or ETV6 (f) silencing. The median is shown (n = 9 in three independent experiments; paired Student’s t-test). g,h, Expression of ETV3, ETV6 and selected ISGs measured by RT–qPCR after ETV3 (g) or ETV6 (h) silencing (n = 5 for ETV3 and 6 for ETV6; paired two-way ANOVA). For all panels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All statistical tests were two sided.