Volume 21, Issue 1, Pages 154-167 (October 2017) Prostaglandin E2 Leads to the Acquisition of DNMT3A-Dependent Tolerogenic Functions in Human Myeloid-Derived Suppressor Cells  Javier Rodríguez-Ubreva, Francesc Català-Moll, Nataša Obermajer, Damiana Álvarez-Errico, Ricardo N. Ramirez, Carlos Company, Roser Vento-Tormo, Gema Moreno-Bueno, Robert P. Edwards, Ali Mortazavi, Pawel Kalinski, Esteban Ballestar  Cell Reports  Volume 21, Issue 1, Pages 154-167 (October 2017) DOI: 10.1016/j.celrep.2017.09.018 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 154-167DOI: (10.1016/j.celrep.2017.09.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Characterization of the In Vitro System to Differentiate MOs to MDSCs (A) Scheme depicting the differentiation system. (B) Analysis of MDSC, DC, and MO surface markers analyzed by flow cytometry, including CD14, HLA-DR, CD33, and CD1a. One representative experiment is shown. (C) Effects on allogeneic CD8+ proliferation of in-vitro-differentiated MDSCs and DCs (DC or MDSC:CD8+ ratio 1:2). CD8+ T cell proliferation was analyzed by flow cytometry considering CD8+ T proliferating cells those in which CFSE staining is decreased (n = 9). One histogram example corresponding to 1:2 ratio is shown. (D) IFN-γ release by allogeneic CD8+ T cells in the presence of DCs or MDSCs and CD8+ cells alone, quantified by ELISA (n = 3). (E) qRT-PCR analysis of selected MDSC-associated genes (ARG1, NOS2, IL10 PTGS2/COX2, IL12B, and IRF8) in MDSCs, DCs, and MOs. Data are relative to HPRT1 and RPL38 (n = 4). (F) Analysis by bisulphite (BS) pyrosequencing of selected genes (MO n = 3, DC n = 4, MDSC n = 4). The CpG sites analyzed for each gene are marked with a red line in the scheme placed on top of each graph, where the TSS is marked with an arrow, the remaining CpG sites with black vertical lines, and CpG islands (CGI) in green. Results are mean ± SEM. Mann-Whitney and t tests were used to determine significance (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n.s., not significant). Cell Reports 2017 21, 154-167DOI: (10.1016/j.celrep.2017.09.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Global Analysis of DNA Methylation Changes in MDSC and DC Differentiation Reveals an MDSC-Specific Gain of DNA Methylation (A) DNA methylation heatmap of three paired samples (D1, D2, and D3) of MOs and their derived MDSCs and DCs. The heatmap includes all CpG-containing probes displaying significant methylation changes (15% of differential of beta values; p < 0.01 and FDR < 0.05). A scale is shown at the bottom ranging from −2 (lower DNA methylation levels, blue) to +2 (higher methylation levels, red). (B) Volcano plots representing –log10(p value) versus log2(fold change). In red are hypermethylated probes and in blue are hypomethylated probes; in gray are probes that do not pass the cutoff criteria. (C) Distribution of differentially methylated CpGs among genomic regions; intergenic, promoter (including 1,500 and 200 upstream of the TSS), gene body (including 5′ UTR and first exon), and 3′ UTR, for MO-to-MDSC and MO-to-DC differentiation. Odds ratios have been used to avoid bias due to DNA methylation array design. (D) Location of differentially methylated CpG sites in the context of CpG islands (CGIs). Odds ratios have been used to avoid bias in the relative distributions due to DNA methylation array design. (E) Analysis of DNA methylation by bisulphite pyrosequencing of selected genes during MDSC and DC differentiation (n = 4). A schematic representation of each gene is depicted. Arrows refer to TSS and transcription direction (in red the analyzed CpGs and in green CGI location). (F) Gene expression heatmap of three paired samples (D1, D2, and D3) of MOs and their derived MDSCs and DCs. (G) Correlation between DNA methylation and gene expression in MDSCs versus MOs and MDSCs versus DCs. Only differentially methylated CpG sites and differentially expressed probes are represented in the graphs. Dot colors refer to genomic location (3′ UTR, gene body, intergenic, or promoter) of analyzed CpG sites. (H) Correlation between DNA methylation and gene expression along the different clusters identified in DNA methylation array. Expression data from DC and MDSC are expressed in comparison with MO expression values (dotted line) of genes located at the previously identified DNA methylation clusters. (I) Gene expression profile of selected genes during MDSC and DC differentiation corresponding to one example of each DNA methylation cluster. Results are mean ± SEM. Mann-Whitney was used to determine significance. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s., not siginificant. Cell Reports 2017 21, 154-167DOI: (10.1016/j.celrep.2017.09.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 MDSCs Generated by Co-culture of MOs with Ovarian and Colorectal Cancer Cells Also Display the MDSC-Specific Hypermethylation Signature (A) Scheme depicting the co-culture differentiation system where control cells were generated in the absence of tumor cells and MDSCs in the presence of tumor cells. (B) Allogeneic CD8+ T cell suppression assay using DCs, generated in the absence of tumoral cells or MDSCs generated in a co-culture of MOs with tumor cell lines (MDSCs [HCT] were generated with HCT116 and MDSCs [OVC] with OVCAR8) for 6 days and myeloid cells isolated with anti-CD33. DCs or MDSCs were then co-cultured with CFSE-stained CD8+ cells for 3 days (1:2 ratio), and then CD8+ cells proliferation was analyzed by flow cytometry considering CD8+ proliferating cells those in which CFSE staining is decreased (n = 3). (C) Effect on IFN-γ release by allogeneic CD8+ cells in the presence of MDSCs or DCs generated using the co-culture protocol (n = 3). (D) Flow cytometry analysis of MOs, MDSCs, and DC surface markers CD1a and CD14. (E) DNA methylation analysis by bisulphite pyrosequencing of selected hypermethylated genes in MDSCs generated by PGE2 and by the co-culture model with HCT116 (HCT) and OVCAR8 (OVC) cells (n = 3). (F) Heatmap of three biological paired samples corresponding to DCs (DC1, DC2, and DC3) and MDSCs (MDSC1, MDSC2, and MDSC3) generated in the absence or presence of OVCAR8 cells and boxplot showing the average methylation levels (beta values) of genes in cluster 1. (G) Analysis by qRT-PCR of the mRNA levels of EP1, EP2, EP3, and EP4 in human monocytes (n = 20). (H) Violin plots showing the average methylation levels (beta values) of genes in cluster 1 (specifically hypermethylated in MDSCs) in MOs, DCs, and MDSCs generated in the absence and presence of anatagonists (5 μM) for EP2 and EP4 (5 μM each), both individually and combined. (I) Bisulphite pyrosequencing of two example genes from cluster 1 (S1PR4 and RUNX1) showing the effects of treatment with EP2 and EP4 antagonists (n = 3). (J) Effect on CD8+ cell proliferation by MDSCs generated from MOs in the presence of GM-CSF/IL-4/PGE2 in the presence or absence of EP2 and EP4 antagonists (n = 3). (K) Effect on CD8+ cell proliferation by MDSCs generated from MOs co-cultured with HCT116 (left panel) or OVCA8 cells (right panel) in the absence or presence of EP2 and EP4 antagonists (n = 3). Results are mean ± SEM. Mann-Whitney test and t test were used to determine significance (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n.s., not significant). Cell Reports 2017 21, 154-167DOI: (10.1016/j.celrep.2017.09.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 DNMT3A Is Responsible for the Acquisition of the MDSC-Specific Hypermethylation Signature and the Immunosuppressive Phenotype (A) Time course qRT-PCR analysis and western blot showing the levels of DNMT3A and DNMT3B in MOs and in-vitro-differentiated DCs and MDSCs. (B) qRT-PCR of the mRNA levels of DNMT3A in MOs, DCs, and MDSCs and MDSCs differentiated in the presence of EP2 and EP4 antagonists. (C) Effects of specific siRNA for DNMT3A (si3a) or control siRNA (SC) on DNMT3A levels analyzed by qRT-PCR and western blot assay. (D) Effects on DNA methylation analyzed by bisulphite pyrosequencing following downregulation of DNMT3A. (E) Effect of DNMT3A downregulation on the expression levels of the DNMT3A gene and selected hypermethylated genes from cluster 1. (F) Effects on CD8+ T cell proliferation by MDSCs following downregulation of DNMT3A. (G) Effect on IFN-γ release by CD8+ T cells following the knockdown of DNMT3A. (H) ELISA showing the levels of secreted IL-12 in cultures of DCs or MDSCs, before and after exposure to LPS+ IFN-γ, using the cells transfected with either scrambled siRNAs (control) or siRNA specific for DNMT3A (3a). (I) Analysis by qRT-PCR of the DNMT3A mRNA levels in MOs, DCs, and MDSCs and MDSCs differentiated in the presence of CREB inhibitor 666-15 (100 nM). (J) Effect of CREB inhibitor 666-15 (100 nM) on some hypermethylated genes from cluster 1. (K) Effects on CD8+ T cells proliferation by MDSCs generated in the presence of CREB inhibitor 666-15 (100 nM). Results are mean ± SEM. To determine significance a t test was used (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n.s., not significant). Cell Reports 2017 21, 154-167DOI: (10.1016/j.celrep.2017.09.018) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Primary MDSCs Isolated from Ovarian Carcinoma Patients Also Display Hypermethylation of Key Myeloid Genes (A and B) Flow cytometry analysis of PDL-1 expression (including isotype controls) (A) or CD14 plus CD15 expression (B) in CD11b+ cells obtained from healthy donors (HD) and from peripheral blood (OvCa-Blood) or ascites (OvCa-Ascites) in ovarian cancer patients. One representative experiment for each condition is shown, as well as the percentage average of positive cells for each condition. Numbers in plots indicate the proportion of positive cells. (C) qRT-PCR analysis showing the levels of DNMT3A and DNMT3B in CD11b+ cells isolated from healthy donors (HD n = 15), ovarian cancer peripheral blood (OvCa-Blood n = 11), and acites (OvCa-Ascites n = 11). (D) Heatmap showing DNA methylation values corresponding to CpG sites from cluster 1 in CD11b+ cells isolated from peripheral blood of healthy donors (HD) and peripheral blood (OvCa-Blood) or ascites (OvCa-Ascites) from ovarian cancer patients. (E) Heatmaps corresponding to DNA methylation levels analyzed by bisulphite pyrosequencing of selected cluster 1 genes in CD11b+ cells obtained from peripheral blood (OvCa-Blood) and ascites (OvCa-Ascites) from ovarian cancer patients. CD11b+ cells isolated from peripheral blood of healthy donors (HD) are also analyzed. The top heatmap corresponds to CpG sites/genes from cluster 1 (including the array ID for analyzed CpG sites). The bottom heatmap includes additional CpG sites of genes in cluster 1 (indicating their genomic location). (F) DNA methylation levels analyzed by bisulphite pyrosequencing of selected example genes from cluster 1. (G) qRT-PCR of selected genes from cluster 1. (H) Allogeneic CD8+ T cells suppression assay using DCs and MDSCs generated in a co-culture of MOs with ascitic supernatants with or without EP2 and EP4 antagonists (10 μM each). DCs or MDSCs were then co-cultured with CFSE-stained CD8+ T cells for 5 days (1:2 ratio), and then CD8+ cells proliferation was analyzed by flow cytometry considering CD8+ proliferating cells those in which CFSE staining is decreased. (I) DNA methylation levels analyzed by bisulphite pyrosequencing of selected example genes from cluster 1 using DNA from DCs or MDSCs generated in a co-culture of MOs with ascitic supernatants with or without EP2 and EP4 antagonists (10 μM each). Results are mean ± SEM. Mann-Whitney test was used to determine significance (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n.s., not significant). Cell Reports 2017 21, 154-167DOI: (10.1016/j.celrep.2017.09.018) Copyright © 2017 The Author(s) Terms and Conditions