Fig 2: c-FLIP protects in vitro generated M-MDSCs from chemotherapy-induced death. a Representative flow cytometry plots of mouse BM-MDSCs after chemotherapy treatment. Untreated Ly6C+ cells are in black squares; in red and in blue, Ly6C+ cells after exposure to the highest dose of chemotherapy that maintains the cell viability (defined as =75% of AnnV-/7AAD- cells). b Suppressive activity of untreated (black bar) or chemotherapy-treated BM-MDSCs (red bars indicate M-MDSC-affecting chemotherapeutics whereas blue bars indicate ineffective chemotherapeutics) was measured by CellTrace dilution in peptide-activated CD8+ T cells. c Expression levels of both c-FLIPL and c-FLIPS in Ly6C+ cells purified from BM-MDSCs treated with different chemotherapy were evaluated by real-time PCR. d Flow cytometry analysis of c-FLIP infected human in vitro generated BM-MDSCs (identified as CD34- cells) after 5-fluorouracil treatment. e Functional assay on luciferase- and c-FLIP-infected BM-MDSCs derived from in vitro differentiation of hCD34+ cells, either left untreated or after exposure to a M-MDSC-affecting dose of 5-fluorouracil. Data are presented either as mean ± s.e.m of three independent experiments (b–e) or as a representative experiment of three independent experiments (a). *P < 0.05, **P < 0.01; ***P < 0.001; n.s., not significant, by Mann–Whitney test

Fig 3: Circulating c-FLIP expressing monocytes increase in PDAC patients. a Enumeration of circulating c-FLIP-expressing monocytes (CD14+ cells) in healthy donors (HD, n = 25) and in neuroendocrine tumor (NET, n = 5) and pancreatic ductal adenocarcinoma (PDAC, n = 25) patients. b PD-L1 expression on HD, NET and PDAC circulating monocytes that express c-FLIP at different levels. c PD-L2 expression on HD, NET, and PDAC circulating monocytes that express c-FLIP at different levels. d Kaplan–Meier curves for overall survival (OS) and disease-free survival (DFS) in PDAC patients (n = 71) by the percentage of FLIP+PD-L1+CD14+ cells and FLIP+PD-L2+CD14+ cells, or e high serum levels of IL-6 (IL-6high) and FLIP+PD-L1+CD14+ cells. The optimal cutoff thresholds were obtained based on the maximization of the Youden’s statistics (J = sensitivity + specificity + 1). *P < 0.05, **P < 0.01; ***P < 0.001; n.s., not significant, by Mann–Whitney test (a–c). Survival curves were compared by log-rank test (d, e)

Fig 4: Infected c-FLIP expressing CD14+ cells activate MDSC-associated molecular programs. a Supervised clustering of c-FLIP arrays using 1,552 differentially expressed genes (FDR <0.01 and absolute fold change >2). b Gene set enrichment analysis (GSEA) for association between c-FLIP overexpression and Hallmark (n = 50) and Biocarta (n = 217) gene sets denoting the activation of specific signaling pathways with a statistical confidence of 95%. NES, normalized enrichment score; FDR, false discovery rate q-value; GeneRatio, the percentage of gene hits before the peak in the running enrichment score used as an indication of the percentage of genes contributing to the enrichment score. Dotplot was generated using the ggplot function of the ggplot2 R package. c Validation of immune markers induced by c-FLIP in monocytes was performed by real-time PCR (c-FLIPL, c-FLIPS, and IDO1), by high-performance liquid chromatography (HPLC) in culture supernatants (kynurenine production), by ELISA quantification of culture media (IL-10) and flow cytometry (CD38, PD-L1, and PD-L2). Data are presented as mean ± s.e.m. of five independent experiments (c). *P < 0.05, **P < 0.01; ***P < 0.001; n.s., not significant, by Mann–Whitney test

Fig 5: c-FLIP-engineered monocytes transfer controls GvHD progression. a Immunosuppressive functions of CD14+ monocytes freshly isolated from buffy coats of healthy donors and infected with either luciferase- (as control) or c-FLIP-expressing lentivirus vector was measured by enumeration of absolute number of human CD3+ T cells collected after co-culture with engineered monocytes. b Kaplan–Meier survival analysis of mice developing xenogeneic GvHD and treated with adoptive cell therapy based on the administration of human engineered, c-FLIP-expressing monocytes: untreated mice, n = 10; luciferase-treated mice, n = 22 and c-FLIP-treated mice, n = 18. c Frequency of circulating human CD3+ T lymphocytes and Treg (CD3+CD4+CD25+FoxP3+ cells) in mice with GvHD treated with the administration of either luciferase- or c-FLIP-expressing monocytes. d Percentage of IFN-?-producing human CD8+ T cells isolated from the spleen of mice with GvHD treated with the administration of either luciferase- or c-FLIP-expressing monocytes and stimulated in vitro with polyclonal activators (PMA/IONO). e Representative histological analysis of tissues (spleen, lung, and skin) of mice developing GvHD and treated with human-engineered monocytes. Analysis was performed at day 70 after PBMC transplantation. f Human CD3+ T cell infiltration in tissues (spleen, lung, and skin) of mice developing GvHD and treated with engineered monocytes. Analysis was performed at day 70 after PBMC transplantation. Data are presented either as mean ± s.e.m of five independent experiments (a) or as mean ± s.e.m of seven independent samples (c–f) or as cumulative data of three independent experiments (b). *P < 0.05, **P < 0.01; ***P < 0.001; n.s., not significant, by Mann–Whitney test (a, c–f) and by log-rank test (b)