In order to confirm that there was a direct link between the increased (ox-)LDL-uptake and the formation of suppressive CAT-DCs, we inhibited the most prominent LDL-uptake receptors LDL-R, LRP1 and CD36

In order to confirm that there was a direct link between the increased (ox-)LDL-uptake and the formation of suppressive CAT-DCs, we inhibited the most prominent LDL-uptake receptors LDL-R, LRP1 and CD36. to enhanced H2O2-production with a consecutive increase in LDL-uptake throughout differentiation of monocytes to monocyte-derived dendritic R112 cells and, as a result, to the induction of distinct immunosuppressive properties. These findings shed new light on the role of LDL-metabolism in tumor-immunology and might help to further improve immunotherapeutic approaches against cancer. Abstract Background and Aims: Induction of myeloid-derived suppressor cells (MDSC) is a critical step in immune cell evasion by different cancer types, including liver cancer. In the liver, hepatic stromal cells orchestrate induction of MDSCs, employing a mechanism dependent on hydrogen peroxide (H2O2) depletion. However, the effects on monocyte-derived dendritic cells (moDCs) are unknown. Methods: Monocytes from healthy donors were differentiated to moDCs in the presence of extracellular enzymatic H2O2-depletion (hereinafter CAT-DCs), and studied phenotypically and functionally. To elucidate the underlying molecular mechanisms, we analyzed H2O2- and LDL-metabolism as they are interconnected in monocyte-driven phagocytosis. Results: CAT-DCs were of an immature DC phenotype, particularly characterized by impaired expression of the costimulatory molecules CD80/86. Moreover, CAT-DCs were able to suppress T-cells using indoleamine 2,3-dioxygenase (IDO), and induced IL10/IL17-secreting T-cellsa subtype reported to exert immunosuppression in acute myeloid leukemia. CAT-DCs also displayed significantly increased NADPH-oxidase-driven H2O2-production, enhancing low-density lipoprotein (LDL)-uptake. Blocking LDL-uptake restored maturation, and attenuated the immunosuppressive properties of CAT-DCs. Discussion: Here, we report a novel axis between H2O2- and LDL-metabolism controlling tolerogenic properties R112 in moDCs. Given that moDCs are pivotal in tumor-rejection, and lipid-accumulation is associated with tumor-immune-escape, LDL-metabolism appears to LSH play an important role in tumor-immunology. = 6 experiments with SD values). (C) Flow-cytometric analysis of CD11c on ex-vivo monocytes, mDCS and CAT-DCs. (D) Exemplary histograms of cell surface markers expressed on mDCs and CAT-DCs after 7 days of culture as analyzed by flow cytometry. Gray plots: isotype controls; MFIs represents the MFIs of all cells R112 analyzed by flow cytometry for the according surface marker (raw data can be found in Figure S3). (E) Flow-cytometric analysis of maturation-marker-expression on CAT-DCs as compared to mDCs. (F) Formation of an adherent, morphologically distinct subpopulation in DCs differentiated and matured in the presence of catalase; exemplary images shown for mDCs (1) and CAT-DCs (3) after 7 days of culture vs. mDCs (2) and CAT-DCs (4) culture dishes after gentle detachment by washing. (G) Comparison of CD163 expression of mDCs, non-adherent CAT-DCs, and adherent CAT-DCs (= 3). General gating strategy for flow-cytometric analysis of DC surface markers with dead cell exclusion using SYTOX blue dead cell stain and definition of mDCs as CD80+/CD86+ and CAT-DCs as CD80low/?/CD86? can be viewed in Figure S1. Statistical analysis was performed with a paired two-tail Students < 0.05, ** < 0.01, *** < 0.001. When maturing monocytes to DCs in the presence of catalase we noticed a change in morphology and adhesive properties (Figure 1F). Untreated mDCs were easily detachable after 7 days in culture (Figure 1(F1): pre-wash, Figure 1(F2): post-wash), but we observed a morphologically distinct, adherent subpopulation that could only be detached by R112 firm washing when DCs were differentiated and matured in the presence of catalase (CAT-DCs) (Figure 1(F3): pre-wash, (F4): post-wash). Interestingly, 54% (< 0.05) of normally detached CAT-DCs and 82% (< 0.001) of adherent CAT-DCs expressed CD163 (Figure 1G), a surface marker indicative of M2-macrophages [27]. All other surface markers analyzed were unaffected in comparison to non-adherent CAT-DCs (Figure S2). 3.2. CAT-DCs Are Capable of Suppressing Pan-T-Cell Proliferation Next, we investigated whether the effects of catalase are of functional relevance, by comparing the immune-stimulatory capacity of CAT-DCs and mDCs in coculture with allogenic Pan-T-cells. In so doing, CAT-DCs showed 5-fold reduced capacity to stimulate T-cells in vitro compared to untreated mDCs in the absence of CD3/CD28-stimulation (Figure 2A). Open in a separate window Figure 2 CAT-DCs suppress T-cell proliferation in a contact dependent, IDO-mediated manner. (A) Analysis of stimulatory capacity of CAT-DCs on PAN T-cells in comparison to mDCs after 5 days of coculture without addition of CD3/28-beads (= 6). (B) Proliferation of CD3/CD28-stimulated T-cells cocultured with CAT-DCs for 5 days. Proliferation of T-cells in CAT-DC-cocultures are shown relative to stimulated control (= 6). (C) Representative flow cytometry plots of T-cell proliferation following CD3/CD28-stimulation after 5 days of CAT-DC coculture. T-cells were stained with Tag-it Violet Proliferation and Cell Tracking Dye to visualize proliferation. (D) Comparison of proliferation of CD8+ T-cells, CD4+ T-cells, and CD3+ PAN-T-cells in coculture with CAT-DCs at a ratio of 4:1 T-cell:CAT-DC following CD3/CD28-stimulation (= 3, representative plots: Figure S4C; General gating strategy for T-cell-analysis can be found in Figure S5). (E) Analysis of CD3/28-bead stimulated T-cell-proliferation applying physical separation (transwell inserts) throughout coculture of T-cells with CAT-DCs in.