Unlike previous publications, we only observed a weak expression of CD244 and CD115 at the surface of Ly6G+ cells.24 These molecules were more expressed at the surface of Gr1+ and Ly6C+ subsets (Fig.?4B). co-stimulatory CD86 and MHCII and more co-inhibitory CD274 molecules in HCC-bearing livers than in control livers. Corresponding to this phenotype, Kupffer cells from HCC-bearing mice were less efficient in their function as antigen-presenting cells. Three CD11b+ cell populations were identified and sorted from HCC-bearing mice. These cells had various phenotypes with different levels of MDSC-specific surface Dynemicin A markers (Ly6Ghigh cells, Gr1high cells, and Ly6Clow cells), and may be considered as bonafide MDSCs given Dynemicin A their suppression of antigen-specific T cell proliferation. Primary isolated Kupffer cells in co-culture with the three MDSC subsets showed a decrease in CCL2 and IL-18 secretion, and an increase in IL-10 and IL-1 secretion, and an increased expression of CD86, CD274, and MHCII. In conclusion, these data demonstrated the existence of three MDSC subsets in HCC-bearing animals. These cells altered Kupffer cell function and may decrease the migration and activation of anticancer effector cells in the Dynemicin A liver. mouse model of HCC, we aimed (1) to assess the phenotype and activation level of Kupffer cells in the presence of HCC, (2) to characterize all involved MDSC subsets in such a model, and (3) to explore the effect of MDSCs on Kupffer cell phenotype and function. Results Kupffer cells in HCC and in the liver parenchyma surrounding the tumor In order to specifically study Kupffer cells (and not circulating macrophages), we have developed a protocol of liver perfusion, non-parenchymal cells isolation, and specific flow cytometry-labeling strategy (Fig.?1A). Liver mononuclear cells were isolated from livers of control and HCC-bearing mice, and F4-80high MHCII+ cells were identified. To exclude Kupffer cell/endothelial cell doublets (some are not excluded in the classical SSC-Height/SSC-Area plot), an anti-CD68 membrane labeling was performed. CD68 is highly expressed at the surface of endothelial cells, and primarily in the cytoplasm of Kupffer cells (19 and data not shown). Single Kupffer cells were therefore selected as CD68low cells. In addition, the selected population did not express Ly6C, while circulating macrophages do express this marker.20,21 Open in a separate window Figure 1. liver digestion and F4-80high MHCII+ cells were assessed. Single Kupffer cells were therefore selected as CD68low and Ly6C? cells. The expression of the positive and negative co-stimulatory molecules CD86 and CD274 (PD-L1) was assessed. (B) CD86, CD274, and MHCII expression levels in Kupffer cells from control HCC-free mouse liver, and from the surrounding parenchyma from mice with tumor of less or more than 0.5?cm diameter, and from the HCC-bearing median lobe. (MFI: Mean Fluorescence Intensity). We further analyzed whether Kupffer cells in the liver lobes harboring Rabbit Polyclonal to FAKD1 HCC expressed positive and negative co-stimulatory molecules differently than Kupffer cells residing in non-tumorous liver lobes (surrounding parenchyma) or in control livers (Fig.?1B). CD86 expression was lower in both tumor-bearing and surrounding liver parenchyma compared to controls (Mean Fluorescence Intensity [MFI]: 75 and 99.9, respectively, compared to 158 in controls). In contrast, CD274 (also known as Programmed Death-Ligand 1 (PD-L1)) was increased both in tumor tissue and surrounding parenchyma compared to control liver parenchyma (MFI: 290 and 370, respectively, compared to 223 in controls). This distinct phenotype was more pronounced when the tumor diameter was greater than 0.5?cm. The capacity of Kupffer cells to present antigen was also assessed (Fig.?1B). Membrane MHCII expression was decreased on Kupffer cells from the tumor surrounding parenchyma compared to cells from control liver (MFI: 108 vs. 529). Kupffer cells from HCC-bearing animals have a decreased antigen-presenting activity Kupffer cells have an important role as antigen-presenting cells, and their efficiency for that purpose is related to the presence of co-stimulatory molecules.4 To determine whether the observed co-stimulatory phenotype was related to cell functionality, Kupffer cells from control and HCC-bearing mouse livers were incubated with CFSE-labeled CD4+ T cells from OT-II mice (Fig.?2). This antigen-presentation assay revealed a decreased proliferation of CD4+ T cells upon antigen presentation by Kupffer cells from HCC-bearing livers as compared to controls (ratio 1C1: 50.23% proliferation using Kupffer cells from controls versus 12.1% using Kupffer cells from HCC-bearing animals). Of note, a 3-h pre-incubation with lipopolysaccharide (LPS) decreased the ability of Kupffer cells to stimulate the antigen-specific proliferation of CD4+ T.