In addition, immunostimulants such as CpG DNA inhibit DC apoptosi

In addition, immunostimulants such as CpG DNA inhibit DC apoptosis 18, whereas the deficiency of pro-apoptotic Bim protein in DC results in autoimmunity 19. Immature DC have the ability to acquire protein complexes or soluble antigen using many different pathways such as macropinocytosis, endocytosis and even through ingestion of entire cells. Despite the importance of DC apoptosis in the immune response, studies have not investigated the

effects of DC death on viable DC. In this study, we show that viable DNA Damage inhibitor immature DC have the ability to uptake apoptotic DC. The uptake of apoptotic DC or necrotic DC is recognized as an immunologically null event. However, it is the uptake of apoptotic DC that suppresses subsequent maturation of viable DC in response to LPS and results in upregulation of TGF-β2 and preferential secretion of TGF-β1, which mediates induction of naïve T cells into Foxp3+ Treg. In contrast, the uptake of apoptotic splenocytes by viable immature DC does not result in TGF-β1 secretion, nor does it result in induction of Foxp3+ Treg. Therefore, it is likely the uptake of apoptotic DC by viable DC that provides a potential to induce Foxp3+ Treg. Bone-marrow-derived DC were treated with UV light, and apoptosis

induction was assessed at 1 and 6 h after UV treatment. Prior to UV treatment, cells were mostly positive for Hoechst 3342 (a cell permeant DNA-binding stain, blue) with very few cells being check details positive for annexin V (a phosphatidylserine-binding protein, green), indicative of live DC. One hour after UV treatment, majority of the cells

were positive for both annexin V and Hoechst 3342, with very few cells positive for ethidium homodimer (EH) (a nuclei probe, impermeant to live Ribonuclease T1 or apoptotic cells, red) (Fig. 1A). In these cells, there was translocation of phosphatidylserine on the membrane as indicated by positive annexin V staining, but the membrane integrity was still maintained, as they were mostly negative for EH stain; hence, they can be classified as apoptotic cells. In contrast, 6 h after UV treatment, there was a pronounced increase in EH positive cells, indicating that the membrane integrity was compromised. However, these cells were also positive for annexin V (Fig. 1A). Therefore, these cells can be classified as late apoptotic cells. In order to further confirm apoptosis in a quantitative manner, 1 or 6 h after UV treatment, DC were stained with annexin V and propidium iodide (PI), and apoptosis was assessed via FACS analysis. Prior to UV treatment, approximately 10% of DC were annexin V+PI–, whereas 1 h after UV treatment approximately 45% of DC were annexin V+PI–, indicative of apoptotic cells and confirming our above findings (Fig. 1B). At 6 h post-UV treatment, approximately 80% of cells were annexin V+PI+, indicating that these cells were in late apoptosis (Fig. 1B).

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