3 In contrast, monocyte-derived DCs (MoDCs) are generated during inflammation.4,5 Dendritic cells have been extensively characterized in a variety of species and protocols for obtaining DC subtypes range from in vitro culture methods to direct isolation of DCs from blood and tissues. Isolation, however, is complicated in humans and large animal species resulting in limited availability of functional studies. In pigs, blood

DCs (BDCs) have only been investigated in a few studies and very little is known about the function of these DCs in antigen presentation and T-cell activation. The objectives of the C59 wnt manufacturer present study were to compare directly isolated porcine BDCs with traditionally generated porcine MoDCs in terms of phenotype and functionality. Various porcine DCs have been described including bone marrow-derived (BM) DCs,6 Langerhans-type cells7 and MoDCs.6–11 The MoDCs are the most widely used subtype and can be phenotyped as CD1+, CD14+/−, CD16+, CD80/86+, CD172+, major histocompatibility complex (MHC) I+, MHC II+, CD4−, CD3−, and CD8−.6,7 Initially RAD001 MoDCs were generated by isolation of peripheral blood

mononuclear cells (PBMCs) followed by overnight plastic adherence. Non-adherent cells were then removed and the remaining monocytes were cultured in the presence of interleukin-4 (IL-4) and granulocyte–macrophage colony-stimulating factor (GM-CSF).6 More recent protocols, however, involve the isolation of monocytes using antibodies against CD1412,13 or CD172a,14 a porcine marker known as SWC3 that is present on myeloid cells15 including cDCs and pDCs.16 Porcine BDCs, on the other hand, comprising pDCs and cDCs, were originally described by Summerfield et al.,16 by flow cytometric analysis of PBMCs as being CD172a+, MHC II+, CD80/86+, CD1+/− and CD14− with pDCs being CD4+ and cDCs being CD4−. Subsequently,

this approach was further developed by isolating BDCs using antibodies against CD172a. However, because ASK1 CD172a is also expressed on monocytes, these enriched BDC populations contained not only different DC subtypes but monocytes as well.17 In the present study, we adapt previous protocols by initially depleting monocytes and subsequently enriching for CD172a to achieve a purer BDC population. These BDCs were compared with MoDCs in terms of antigen uptake, activation and maturation. DC maturation occurs upon recognition of microbe-associated molecule patterns and is characterized by up-regulation of co-stimulatory molecules such as CD80/86 and MHC II, various cytokines and the chemokine receptor CCR7.18,19 The process of maturation occurs as DCs migrate towards the lymph nodes where they encounter naive or primed T cells. In porcine MoDCs, stimulation with lipopolysaccharide (LPS) was demonstrated to decrease the expression of CD16, up-regulate the expression of CD80/866,20 and either increase7 or have no effect6,20 on expression of MHC II.