Supplementary Materialssupplement. et al., 2002; Segura et al., 2013). Human CD141+ and CD1c+ cDCs were originally identified in blood (Dzionek et al., 2000), and have been ontogenetically aligned to mouse cDC1 and cDC2, respectively (Bachem et al., 2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al., 2010). Human DC subsets have been characterized in tissues (Guilliams et al., 2016; Haniffa et al., 2012; Heidkamp et Baricitinib inhibitor al., 2016; Watchmaker et al., 2014), however, tissues were derived from isolated surgical explants from a small number of individuals at different life stages. The ability to adapt DC therapies to the diverse human population requires a comprehensive analysis of DC populations in multiple tissues at different life stages. A key paradigm in cDC biology is their capacity to acquire antigens in peripheral tissues, deliver them to draining lymph nodes (LNs), and undergo maturation through upregulation Baricitinib inhibitor of major histocompatibility (MHC) and co-stimulatory molecules required for T cell activation (Banchereau and Steinman, 1998; Worbs et al., 2016). Peripheral tissue cDC1 and cDC2 in mice both display a capacity to mature and migrate to the draining LNs during homeostasis or inflammation (Hammer and Ma, 2013). Human tissue-migratory cDCs have been described based on phenotype or migration assays in LN samples and skin (Haniffa et al., 2012; Segura et al., 2012); however, little is known about human cDC migration and maturation between peripheral tissues and their draining LNs, due to the difficulty of obtaining these complementary sites for research. Thus, the defining characteristic of human cDCs as tissue sentinels remains poorly understood. We have established a unique human tissue resource through collaborations with the local organ procurement agency, LiveOnNY, to obtain physiologically healthy lymphoid and mucosal tissues from human organ donors (Sathaliyawala et al., 2013; Thome et al., 2016a; Thome et al., 2014). Our ability to obtain Rabbit polyclonal to MBD3 Baricitinib inhibitor multiple tissues sites from individual donors of all ages has revealed insights into the compartmentalization of T cells, their homeostatic maintenance over life, and genesis and function in tissues during early life (Thome et al., 2016a; Thome et al., 2014). Here, we asked whether tissue compartmentalization of immune responses was controlled at the level of tissue surveillance by DC. We developed a robust phenotyping scheme combining well-established (CD1c, CD141) and tissue-optimized (CD13, CD64) markers, to distinguish cDC subsets in 14 diverse tissues sites, including lung, intestines, mucosal-draining and peripheral LNs, spleen and gut-associated lymphoid tissues (GALT). Our analysis reveals new insights into human DC biology: that cDC subset composition is largely a function of the tissue site; that LNs differ in the extent of DC maturation with lung-draining LN having the highest proportion of mature DC compare to other LN sites, and that the cDC2 subset exhibits predominant maturation phenotypes within LNs compared to cDC1. This localization of DC maturation is largely maintained throughout life, with site-specific variation in subset distribution in early life and an overall increase in maturation throughout many sites in later life. Our findings provide new insights into the dynamic processes underlying adaptive immunity through tissue-specific DC maturation. RESULTS Analysis of DC subsets in multiple human tissues We obtained multiple lymphoid and mucosal tissues from research-consented human organ donors as previously described (Sathaliyawala et al., 2013; Thome et al., 2016a; Thome et al.,.