Given that human monocytes are HLA-DR+, the cells were initially divided by hCD14 and hCD16 expression to delineate three monocyte subpopulations (Figure 1d). resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCD80 and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors. hematopoiesis when murine spleen- or bone marrow-derived hematopoietic stem/progenitor cells are cocultured with the splenic stromal cell line 5G3.10,11,12,13,14 The L-DCs produced are functionally distinct in their ability to activate CD8+ but not CD4+ T cells. Currently, L-DCs are thought to reflect a tissue-specific APC derived from progenitors endogenous to spleen.15 The currently available evidence supports the possible role of L-DCs in antigen presentation of blood-borne antigens for CD8+ T-cell activation. DC development in the human spleen has not been as extensively studied as it has in the murine spleen. In humans, DC subsets have been investigated mainly in blood due to greater accessibility of cells. Human blood DCs are HLA-DR+CD11c+ cells, and four subsets have been differentiated according to the expression of hCD16, hCD1b/c, hCD304 and CD141.16,17,18 The hCD16+ DC subset has been identified in both spleen and blood.19 These cells resemble murine monocyte-derived DCs and are identifiable as hCD11c+hCD11b+HLA-DR+hCD16+ cells.19,20 The hCD1c+ DCs, delineated as hCD11c+hCD11b+HLA-DR+hCD1c+ cells, resemble murine myeloid DCs.21 These cells are the homolog of murine CD11b+CD4+ DCs.22,23 The pDC subset comprises cells with a hCD11c+hCD11bloHLA-DR+hCD123+ phenotype, which are the homolog of cells found in murine spleen and blood.24,25 The hCD141+ DC, Piribedil D8 identified as Lin?hCD11c+HLA-DR+hCD141+ cells, are the murine homologue of CD141+ DCs.22,26 Information about DC subsets in the human spleen has been limited by tissue availability. Recently, Mittag and colleagues20 identified the same four subsets of DC in human spleen and showed that they resemble subsets that were previously identified in human peripheral Piribedil D8 blood. However, the presence of a human equivalent of the L-DC subset described in murine spleen has not yet been investigated. Here, human spleens were analyzed for DC and monocyte subsets using antibody staining and flow cytometric analysis. Human spleens were also tested for the presence of L-DC progenitors by coculturing splenocytes with the murine 5G3 splenic stromal line which supports the hematopoiesis of murine splenocytes. Cells produced in cocultures were phenotypically and functionally characterized, and compared with murine L-DCs. Materials and methods Splenic stromal cultures The 5G3 splenic stromal line supports hematopoiesis in cocultured bone marrow and splenocytes.27,28,29 The 5G3 stromal cells were maintained by scraping up adherent cells to passage every 3C4 days. Stromal cells were cultured at 37C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 Piribedil D8 mg/ml glucose, 6 g/ml folic acid, 36 g/ml at 4C for 5 min. For the lysis of red blood cells, cells were resuspended in lysis buffer (140 mM NH4Cl, 17 mM Tris base, pH 7.5) at room temperature for 5 min; then cells were washed twice with DMEM by centrifugation at 300at 4C for 5 min and resuspended at the desired concentrations. Preparation of T and B cell-depleted spleen Splenocytes were depleted of T and B cells using MACS methodology (Miltenyi Biotec: North Ryde, NSW, Australia). Biotinylated antibodies specific for hCD19 (HIB18; Biolegend, San Gabriel, CA, USA) and hCD3 (OKT3; Biolegend) were added to 107 cells and incubated on ice for 20 min. The cell suspension was Piribedil D8 then washed twice with labeling buffer (degassed PBS/pH 7.2, 0.5% bovine serum albumin and 2 mM EDTA) by centrifugation at 300at 4C for 5 min. The supernatant was discarded, and the cells were resuspended in 20 l of MACS anti-biotin microbeads. The cells were further incubated on ice for 20 min and then washed twice by centrifugation. The cells were resuspended in 500 l labeling buffer and transferred to a MACS MS column placed in GRK4 a SuperMACS II Separator (Miltenyi Biotec). The column was washed three times with 3 ml of labeling buffer, and the flow-through cells were collected and washed. This procedure routinely gave 95% purity. Antibody staining For antibody staining, cells were washed and resuspended in fluorescence-activated cell sorting (FACS) buffer (DMEM, 0.1% sodium azide, 1% fetal calf serum) at 1105C5105 cells/100 l. A.