Cellular & Molecular Immunology (2016) 13, 443–450 ß 2016 CSI and USTC. All rights reserved 1672-7681/16 $32.00 www.nature.com/cmi RESEARCH ARTICLE Delineation of a novel dendritic-like subset in human spleen Sawang Petvises1, Dipti Talaulikar2,3,4 and Helen C O’Neill1 Dendritic cells (DCs) and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c1hCD11b1HLA-DR2hCD861 subset in the spleen, along with the previously described subsets of hCD1c1 DCs, hCD1231 plasmacytoid DCs (pDCs), hCD161 DCs and hCD1411 DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c1HLA-DR1 and hCD11c1HLA-DR2 cells were produced. The latter 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. Cellular & Molecular Immunology (2016) 13 , 443–450;doi:10.1038/cmi.2015.16;published online 20 April 2015 Keywords: dendritic cells; myelopoiesis; spleen; stroma INTRODUCTION subsets with similar functions. The splenic microenvironment The spleen is a secondary lymphoid organ that is essential in the also supports development of dendritic and myeloid cells immune response against blood-borne antigens. Hematopoiesis from bone marrow-derived progenitors that enter the spleen in the spleen is mainly restricted to the production of erythro- through blood. cytes, although hematopoiesis can be triggered at times of stress Dendritic cell (DC) subsets in the murine spleen have been or upon ablation of bone marrow.1 A small number of HSCs broadly classified as conventional DCs (cDCs) and plasmacy- can migrate through the blood and lymphatic system to enter toid DCs (pDCs), and the cDC population can be further the spleen and other tissues.2,3 However, it is not yet known divided into two subsets: CD81 cDCs and CD82 cDCs. whether hematopoietic stem cells in the steady-state spleen can CD82 cDCs are capable of inducing a CD41 T-cell response self-renew and differentiate into a specific lineage of hemato- with production of inflammatory cytokines,5 while the CD81 poietic cells. cDC subset is superior in cross-presenting antigen to CD81 T Human and murine spleens are very similar, but minor cells.6 pDCs are the major producers of type I interferon.7 structural differences in the organization of the white pulp Previously, this lab identified a novel splenic dendritic-like cell are evident. The human spleen differs in that it contains a type called L-DCs in murine spleen with high potential to perifollicular region external to the marginal zone in the red induce CD81 T-cell responses but with no capacity to induce pulp, which is absent in murine spleen.4 In general, human and CD41 T-cell responses.8,9 Murine L-DCs are distinguishable by murine spleens both contain equivalent lymphoid and myeloid very high endocytic capacity and a lack of MHC-II expression. 1Research School of Biology, The Australian National University, Canberra, Australia; 2John Curtin School of Medical Research, The Australian National University, Canberra, Australia; 3ANU Medical School, College of Medicine Biology and Environment, The Australian National University, Canberra, Australia and 4Department of Haematology, Canberra Hospital, Canberra, Australia Correspondence: Professor HC O’Neill, Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia. E-mail: [email protected] Received: 18 December 2014; Revised: 1 February 2015; Accepted: 1 February 2015 Human splenic dendritic cells S Petvises et al 444 Recent studies have shown that these same cells can be gener- Preparation of human splenocytes ated through in vitro hematopoiesis when murine spleen- or Fresh and/or cryopreserved human spleen samples were bone marrow-derived hematopoietic stem/progenitor cells are obtained from the ACT Haematology Research Tissue Bank cocultured with the splenic stromal cell line 5G3.10–14 The L- (Canberra Hospital, Canberra, Australia) with informed con- DCs produced are functionally distinct in their ability to acti- sent from patients undergoing splenectomies for suspected or vate CD81 but not CD41 T cells. Currently, L-DCs are thought known immune thrombocytopenia and/or lymphoma. Spleens to reflect a tissue-specific APC derived from progenitors endo- selected for this study had no histological evidence of lym- genous to spleen.15 The currently available evidence supports phoma. Spleen tissues were kept aseptically on ice and pro- the possible role of L-DCs in antigen presentation of blood- cessed within 12 h of removal. The tissues were chopped into borne antigens for CD81 T-cell activation. small pieces of 0.5–1.0 cm. Cell suspensions were obtained by DC development in the human spleen has not been as exten- forcing the tissue through a fine mesh sieve into DMEM with sively studied as it has in the murine spleen. In humans, DC washing by centrifugation at 300g at 4uC for 5 min. For the lysis subsets have been investigated mainly in blood due to greater of red blood cells, cells were resuspended in lysis buffer 1 1 accessibility of cells. Human blood DCs are HLA-DR CD11c (140 mM NH4Cl, 17 mM Tris base, pH 7.5) at room temper- cells, and four subsets have been differentiated according to the ature for 5 min; then cells were washed twice with DMEM by expression of hCD16, hCD1b/c, hCD304 and CD141.16–18 The centrifugation at 300g at 4uC for 5 min and resuspended at the hCD161 DC subset has been identified in both spleen and desired concentrations. blood.19 These cells resemble murine monocyte-derived DCs and are identifiable as hCD11c1hCD11b1HLA-DR1hCD161 Preparation of T and B cell-depleted spleen cells.19,20 The hCD1c1 DCs, delineated as hCD11c1hCD11b1 Splenocytes were depleted of T and B cells using MACS meth- HLA-DR1hCD1c1 cells, resemble murine myeloid DCs.21 These odology (Miltenyi Biotec: North Ryde, NSW, Australia). cells are the homolog of murine CD11b1CD41 DCs.22,23 The Biotinylated antibodies specific for hCD19 (HIB18; Biolegend, pDC subset comprises cells with a hCD11c1hCD11bloHLA- San Gabriel, CA, USA) and hCD3 (OKT3; Biolegend) were 7 DR1hCD1231 phenotype, which are the homolog of cells added to 10 cells and incubated on ice for 20 min. The cell found in murine spleen and blood.24,25 The hCD1411 DC, suspension was then washed twice with labeling buffer (degassed identified as Lin2hCD11c1HLA-DR1hCD1411 cells, are the PBS/pH 7.2, 0.5% bovine serum albumin and 2 mM EDTA) by murine homologue of CD1411 DCs.22,26 centrifugation at 300g at 4uC for 5 min. The supernatant was Information about DC subsets in the human spleen has discarded, and the cells were resuspended in 20 mlofMACSanti- been limited by tissue availability. Recently, Mittag and biotin microbeads. The cells were further incubated on ice for colleagues20 identified the same four subsets of DC in human 20 min and then washed twice by centrifugation. The cells were spleen and showed that they resemble subsets that were pre- resuspended in 500 ml labeling buffer and transferred to a MACS viously identified in human peripheral blood. However, the MS column placed in a SuperMACS II Separator (Miltenyi presence of a human equivalent of the L-DC subset described Biotec). The column was washed three times with 3 ml of label- in murine spleen has not yet been investigated. Here, human ing buffer, and the flow-through cells were collected and washed. spleens were analyzed for DC and monocyte subsets using This procedure routinely gave 95% purity. antibody staining and flow cytometric analysis. Human Antibody staining spleens were also tested for the presence of L-DC progenitors For antibody staining, cells were washed and resuspended in by coculturing splenocytes with the murine 5G3 splenic stro- fluorescence-activated cell sorting (FACS) buffer (DMEM, mal line which supports the hematopoiesis of murine sple- 0.1% sodium azide, 1% fetal calf serum) at 13105–53105 nocytes. Cells produced in cocultures were phenotypically cells/100 ml. A 100 ml volume of cells was plated into a flexible and functionally characterized, and compared with murine 96-well microtiter plate and centrifuged at 300g at 4uC for L-DCs. 5 min. The cells were resuspended in 25 ml of FACS buffer containing 40 mg/ml of Fc block specific for FccII/IIIR MATERIALS AND METHODS (eBioscience, San Diego, CA, USA) for 15mins on ice before Splenic stromal cultures washing by centrifugation and discarding the supernatant. A The 5G3 splenic stromal line supports hematopoiesis in cocul- 25 ml aliquot of diluted primary antibody was added to the cells, tured bone marrow and splenocytes.27–29 The 5G3 stromal cells which were then incubated in the dark on ice for 25 min. The were maintained by scraping up adherent cells to passage every cells were washed twice with 150 ml of FACS buffer by centrifu- 3–4 days.