ARTICLES Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo Shalin H Naik1,2, Priyanka Sathe1,3, Hae-Young Park1,4, Donald Metcalf1, Anna I Proietto1,3, Aleksander Dakic1, Sebastian Carotta1, Meredith O’Keeffe1,4, Melanie Bahlo1, Anthony Papenfuss1, Jong-Young Kwak1,4,LiWu1 & Ken Shortman1 The development of functionally specialized subtypes of dendritic cells (DCs) can be modeled through the culture of bone marrow with the ligand for the cytokine receptor Flt3. Such cultures produce DCs resembling spleen plasmacytoid DCs (pDCs), http://www.nature.com/natureimmunology CD8+ conventional DCs (cDCs) and CD8– cDCs. Here we isolated two sequential DC-committed precursor cells from such cultures: dividing ‘pro-DCs’, which gave rise to transitional ‘pre-DCs’ en route to differentiating into the three distinct DC subtypes (pDCs, CD8+ cDCs and CD8– cDCs). We also isolated an in vivo equivalent of the DC-committed pro-DC precursor cell, which also gave rise to the three DC subtypes. Clonal analysis of the progeny of individual pro-DC precursors demonstrated that some pro-DC precursors gave rise to all three DC subtypes, some produced cDCs but not pDCs, and some were fully committed to a single DC subtype. Thus, commitment to particular DC subtypes begins mainly at this pro-DC stage. Dendritic cells (DCs) are antigen-presenting cells crucial for the innate macrophages12. Further ‘downstream’, ‘immediate’ precursors have and adaptive response to infection as well as for maintaining immune been identified for several DC types, including Ly6Chi monocytes as 3,4,6 13 Nature Publishing Group Group Nature Publishing tolerance to self tissue. Among the cells classified as DCs there are precursors of inflammatory DCs and Langerhans cells . A non- 7 many subtypes that, despite having many common features, have monocyte immediate precursor of cDCs (a ‘pre-cDC’) that had 200 distinct immune functions1. The DC subtypes present in the steady branched away from pDC development has been isolated from © state, before infection, include type 1 interferon–producing plasma- spleen4. This population includes CD24hi pre-cDCs producing only cytoid DCs (pDCs), ‘migratory’ DCs such as Langerhans cells, and CD8+ cDCs, and CD24lo pre-cDCs producing only CD8– cDCs4. ‘lymphoid tissue–resident’ conventional DCs (cDCs)1. The lymphoid A more detailed and sequential map of the pathway of DC tissue–resident cDCs consist of different subtypes, including CD8+ development would be possible with a suitable culture model of the cDCs and CD8– cDCs2. More DC subtypes develop after infection or process. Culture of precursor cells with granulocyte-macrophage inflammation, such as the monocyte-derived ‘inflammatory’ DCs3–6. colony-stimulating factor has led to models of the development of Despite recent insights, the pathways of development of these distinct migratory DCs and of monocyte-derived inflammatory DCs1,14. DC subtypes have not been adequately mapped. However Flt3 ligand (Flt3L), rather than granulocyte-macrophage DCs, like other blood cells, derive from hematopoietic stem cells colony-stimulating factor, is the limiting cytokine for the steady- through early progenitor cells. However, there is little evidence of state development of spleen DCs15,16, so culture of bone marrow commitment to DC subtype at the ‘early’ progenitor stage, as either precursor cells with Flt3L1,17–19 is a more appropriate model for common myeloid or common lymphoid progenitors have the ability studying the origin of pDCs and of lymphoid tissue–resident cDCs. to form pDCs, CD8+ cDCs and CD8– cDCs7,8 as long as the Despite the use of Flt3L in amounts well above steady state amounts20, progenitors express the cytokine receptor Flt3 (refs. 9,10). ‘Down- the DCs produced by Flt3L culture closely resemble the immature stream’ of these early progenitors, there is now evidence for restriction steady-state spleen pDCs, CD8+ cDCs and CD8– cDCs19. at ‘intermediate’ precursor stages. Precursor cells that can form cDCs Here we evaluate early stages of Flt3L-stimulated bone marrow but no longer produce pDCs have been found in bone marrow11,12, cultures to demonstrate two successive DC-restricted precursor cells, including one precursor type that produces both cDCs and ‘pro-DCs’ and pre-DCs, en route to the generation of all three DC 1The Walter and Eliza Hall Institute, Parkville, Victoria 3050, Australia. 2The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands. 3Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia. 4Present addresses: Department of Biochemistry, College of Medicine and Medical Research for Cancer Molecular Therapy, Dong-A University, Busan 602-714, Korea (H.-Y.P. and J.-Y.K.) and Bavarian Nordic, Munich 82152, Germany (M.O.). Correspondence should be addressed to S.H.N. ([email protected]) or K.S. ([email protected]). Received 14 May; accepted 18 September; published online 7 October 2007; corrected online 19 October 2007 (details online); doi:10.1038/ni1522 NATURE IMMUNOLOGY VOLUME 8 NUMBER 11 NOVEMBER 2007 1217 ARTICLES Figure 1 DC precursors in cultures of bone a b marrow stimulated with Flt3L. (a) Flow cytometry 104 104 CD8+ cDC E ) 1.4 6 of bone marrow cells cultured for 9 d with mouse 3 3 10 1.2 Flt3L and then analyzed for expression of CD11c 10 pDC 10 × 1.0 (to segregate DCs as CD11c+), CD45RA (to 0.8 + 102 102 segregate pDCs as CD45RA ) and CD24 and + CD24 0.6 Sirp-a (CD172a; to segregate CD8 cDC– CD45RA cDC 101 101 0.4 equivalent cells (CD45RA–CD24hiSirp-a–)and – CD8 cDCE 0.2 CD8– cDC–equivalent cells (CD45RA–CD24loSirp- 100 100 ( cell number Total 0.0 a+)). Similar results were obtained over 50 times. 100 101 102 103 104 100 101 102 103 104 02468 (b) Total number of viable cells versus time in the CD11c Sirp-α Time (d) culture conditions in a. This result was obtained twice. (c) Flow cytometry of bone marrow cells cdDay 3, depleted e 104 labeled with CFSE and anti-CD11c on days 1, 3 Day 1 104 CD11c+ 103 and 8 of the culture conditions in a.Three precursors experiments gave similar results. (d)Flow 102 3 10 8 100 cytometry and sorting of CFSEloCD11c+ and 1 10 lo – – 2 80 CFSE CD11c Ly6C DC precursors. Similar 100 CD11c 10 6 0 1 2 3 4 results were obtained over 30 times. 10 10 10 10 10 60 + – 1 (e) Proliferation of the CD11c and CD11c 104 10 4 Day 3 40 precursor populations from d recultured for 103 DCs (%) 102 103 104 105 2 a further 5 d in conditioned medium. The left bar 2 (fold) Proliferation 20 10 CFSE graph shows the extent of cell expansion and the 101 4 0 0 right bar graph shows the percentage of DCs 10 – + – + 100 among the cells produced. A second experiment 100 101 102 103 104 3 10 CD11c CD11c CD11c CD11c gave similar results. 104 – Day 8 CD11c http://www.nature.com/natureimmunology 102 precursors 103 CD11c 2 10 101 with cell division. Labeling the cells with 101 CFSE did not affect the development of 100 100 CD11c 0 1 2 3 4 Flt3L-DCs relative to that of unlabeled con- 100 101 102 103 104 10 10 10 10 10 CFSE Ly6C trol cells (data not shown). We then moni- tored the dilution of CFSE in the cells and the expression of CD11c during the culture subtypes: pDCs, CD8+ cDCs and CD8– cDCs. We also identify in vivo (Fig. 1c). After 1 d, only a few cells had divided; by day 3, a higher equivalents of these precursor cells. Clonal analysis of both in vitro and proportion of cells had undergone several divisions and some of these in vivo pro-DCs demonstrated that in this population, some indivi- expressed CD11c. By day 8, most cells were CD11chi DCs and had low Nature Publishing Group Group Nature Publishing dual precursor cells produced clones of all three DC subtypes, some CFSE fluorescence, demonstrating that they were derived from exten- 7 produced clones of two DC subtypes, and others produced clones of sive precursor cell division. We therefore reasoned that selection of 200 only one DC subtype. Earlier bone marrow progenitors were more dividing cells with low CFSE fluorescence during the course of the © likely to produce clones containing all DC subtypes. Our data show culture would enrich the cultures for DC precursors. Furthermore, day that the process of commitment to DC subtype occurs mainly at the 3.5 of culture, when most irrelevant cells had died, would be the dividing, pro-DC stage. optimal time to select precursor cells that still had substantial proliferative potential. We used two separate assays to assess the RESULTS activity of DC precursors: an in vivo assay4 involving transfer into Dividing DC precursors in Flt3L cultures unirradiated recipient mice followed by quantification of donor- When we cultured bone marrow cells with Flt3L for 8–10 d, three derived DCs in the recipient spleens after 5 d; an in vitro assay populations of DCs (Flt3L-DCs) were generated in good yield, as involving the reculture of cells in conditioned medium retained from reported before19. We segregated Flt3L-DCs as CD11c+CD45RAhi the Flt3L bone marrow cultures at day 3.5 (called ‘conditioned pDCs and CD11c+CD45RAlo cDCs (Fig. 1a). We then separated the medium’ here), with quantification of population expansion and cDCs into CD24hiSirp-alo and CD24loSirp-ahi subpopulations Flt3L-DC production 5 d later. (Fig. 1a), shown before to be equivalent to CD8+ and CD8– spleen cDCs, respectively, despite the lack of expression of CD8a in culture19. Isolation of DC precursors We therefore called these ‘CD8+ cDC–equivalent cells’ and ‘CD8– We first labeled bone marrow cells with CFSE, then cultured them cDC–equivalent cells’, respectively.