Dendritic cell potentials of early lymphoid and myeloid progenitors by Markus G. Manz, David Traver, Toshihiro Miyamoto, Irving L. Weissman, and Koichi Akashi Blood Volume 97(11):3333-3341 June 1, 2001 ©2001 by American Society of Hematology

Morphology of in vitro–generated DCs from CLPs and CMPs Morphology of in vitro–generated DCs from CLPs and CMPs.(A) Typical clusters of DCs developing from 1 × 103 CLPs cultured with IL-1β, IL-3, IL-4, IL-7, SLF, TNF-α, and Flt3-L. Morphology of in vitro–generated DCs from CLPs and CMPs.(A) Typical clusters of DCs developing from 1 × 103 CLPs cultured with IL-1β, IL-3, IL-4, IL-7, SLF, TNF-α, and Flt3-L. Clusters were photographed directly in the Terasaki tray on an inverted microscope (objective × 40) on day 4 of culture. (B, C) Cells developing from 100 CLPs in the above cocktail on day 4 or from 5000 CMPs in IL-3, IL-4, SLF, TNF-α, Flt-3L, and GM-CSF on day 6 were dissociated with EDTA, spun onto slides, and stained with Giemsa (objective × 60 oil). Almost all CLP-derived cells (B) and a substantial fraction of CMP-derived cells (C) displayed typical DC morphologies varying in size and nuclear shape. Markus G. Manz et al. Blood 2001;97:3333-3341 ©2001 by American Society of Hematology

Phenotypic characterization of in vitro CLP- and CMP-derived DCs Phenotypic characterization of in vitro CLP- and CMP-derived DCs.Cells were cultured as described in “Material and methods.” All cells were stained with anti–MHC class II and anti-CD11c (contour plot) plus 1 or 2 additional markers (histograms) by day 4 (CL... Phenotypic characterization of in vitro CLP- and CMP-derived DCs.Cells were cultured as described in “Material and methods.” All cells were stained with anti–MHC class II and anti-CD11c (contour plot) plus 1 or 2 additional markers (histograms) by day 4 (CLP cultures) or day 6 (CMP cultures), respectively. Filled histograms show specific staining, and open histograms show isotype-matched controls of gated population in the contour plot (MHC class II+CD11c+). CLPs and CMPs do not express MHC class II (not shown). Markus G. Manz et al. Blood 2001;97:3333-3341 ©2001 by American Society of Hematology

Gene expression in CLPs and DCs Gene expression in CLPs and DCs.RT-PCR analysis of CLPs, CLP-derived DCs in culture, CD8α+ and CD8α− splenic DCs, total splenic cells, and CD8α+ thymic DCs sorted from healthy animals. Gene expression in CLPs and DCs.RT-PCR analysis of CLPs, CLP-derived DCs in culture, CD8α+ and CD8α− splenic DCs, total splenic cells, and CD8α+ thymic DCs sorted from healthy animals. Although CLPs do not express CIITA, ELC, andRelB, CLP-derived DCs in culture, splenic and thymic DCs express these genes. No DC subsets expressed CD3. Uncultured splenic and thymic DCs expressed CD8α+ mRNA. PCR conditions were as described in “Materials and methods.” Markus G. Manz et al. Blood 2001;97:3333-3341 ©2001 by American Society of Hematology

CLPs give rise to DCs in vivo CLPs give rise to DCs in vivo.CLPs were competitively transplanted into lethally irradiated CD45 congenic animals, together with host-type whole bone marrow (A). CLPs give rise to DCs in vivo.CLPs were competitively transplanted into lethally irradiated CD45 congenic animals, together with host-type whole bone marrow (A). CD45 expression was used to gate on donor-type (CD45.1) and host-type (CD45.2) CD11c+-enriched, live splenocytes on day 15 after transplantation (B). Both CLP (upper panel) and host-derived (lower panel) cells were analyzed for DC phenotype by MHC class II+ and CD11c+ expression (C, contour plots). CLP-derived MHC class II+CD11c+ cells account for approximately 10% of donor-derived MHC class II+CD11c+ cells in a CD11c+-enriched sample. MHC class II+CD11c+ cells were further analyzed for CD8α, CD40, CD80, and CD86 expression (C). Closed hisograms represent specific staining, and open histograms represent isotype-matched controls of the MHC class II+CD11c+ cells. On day 15, 69% of CLP-derived DCs and 63% of host-derived DCs were CD8α+. Both CLP and host-derived DCs expressed low to intermediate levels of CD40, CD80, and CD86. Details are given in “Materials and methods.” Markus G. Manz et al. Blood 2001;97:3333-3341 ©2001 by American Society of Hematology

DCs derived from CLPs and CMPs were functionally active in allogeneic mixed leukocyte reactions.(A) Stimulation of 105 allogeneic BALB/c lymph node cells by graded numbers (x-axis) of in vitro CLP-derived DCs (day 5 of culture) (closed diamonds) and by bone... DCs derived from CLPs and CMPs were functionally active in allogeneic mixed leukocyte reactions.(A) Stimulation of 105 allogeneic BALB/c lymph node cells by graded numbers (x-axis) of in vitro CLP-derived DCs (day 5 of culture) (closed diamonds) and by bone marrow (Lin−)-derived DCs (day 5 of culture) (open squares). Cultures were grown and MLR was performed as described in “Materials and methods.” Results are the means ± SE each with 3 to 6 wells per point. (B) MLR of in vivo CLP-, CMP-, and host-derived DCs sorted by high expression of CD11c and MHC class II. Sorted DCs were cultured in numbers as indicated for 12 hours in complete media without cytokines, and 2 × 105 nucleated BALB/c splenocytes were added. MLR was performed as described in “Materials and methods.” (A, B) Results of 3 representative experiments are shown. Markus G. Manz et al. Blood 2001;97:3333-3341 ©2001 by American Society of Hematology

Proposed differentiation pathways from HSC to DC Proposed differentiation pathways from HSC to DC.The earliest CLPs and CMPs in bone marrow are capable of developing into DCs of both CD8α+ and CD8α−phenotypes. Proposed differentiation pathways from HSC to DC.The earliest CLPs and CMPs in bone marrow are capable of developing into DCs of both CD8α+ and CD8α−phenotypes. DC development potential is preserved in early T-cell progenitors and declines along with T-cell maturation as well as during granulocyte-macrophage commitment but is absent in B-cell progenitors and megakaryocyte-erythrocyte progenitors. Markus G. Manz et al. Blood 2001;97:3333-3341 ©2001 by American Society of Hematology