The concerted action of GM-CSF and Flt3-ligand on in vivo dendritic cell homeostasis by Dior Kingston, Michael A. Schmid, Nobuyuki Onai, Aya Obata-Onai, Dirk Baumjohann, and Markus G. Manz Blood Volume 114(4):835-843 July 23, 2009 ©2009 by American Society of Hematology

Flt3, Gm-csfr, and M-csfr mRNA expression in progenitor and myeloid cells. Flt3, Gm-csfr, and M-csfr mRNA expression in progenitor and myeloid cells. Real-time PCR analysis of mRNA expression of Flt3 (A), Gm-csfr (B), and M-csfr (C) in cell populations sorted from WT mice based on the following markers: c-kithi (lin−c-kithi); MDPΔ (lin−M-CSFRhi); CDP (lin−c-kitintFlt3+IL7Rα−); spleen (Sp) and LN cDC (CD19−MHCII+CD11c+) and pDC (CD19−CD45RA+CD11c+); LN CD40hi DC (CD19−CD40hiCD11cint); Langerhans cells (LC from epidermis) and dermal DC (MHCII+CD45+); blood (Bl), spleen, and bone marrow (BM) monocytes (mo) (M-CSFR+CD11b+ and Gr-1+ or Gr-1−). Data shown are averages of 3 independent experiments. Dior Kingston et al. Blood 2009;114:835-843 ©2009 by American Society of Hematology

Dendritic cell progenitors are significantly reduced in the absence of GM-CSF and FL. Quantification of total cell numbers of lineage-negative (lin−) cells (A) and for each progenitor population (B) (n = 5-7 mice/group). *P < .05; **P < .01; ***P < .001. Dendritic cell progenitors are significantly reduced in the absence of GM-CSF and FL. Quantification of total cell numbers of lineage-negative (lin−) cells (A) and for each progenitor population (B) (n = 5-7 mice/group). *P < .05; **P < .01; ***P < .001. (C) Representative fluorescence-activated cell sorting (FACS) plots of stained BM cells from WT, GM-CSF−/−, FL−/−, and DKO mice. Gating of lin− cells (dead cells excluded by PI staining; first row). Second and third rows were first gated on lin− cells. MDPΔ gate: M-CSFRhi (second row). CDP and Flt3+ CLP gates were as follows: CDP, c-kitintFlt3+M-CSFR+IL7Rα−; Flt3+ CLP, c-kitintFlt3+M-CSFRlo/−IL7Rα+ (third and fourth rows). Numbers in or beside boxes indicate percentage of cells shown. Dior Kingston et al. Blood 2009;114:835-843 ©2009 by American Society of Hematology

Analysis of cell subsets in the spleen of DKO mice in steady state. Analysis of cell subsets in the spleen of DKO mice in steady state. (A) Total cellularity of spleens from WT, GM-CSF−/−, FL−/−, and DKO mice. Horizontal lines indicate mean values of the results. (B) Percentage of CD19+ and CD3+ cells in the spleen. (C) Percentage of myeloid cell subsets: Monocytes were first gated as M-CSFR+CD11b+ and separated into Gr1+ or Gr1− cells; NK cells were gated as CD3−NK1.1+; DCs were divided into cDCs (MHCII+CD11c+) and pDCs (CD19−CD45RA+CD11c+) with representative FACS plots shown (D). CD45RA versus CD11c plots were first gated on CD19− cells. Numbers in FACS plots are percentages of total nucleated cells. *P < .05; **P < .01; ***P < .001. Dior Kingston et al. Blood 2009;114:835-843 ©2009 by American Society of Hematology

Dermal DCs are significantly reduced in the absence of GM-CSF and FL in steady state. Dermal DCs are significantly reduced in the absence of GM-CSF and FL in steady state. DCs were analyzed from WT, GM-CSF−/−, FL−/−, and DKO mice. (A) Immunofluorescence microscopy of MHCII-stained (red) epidermal sheets. MHCII+ cells were counted on images taken from multiple fields per mouse (n = 4/group). Epidermal sheets were stained with PE-conjugated anti-MHCII antibody and mounted on slides with the use of Eukitt mounting medium. Images were taken at room temperature on a Nikon Eclipse E800 microscope with a CCD Qimaging camera using a Nikon Plan Apo 20×/0.75 NA objective lens and acquired with the use of OpenLab software. Scale bar represents 10 μm. (B-C) Flow cytometry of ex vivo–isolated dermal-derived cells. Representative FACS plots from WT, GM-CSF−/−, FL−/−, and DKO mice are shown. (B) Percentage of all CD45+ cells shown by outer gate. Inner gate represents percentage of MHCII+ cells within CD45+ gate. (C) Dermal DCs were pregated on MHCII+CD45+, followed by gating on CD11b+ and CD11b− populations. (D) Percentages of total dermal-derived MHCII+CD45+ cells, as well as CD11b− and CD11b+ subsets are shown. Results are given as the percentage of CD45+ cells (n = 4-6 mice/group). *P < .05; **P < .01. Dior Kingston et al. Blood 2009;114:835-843 ©2009 by American Society of Hematology

Skin-derived DCs in the draining lymph nodes are significantly reduced in GM-CSF– and FL-deficient mice in steady state. Skin-derived DCs in the draining lymph nodes are significantly reduced in GM-CSF– and FL-deficient mice in steady state. (A) Flow cytometry and analysis of DC subsets in skin-draining LNs (pooled inguinal, axillary, and cervical LNs). Horizontal lines indicate mean values of the results. LN DC subsets gated as CD40hiCD11cint, CD40+CD11chi, and CD40−CD11cint. Total LN cell numbers (A) and percentage of each LN DC subset (B) are shown in graphs. *P < .05; **P < .01. Dior Kingston et al. Blood 2009;114:835-843 ©2009 by American Society of Hematology

Reduced T-cell and antibody responses after subcutaneous immunization in the absence of GM-CSF and FL. (A) T-cell proliferation assay. Reduced T-cell and antibody responses after subcutaneous immunization in the absence of GM-CSF and FL. (A) T-cell proliferation assay. Naive OT-II CD4+CD45.1+ T cells were sorted and labeled with CSFE before transfer into CD45.2+ WT, GM-CSF−/−, FL−/−, and DKO mice (6 × 104 cells/recipient). The next day, mice were immunized subcutaneously in the right flank with 2 μg OVA protein and 4 μg MPL. Draining LNs (right axillary and inguinal) and nondraining LNs (shown as representative WT control) were analyzed 3 days later. The proliferation of the transferred OT-II T cells was analyzed by gating on CD45.1+CD3+CD4+ cells. Histograms show the percentage of OT-II T cells having diluted the CFSE label. Results are representative of 2 independent experiments with a total of 3 to 7 mice/group. (B) Production of OVA-specific IgG antibodies. WT, GM-CSF−/−, FL−/−, and DKO mice were immunized in the footpads with 2 μg whole OVA protein and 4 μg MPL, and serum was collected at the indicated time points. A second immunization was given at day 21. OVA-specific IgG antibodies were measured by ELISA (n = 7-8 mice/group). ***P < .001. Dior Kingston et al. Blood 2009;114:835-843 ©2009 by American Society of Hematology