Figure 1
Figure 1. DOCK2−/− plasmacytoid dendritic cells (pDCs) are impaired in their homing to and localization within secondary lymphoid organs. (A,B) Bone marrow (BM), spleen, and lymph node (LN) cells from B6 or DOCK2−/− mice were stained with fluorescein isothiocyanate (FITC)-labeled anti-B220, phycoerythrin (PE)-labeled anti-CD11c and biotinylated anti-mPDCA-1 antibodies followed by allophycocyanin (APC)-conjugated streptavidin. Before staining, BM and splenic dendritic cells (DCs) were enriched with anti-CD11c microbeads. (A) Expression of B220 and mPDCA-1 on CD11c+ BM or splenic DCs are shown. Numbers in quadrants indicate the percentage of cells in each after gating on CD11c+ cells. Data are representative of 4 independent experiments. (B) The number of myeloid dendritic cells (mDCs; CD11c+B220−mPDCA-1−) and pDCs (CD11c+B220+mPDCA-1+) in the BM, spleen, peripheral LN (PLN) and mesenteric LN (MLN) were compared between B6 (□, n = 4) and DOCK2−/− (■, n = 4) mice. Data are mean plus or minus SD; *P < .01. (C) Spleen and PLN tissue sections from B6 and DOCK2−/− mice were stained for B220 (Alexa Fluor 546; red) and mPDCA-1 (FITC; green). Scale bars, 100 μm. Data are representative of 3 independent experiments with different mice. (D-F) BM-derived pDCs from B6 and DOCK2−/− mice were labeled with PKH-26 (green) and PKH-67 (red) dyes, respectively, and were mixed in equal numbers and intravenously injected into B6 mice. (D) The ratios of DOCK2−/− pDCs (■) to B6 pDCs (□; set as an arbitrary value of 1) in the spleen, PLN and MLN of the recipient mice were analyzed at 24 hours after transfer (n = 3, *P < .05). (E) Spleen sections were prepared at 24 hours after transfer and stained for metalophilic macrophages with anti-MOMA1 antibody (Alexa Fluor 647; blue). Scale bar, 100 μm. Data are representative of 2 independent experiments with different mice. (F) The ratios of DOCK2−/− pDCs to B6 pDCs in the spleen were analyzed at indicated time points after transfer. Data are means plus or minus SD (n = 3). Images in panels C and E were acquired using an LSM 510 META confocal microscope (Carl Zeiss, Gottingen, Germany) equipped with a 20×/0.75 (C) or 10×/0.45 (E) NA Plan-Apochromat objective lens (Carl Zeiss).

DOCK2−/− plasmacytoid dendritic cells (pDCs) are impaired in their homing to and localization within secondary lymphoid organs. (A,B) Bone marrow (BM), spleen, and lymph node (LN) cells from B6 or DOCK2−/− mice were stained with fluorescein isothiocyanate (FITC)-labeled anti-B220, phycoerythrin (PE)-labeled anti-CD11c and biotinylated anti-mPDCA-1 antibodies followed by allophycocyanin (APC)-conjugated streptavidin. Before staining, BM and splenic dendritic cells (DCs) were enriched with anti-CD11c microbeads. (A) Expression of B220 and mPDCA-1 on CD11c+ BM or splenic DCs are shown. Numbers in quadrants indicate the percentage of cells in each after gating on CD11c+ cells. Data are representative of 4 independent experiments. (B) The number of myeloid dendritic cells (mDCs; CD11c+B220−mPDCA-1−) and pDCs (CD11c+B220+mPDCA-1+) in the BM, spleen, peripheral LN (PLN) and mesenteric LN (MLN) were compared between B6 (□, n = 4) and DOCK2−/− (■, n = 4) mice. Data are mean plus or minus SD; *P < .01. (C) Spleen and PLN tissue sections from B6 and DOCK2−/− mice were stained for B220 (Alexa Fluor 546; red) and mPDCA-1 (FITC; green). Scale bars, 100 μm. Data are representative of 3 independent experiments with different mice. (D-F) BM-derived pDCs from B6 and DOCK2−/− mice were labeled with PKH-26 (green) and PKH-67 (red) dyes, respectively, and were mixed in equal numbers and intravenously injected into B6 mice. (D) The ratios of DOCK2−/− pDCs (■) to B6 pDCs (□; set as an arbitrary value of 1) in the spleen, PLN and MLN of the recipient mice were analyzed at 24 hours after transfer (n = 3, *P < .05). (E) Spleen sections were prepared at 24 hours after transfer and stained for metalophilic macrophages with anti-MOMA1 antibody (Alexa Fluor 647; blue). Scale bar, 100 μm. Data are representative of 2 independent experiments with different mice. (F) The ratios of DOCK2−/− pDCs to B6 pDCs in the spleen were analyzed at indicated time points after transfer. Data are means plus or minus SD (n = 3). Images in panels C and E were acquired using an LSM 510 META confocal microscope (Carl Zeiss, Gottingen, Germany) equipped with a 20×/0.75 (C) or 10×/0.45 (E) NA Plan-Apochromat objective lens (Carl Zeiss).

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