Homing and in vivo expansion of donor pDCs and T cells in the spleen and GVHD target organs. (A) The bioluminescent image was taken 7 days after Balb/c mice were transplanted with 5 × 104 FACS-purified luciferase pDCs or 1 × 106 luciferase T cells, in combination with 5 × 103 FACS-sorted HSCs from C57BL/6 mice. Mice were sacrificed, and thymus (an anatomic site for tolerogenic pDC),53 lung, GI tract, spleen, paw, and liver were collected. Left side radiance scale bar (200- 500) is for the whole-body image; right side radiance scale bar (100-350) is for the organ images. (B) Fourteen days after the transplant, bioluminescent images of anesthetized mice and isolated organs were obtained. The highest bioluminescence signals are seen in the spleen, GI tract, and lung. Left side radiance scale bar (103 to 104 p/sc/cm2/sr) is for the whole-body image of both groups; right side radiance scale bar (104 to 105) is for the organ images. (C) Mean ± standard deviation for bioluminescent signals in organs following transplantation of luciferase+ T cells; N = 6 biological replicates per group. (D) Mean ± standard deviation for bioluminescent signals in organs following transplantation of luciferase+ pDCs recipients; N = 4 biological replicates per group. (E) Correlation of mean bioluminescent signals per organ comparing recipients of luciferase+ donor T cells and donor pDCs (R2 = 0.93). N = 4 biological replicates in the luc+ pDCs + HSC group, and n = 6 in luc+ T cells and HSC group. Statistics: power linear progression. (F) The small intestine from B10.BR recipients was collected 8 days after allo-HSCT from a C57BL/6 donor graft containing 5 × 103 HSCs, 5 × 104 pDCs, and 1× 106 GFP+ T cells. Recipients were sacrificed on day 8 posttransplant. GI tract was stained for rat anti-PDCA-1, followed by staining with an anti-rat AF568-conjugated antibody. Images were analyzed with an Olympus FV10000 Confocal Laser Scanning Biological Microscope. An image showing the presence of pDCs and donor T cells is shown from 4 images with detectable donor cells out of 40 images examined. Magnification: ×20. Bar indicates 50 μm. (G) Immunofluorescence staining with anti-H-2Kb and anti-PDCA-1 antibodies in optimal cutting temperature compound–embedded, 4% paraformaldehyde-fixed small intestine of Balb/c mice 14 days after lethal irradiation and transplantation with 5 × 104 donor GFP+ pDC, 5 × 103 HSC, and 5 × 104 T cells from C57BL/6 donors. Identity of donor pDCs in the gut section was established by coexpression of H-2Kb and PDCA-1. White arrows show an exemplar of 3 cells that coexpressed H-2Kb and PDCA-1. Magnification: ×40. Bar indicates 50 μm. (H) Immunofluorescence staining of the same sections as in panel G with anti-GFP and anti-PDCA-1 antibodies. Identity of donor pDCs in the gut section was established by coexpression of GFP and PDCA-1. Gray arrows show 2 cells that coexpressed GFP and PDCA-1. Anti-GFP Ab was applied to eliminate the possible confounding effects of autofluorescence in the GI tract with GFP signals. Magnification: ×40. Bar indicates 50 μm.