Development of donor-derived DCs in the spleens from CD8α⁺CD11c⁻Lin⁻ cells in vivo.

(A) CD8α⁺CD11c⁻Lin⁻ cells isolated from the spleens of Ly5.2 B6 mice were intravenously transferred to the lethally irradiated congenic Ly5.1 B6 recipient mice, accompanied by Ly5.1-type BM cells to rescue the recipient mice from lethal irradiation. Mice transferred with Ly5.1-type BM cells only were used as negative control, whereas mice transferred with Ly5.2-type BM-derived Lin⁻c-kit⁺ HPCs were used as positive control. At day 14, splenocytes were isolated and enriched with CD11c-conjugated microbeads, as described in “Materials and methods” and then stained with FITC-conjugated anti-Ly5.2 and PE-conjugated anti-Ia. The quads were set up on the isotype-matched control dot plot. The results are representative of 6 independent experiments. (B) Giemsa staining was performed on sorted Ly5.2⁺CD8α⁺Ia⁺ cells from CD8α⁺CD11c⁻Lin⁻cell–reconstituted mice, which were isolated freshly or cultured overnight with GM-CSF. The results are representative of 3 independent experiments. Original magnification × 400. (C) FACS analyses of CD8α⁺CD11c⁻Lin⁻cell–derived DCs by tricolor staining. Cells were collected as described in (A) and then stained with biotin-conjugated anti-Ly5.2, followed by APC-streptavidin, as well as PE-conjugated anti-Ia, FITC-conjugated anti-CD11c, anti-CD8α, anti-CD40, anti-CD86, or anti-CD11b. As for the staining of DEC-205, rat DEC-205 was used as first antibody, followed by FITC-conjugated goat F(ab′)₂antirat IgG (H&L). Data from mice transferred with Ly5.1-type BM cells only were shown as negative control. The quads were set up on the isotype-matched control dot plot. The results are representative of 6 independent experiments.