Development of donor-derived DCs in the thymi 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 were used as negative control, whereas mice transferred with Ly5.2-type BM cells were used as positive control. At day 14 after transplantation, thymi were digested with collagenase D. Collected thymocytes were enriched with CD11c-conjugated microbeads 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 3 independent experiments. (B) Phenotype characterization of CD8α⁺CD11c⁻Lin⁻ cell–derived thymic DCs. 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 DEC205, rat DEC205 was used as first antibody, followed by FITC-conjugated goat F(ab′)₂ antirat IgG (H&L). Solid and dotted lines indicated the histograms of specific stainings and isotope-matched controls gated on the Ly5.2⁺Ia⁺ cells, respectively. The results are representative of 3 independent experiments.