Development of donor-derived DCs in the lymph nodes 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, combined axillary, cervical, inguinal, and mesenteric lymph nodes were collected and digested with collagenase D. Cells 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 DCs in the lymph nodes. 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 represent 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.