No deficiency of pre-cDCs, but a reduced half-life of CD4⁺ cDCs in the spleen of SIRPα MT mice. (A) Splenocytes from WT or SIRPα MT mice were incubated with a biotin-conjugated mAb to Flt3 or an isotype-matched control mAb and were also stained with an FITC-conjugated mAb to I-A, PE-conjugated streptavidin, PE-Cy5–conjugated mAbs to lineage markers (CD3ϵ, CD19, B220, NK1.1, and TER-119), an APC-conjugated mAb to CD11c, and PI. The expression of CD11c and I-A on lineage- and PI-negative (Lin⁻PI⁻) cells (top left panel) or that of Flt3 on CD11c⁺ I-A⁻ cells (open traces; filled traces represent staining with the isotype control; bottom left panel) was analyzed by 4-color flow cytometry. The relative numbers of CD11c⁺ I-A⁻ cells or CD11c⁺ I-A⁺ cells (top left panel) and those of Flt3⁺ cells (pre-cDCs, bottom left panel) are expressed as a percentage of all Lin⁻PI⁻ splenocytes (top left panel) or of CD11c⁺ I-A⁻ cells (bottom left panel) on each plot. The percentage of pre-cDCs among total viable splenocytes was also determined (right panel); data are means ± SE from 3 separate experiments. (B) Pre-cDCs that had been sorted from BM cells of WT or SIRPα MT mice (see supplemental Figure 2) were injected intravenously into B6-Ly5.1 mice. Eight days after injection, splenocytes from recipient mice were stained as in Figure 3A. The expression of Ly5.1 on cDCs (top left panel) or that of CD4 and CD8 on Ly5.1⁻ cDCs (derived from transferred cells of Ly5.2⁺ WT or MT mice; bottom left panel) was analyzed by 6-color flow cytometry. The relative numbers of Ly5.1⁻ cells (top left panel) and of CD8⁺, CD4⁺, or DN cDCs (bottom left panel) are expressed as a percentage of all PI-negative cDCs (top left panel) or of Ly5.1⁻ cDCs (bottom left panel) on each plot. The percentages of donor (Ly5.1⁻)–derived CD8⁺, CD4⁺, or DN cDCs among total cDCs were also determined (right panel); data are means ± SE from 3 independent experiments. **P < .01 (Student t test). (C) WT or SIRPα MT mice were injected intraperitoneally with 1 mg of BrdU and then continuously supplied with BrdU (0.8 mg/mL) in sterile drinking water. At various times after BrdU administration, splenocytes were isolated from the mice and stained with a PE-conjugated mAb to CD8, a PE-Cy7–conjugated mAb to CD4, an APC-conjugated mAb to CD11c, and an APC-Cy7–conjugated mAb to B220. The cells were then fixed, permeabilized, and stained with an FITC-conjugated mAb to BrdU. The percentage of BrdU-positive cells among total CD8⁺, CD4⁺, or DN cDCs in the spleen at each time point was then determined (left panels), and from these data, the half-lives of CD8⁺, CD4⁺, and DN cDCs were estimated (right panel). Data are means ± SE from 3 independent experiments. *P < .05 (Student t test).