Increased differentiation of preexisting DC precursors in spleen after transfer into DC-depleted mice. (A) Experimental layout. Ten million of CD45.1⁺ CD11c⁻ splenocytes (CD11c⁺ MACS-depleted splenocytes) obtained from untreated mice were transferred into CD45.2⁺ recipient mice with either normal DC numbers (B6 +DT) or reduced (CD11c.DOG +DT). In recipient mice, DT was applied daily from 1 day before precursor cell transfer until the end of the experiment. (B) Intrasplenic DC precursors differentiate into higher numbers of splenic DCs after transfer into DC-deficient environment. The number of donor-derived cDCs per donor spleen was calculated at 5, 7, and 10 days after adoptive transfer by flow cytometry using the CD45 congenic marker. cDC gate was set as in Figure 1A. Results are expressed as mean value ± SEM; *P < .05; ***P < .005, ANOVA. (C) Splenic DC precursors develop into all 3 major cDC populations in the spleen after transfer into DC-deficient environment. Shown are representative FACS diagrams of splenocytes derived from untreated B6 mice (B6, no DT) and donor-derived cells in DC-depleted mice (CD11c.DOG, +DT, day 10 after transfer). B and T cells were depleted before analysis. The experiment was repeated 4 times with comparable results. (D) The enhanced differentiation of splenic DC precursors in mice with reduced DC numbers is Flt3-L dependent. Similar experiment to that in panel B using different sets of recipient mice as indicated and injected with DT for 10 consecutive days. Results are expressed as mean value ± SEM; ***P < .005; #not statistically significant, ANOVA.