DCs develop in the BM from MDPs without monocytic intermediates and are not essential for activation of naïve CD8⁺ T cells. (A) Flow cytometry of BM cells from wild-type mice reconstituted with BM from CD11c-DTRtg mice. Chimeric mice were left untreated or received an intraperitoneal injection of DTx. Twenty-four hours after treatment, the percentage of CD11c^hi MHC-II⁺ DCs and Lin⁻ CD11c^hi MHCII^- Flt3⁺ SIRPα⁻ pre-DCs was calculated. Numbers adjacent to outlined areas indicate DC or pre-DC percentage. Data are from 1 representative experiment out of 3. (B) Images of the cranial BM cavities of [CD11c-DTRtg×Cx3cr1^gfp/+ into wild-type] chimeric mice treated (right) or not (left) with DTx. The treatment thoroughly depletes DCs but spares round monocytes (middle) and spindle-shaped macrophages (right). GFP⁺ cells appear green and blood vessels appear red. Data are from 1 representative experiment of 3. Bars represent 10, 10, and 25 µm. (C) BM images of DTx-treated CD11c-DTRtg into wild-type chimeric mice after transfer of monocytes or MDPs. Fourteen days after transfer, monocytes did not give rise to DCs (left) while MDPs efficiently generated DCs in the BM (right). Data are from 1 representative experiment of 2. Bars represent 40 µm. (D) Chimeric mice (CD11c-DTRtg×Cx3cr1^gfp/⁺ into wild-type) were treated with DTx and injected with 0.5 mg OVA. Sixteen hours later, mice were transferred with CMTMR-labeled OT-I T cells (yellow) and CFP⁺ polyclonal CD8⁺ T cells (blue) to be imaged 4 hours later using 2pm. Despite DC depletion, OT-I cells formed antigen-dependent clusters (indicated). Green cells are monocytes spared in the BM after DC ablation. Data are from 1 representative experiment of 2. Bar represents 50 µm. See also supplemental Video 8. (E) Velocities of polyclonal and OT-I CD8⁺ T cells transferred into chimeric mice that were injected with 0.5 mg OVA and either treated or not with DTx as described in panel A. Data points represent individual cells. The mean velocity of polyclonal T cells and OT-I cells in non–DTx-treated mice was 8.7 vs 3.8 μm/min (n = 40 and 67, respectively); in DTx-treated mice, the mean velocity was 5.7 vs 2.47 μm/min (n = 45 and 37). Individual 2-tailed t tests compared OT-I and polyclonal T cells within each treatment. Data are pooled from 3 untreated mice and 2 DTx-treated mice. *P < .001. (F) Proliferation of OT-I T cells in DC-depleted or intact mice. Chimeric mice (CD11c-DTRtg into wild-type) were treated or not with DTx and transferred with CFSE-labeled CD45.1 OT-I cells. Sixteen hours later, the mice were injected or not with 0.1 mg OVA. Forty-eight hours later, T-cell division in the BM, spleen, and LNs was analyzed using CFSE dilution. Histograms represent transferred cells gated based on CD45.1 and CD8 expression. (G) Analysis of proliferative capacity. The bars represent DI calculated from pooled data from 2 independent experiments involving 5 mice in each immunization condition (for which representative plots are shown in panel F). DC depletion abrogated proliferation in the spleen (DI of 0.3) but not in the BM (DI of 1.6). *P < .05; **P < .01; ***P < .001. Two-way ANOVA was used to compare, within each organ, OVA-treated mice with either untreated or DTx and OVA-treated mice. Error bars represent SEM. SP, spleen.