Activation of naïve CD8⁺ T cells in the BM does not depend on crosspresentation by stromal cells or B cells but is abrogated by ablation of monocytes, macrophages, and DCs using DTx and CLL. (A) Proliferation of OT-I T cells when either stromal or hematopoietic cells can crosspresent antigen. The indicated chimeric mice were transferred with CFSE-labeled CD45.1 OT-I cells, either treated or not with DTx, injected with 0.1 mg OVA, and analyzed by flow cytometry 48 hours later. Histograms represent CFSE profiles of cells gated according to CD45.1 and CD8 expression. In the BM, presentation was carried out by hematopoietic cells but not only DCs. Data are from 1 representative experiment of 3. (B) Proliferation of OT-I T cells in the absence of B cells. Flow cytometry of transferred OT-I cells into CD11c-DTRtg and CD11c-DTRtg×JH^−/− mice treated as in (A). T cell proliferation in the BM persisted in the combined absence of B cells and DCs. Data are from 1 representative experiment of 2. (C) Depletion of mononuclear phagocytes using CLL. Flow cytometric analysis of monocytes (CX₃CR1-GFP^hi, CD11c^int, F4/80⁺, MHC-II⁻), macrophages (CX₃CR1-GFP^hi, CD11c^int, F4/80⁺, MHC-II⁺), DCs (CD11c^hi MHC-II^hi), pDCs (CD11c^int mPDCA-1^hi), and pre-DCs (Lin⁻ CD11c^hi MHCII^- Flt3⁺ SIRPα⁻) 1 day after intravenous injection of CLL. Bars represent total numbers of cells isolated from 2 tibiae and 2 femurs. CLL treatment efficiently depleted almost all BM monocytes and macrophages alongside with half of the DCs. Individual 2-tailed t tests were conducted for each cell type. *P < .05; **P < .0001, Error bars represent SEM. Data are from 1 representative experiment of 2 (n = 3 in each group). (D) Proliferation of OT-I T cells in the absence of mononuclear phagocytes. Chimeric mice [CD11c-DTRtg into wild-type] were transferred with CFSE-labeled CD45.1 OT-I cells; either treated or not with DTx, CLL, or both; injected with 0.1 mg OVA; and analyzed by flow cytometry 48 hours later. Histograms represent CFSE profiles of cells gated according to CD45.1 and CD8 expression. Data are from 1 representative experiment of 2. (E) Analysis of proliferative capacity. The bars represent DI calculated from pooled data from 2 independent experiments involving 2 to 6 mice in each condition (for which representative plots are shown in panel D). T-cell proliferation in the BM was abrogated when all mononuclear phagocytes were depleted. *P < .001. Two-way ANOVA was used to compare, within each organ, OVA-treated mice with each of the other treatment groups. Error bars represent SEM. (F) Images of GFP⁺ OT-I cells (green) transferred into OVA-injected CD11c-DTRtg into wild-type chimeric mice that had been left intact (left) or treated with DTx and CLL (right). T cells were analyzed by 2pm 48 hours after OVA challenge. The BM microvasculature was visualized by Quantum dots (red). T cells in the BM of OVA-injected mice depleted of all mononuclear phagocytes were sparser and failed to divide. Data are from 1 representative experiment of 2. Bar represents 50 µm. See also supplemental Video 9. cDCs, conventional DCs.