T-cell priming following immunization with blood-borne antigen and adjuvant. (A) Images of in vivo antigen uptake by BM cells of a Cx3cr1^gfp/+ mouse at different time points after injection of 0.5 mg fluorescently tagged OVA (yellow) and 10 nmol CpG. An hour after injection, the antigen had been taken up by many cell types, including green CX₃CR1-GFP⁺ cells (arrows, insets, middle). After 24 hours, the fluorescent signal (arrows, right) persisted only in flat cells that line blood vessels (red), consistent with endothelial or perivascular cells (insets, right). Data are from 1 representative experiment of 2. Bar represents 50 µm. (B) Flow cytometric analysis of untreated BM or BM harvested 2 hours after intravenous injection of 0.5 mg fluorescently tagged OVA–Texas red with or without CpG. Histograms depict OVA acquisition, as indicated by the Texas red signal in pDCs (CD11c^int mPDCA-1^hi), T cells (CD3^hi), B cells (CD19^hi), conventional DCs (cDCs) (CD11c^hi MHC-II⁺), macrophages (CX₃CR1-GFP^hi, CD11c^int, F4/80⁺, MHC-II⁺), Ly6C^lo monocytes (CX₃CR1-GFP^hi, CD11c^int, F4/80⁺, MHC-II⁻), and Ly6C^hi monocytes (CX₃CR1-GFP^hi, CD11c⁻). Phagocytic capacity was slightly increased in CpG-treated mice, especially in the case of B cells. (C) Chimeric mice (CD11c-DTRtg×Cx3cr1^gfp/⁺ into wild-type) were pretreated with DTx (right) or not (left) and then immunized with 0.5 mg OVA and 10 nmol CpG. Sixteen hours later, mice were transferred with GFP OT-I T cells (green) and CFP⁺ polyclonal CD8⁺ T cells (blue) to be imaged 4 hours later using 2PM. Regardless of DC depletion, OT-I cells formed antigen-dependent clusters. See also supplemental Video 10. Bar represents 50 µm. (D) Velocities of polyclonal and OT-I CD8⁺ T cells transferred into chimeric mice that had been injected with OVA + CpG and either treated or not with DTx as described in panel C. Data points represent individual cells. The mean velocity of polyclonal T cells and OT-I cells in untreated mice was 5.9 vs 2.8 μm/min (n = 51 and 54, respectively); in DTx-treated mice, the mean velocity was 6.4 vs 3.4 μm/min, (n = 63 and 74, respectively). Regardless of DC depletion, OT-I cells decelerated. Individual 2-tailed t tests compared OT-I and polyclonal T cells within each experimental condition. *P < .001. (E) Analysis of proliferative capacity following immunization with 0.5 mg OVA + CpG. The bars represent DI calculated from pooled data from 2 independent experiments involving 5 mice in each immunization condition. DC depletion significantly reduced proliferation in the spleen (DI of 0.6) but not in the BM (DI of 1.6). *P < .001. Two-way ANOVA was used to compare, within each organ, OVA + CpG–treated mice with the 2 other groups. Error bars represent SEM. (F) Images of GFP⁺ OT-I cells (green) transferred into OVA + CpG–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. T cells in the BM of OVA-injected mice depleted of all mononuclear phagocytes were sparser and failed to divide. Insets depict a dividing cell. See also supplemental Video 11. Blood vessels were counterstained in red. Bar represents 50 µm. (G). Densities of OT-I cells in the BM, as calculated from several fields such as the ones depicted in panel F. T cells in the BM of OVA-injected mice depleted of all mononuclear phagocytes were significantly sparser. *P < .01. Error bars represent SEM. (H) In vivo killing ability of OT-I CTLs primed in the presence or absence of mononuclear phagocytes. 5000 Naïve OT-I -CFP T cells were transferred into CD11c-DTRtg into wild-type chimeric mice prior to treatment with DTx either with or without CLL. Mice were then immunized with 0.5 mg OVA and 10 nmol CpG. Eight days later, recipient mice were injected with equal numbers of CD45.1 CFSE^hi splenocytes pulsed with OVA peptide and CFSE^lo unpulsed cells. Antigen-specific killing was tested in the BM and spleens of recipient mice 1 day later. Combined depletion, but not DC depletion alone, annihilated target cell killing in both the spleen and BM. Bars show normalized percentage specific killing. *P < .05; **P < .01; ***P < .001. A 2-way ANOVA was used to compare, within each organ, OVA + CpG–treated mice with the 3 other groups. Error bars represent SEM. cDCs, conventional DCs; SP, spleen.