Naïve CD8⁺ T cells are efficiently activated in the BM upon antigenic challenge. (A) Average velocities of transferred polyclonal CFP⁺ CD8⁺ T cells and GFP⁺ OT-I cells, collected from the spleen and LNs, in the presence of antigenic protein. During BM imaging, the mice were challenged with 0.1 mg OVA–Alexa 594 and were continuously imaged for 5 hours. Cell speed gradually decreased in an antigen-specific manner. Data points represent individual cells taken from 1 representative experiment out of 3. *P < .05; **P < .001 (based on a comparison between polyclonal and OT-I T cells within the same time point). Multiple comparisons analysis was done using Kruskal-Wallis test with Dunn’s comparisons. (B) The 4-hour time point from the experiment depicted in panel A. Polyclonal CFP⁺ CD8 T cells (blue) and GFP⁺ OT-I cells (red) were followed in the BM after administration of OVA–Alexa 594 (yellow). Cell tracks (white) indicate continued motility of polyclonal cells (left), while OT-I cells arrest (right). Bars represent 50 µm. See also supplemental Video 4. (C) Three-dimensional paths of individual polyclonal CD8⁺ T and OT-I cells 4 hours after injection of OVA showing shorter paths for OT-I cells. (D) CFP⁺ polyclonal CD8⁺ T cells (blue) and CMTMR-labeled OT-I T cells (yellow) were transferred into a mouse harboring Cx3cr1-GFP⁺ cells (green) that had been injected with 0.5 mg OVA 18 hours before. T cells were analyzed 4 hours after transfer. The BM microvasculature was visualized by Quantum dots (red). Data are from 1 representative experiment of 3. Unlike polyclonal T cells, OT-I cells cluster in the vicinity of GFP⁺ cells. Bar represents 50 µm. See also supplemental Video 5. (E) T-cell activation in various lymphoid organs following challenge with blood-borne antigen. Wild-type C57BL/6 CD45.2 mice were transferred with OT-I CD45.1 naïve T cells and challenged intravenously with 0.5 mg OVA protein or served as controls. The bars represent the percentage of CD69⁺ T cells (gated as in supplemental Figure 2) in the BM, spleen, and LNs 2 to 3 hours after OVA injection, as determined by flow cytometry. Repeated-measure ANOVA (n = 7) indicated a significant effect of OVA treatment (P < .0001) and of the organ of origin (P < .0001) and a significant interaction (P < .0001). Scheffé’s post hoc comparisons indicated that OVA challenge increased T-cell activation in all organs (P < .0001 for all) and that T-cell activation was greater in the BM than in the spleen and LNs, both at the steady state and after OVA challenge (P < .001 for all). T-cell activation was greater in the spleen than in LN after OVA challenge (P < .001) but not at steady state (P = .65). Data are from 2 independent experiments. Asterisk denotes significant differences; error bars represent SEM. (F) Proliferation of OT-I T cells in the BM following OVA challenge. Wild-type mice were transferred with OT-I cells injected with 0.1 mg OVA 18 hours later and analyzed 48 hours later. Representative FACS histograms show CFSE dilution in CD45.1 OT-I cells; data are from 1 representative experiment out of 5. (G) Levels of Ki-67 (an early indicator of proliferation) in OT-I T cells retrieved from the spleen, BM, and blood of mice at various times after challenge with 0.1 mg OVA. The lines follow the percentage of Ki-67⁺ cells (non-G₀ cells) among OT-I cells. Ki-67 levels in the BM and spleen showed a similar pattern, rising by 24 hours and plateauing at 36 hours, while blood levels lagged by a day. Error bars represent SEM. (H) The frequency of CD69⁻ and CD69⁺ OT-I cells in the blood, spleen, and BM of the host mice analyzed in panel G. Curves show the percentage of CD69⁺ OT-I cells out of CD8⁺ cells stacked on top of the percentage of CD69⁻ OT-I cells. Blood levels of CD69⁺ OT-I cells remained low. While CD69⁺ cells accumulated gradually in the spleen and BM, these cells downregulated CD69 only 48 hours after antigen challenge. (I) Snapshots from 2pm movies (supplemental Videos 6 and 7) show transferred GFP⁺ OT-I cells (green) in mice injected (right) or not (left) with 0.1 mg OVA 48 hours before. Blood sinusoids appear red. Cell volume increased and, as shown in the insets, cells divided locally in the BM in response to blood-borne antigen. Data are from 1 representative experiment of 2. Bar represents 50 µm. SP, spleen.