Figure 4. Induction of negative selection by Sirpα⁺ cDCs in tumor-bearing mice.

(A) Thymus was collected at 14 days after Col26 tumor inoculation. Percentage of DC-SIGN⁺Sirpα⁺ and total Sirpα⁺ cells in thymic CD11c^high DCs in WT mice bearing tumor or mice without tumor are shown. (B) A double-color fluorescent immunostaining for Sirpα (red) and Col IV (green). Single color image for Sirpα and merged image are shown on the upper and lower, respectively. Scale bar, 50 µm. (C) A double-color fluorescent immunostaining for Col IV (red) and CCL2 (green). The right panel shows the thymus which was collected at 1 hr after intravenous injection of rCCL2 (5 μg). Single color image for CCL2 and merged image are shown on the upper and lower, respectively. Scale bar, 50 µm. (D) Parental Col26 and Col26-7ND were injected into WT mice. Subsequently, OVA₆₄₇ was intravenously injected at 14 days after tumor inoculation. Uptake of OVA₆₄₇ in Sirpα⁻ (left) and Sirpα⁺ cDCs (right) were analyzed at 4 hrs after OVA injection. Parental Col26-, Col26-7ND-bearing mouse, and mouse without tumor were represented by a solid-lined, dash-lined, and gray-filled histogram, respectively. Representative results from 3 independent experiments are shown here. (E) Enhancement of the capability of OVA uptake by Sirpα⁺ cDCs in tumor-bearing mice. Enhancement of OVA uptake  =  MFI of OVA₆₄₇ captured by Sirpα⁺ DCs in WT or CCR2^−/− mice bearing parental Col26 or Col26-7ND tumor/that in WT or CCR2^−/− mice without tumor. The right unfilled column shows the fold-enhancement of OVA₆₄₇, when OVA₆₄₇ was injected at 1 day after three times of daily injections of rCCL2 (2.5 μg/injection). (F) Two mg OVA protein was intravenously injected into DO11.10 mice at 14 days after tumor inoculation and 2 days later, expression of CD4 and CD8 in DO11.10⁺ thymocytes was analyzed. Percentage of DP thymocytes is shown in each panel. Data represent mean ± SD from three independent experiments. (G) The numbers of total thymocytes (upper graph) and DP thymocytes (lower graph) are shown. Representative results from three (B and C) or four (D and F) independent experiments are shown here. Data represent mean ± SD from four independent experiments (A, E, and G). *, p<0.01.