Figure 5. Contact-mediated antigen handoff potentiates stepwise tumor antigen cascade.

(A) Imaging showing ZsGreen⁺CD103⁺ DC (green) sorted from B16ZsGreen tumor-draining lymph nodes (tdLN) and a nuclear-tdTomato⁺ (nT) CD8α⁺ resident DC (red) sorted from steady state nTnG mouse. 2 cells marked with dotted lines. Transfer event is in middle panel. Scale bar = 5 μm. (B) ZsGreen acquisition mode of uptake (debris uptake versus DC:DC transfer) quantified from live imaging as in A. n = 6. (C) Multiphoton imaging of tdLN from B16ZsGreen, XCR1-Venus; CD11c-mCherry; MacBlue mice. Only Venus and mCherry channels shown. ZsGreen⁺ vesicle is surfaced (green); timecourse shown. Scale bar = 10 μm. (D) ZsGreen acquisition mode of uptake (debris uptake versus DC:DC transfer) quantified from live imaging as in C. n = 3. (E) Inducible ZsGreen B16 tumor experiments in F-G. (F-G) ZsGreen accumulation within cells of the tumor (F) and tdLN (G) following induced expression of ZsGreen. Days indicate initiation of tamoxifen treatment prior to sacrifice. % Max. ZsGreen calculated as mean frequency of ZsGreen cells normalized to the average maximum % of ZsGreen accumulation (set to 100%) for each cell type. Plots are mean % Max. ZsGreen +/− SEM. n = 6–10. Representative of 3 independent experiments. Statistical analysis is single sample, two-sided, Student’s t test to determine on which day a cell type’s ZsGreen amount reached statistically above 0%, *p < 0.05, **p < 0.01. See also Videos S6–S8.