Exploring potential mechanism of enhanced cross-presentation by YOD1-C160S APCs. (A) Control and YOD1-C160S BMDCs were pulsed with 50 μg/mL of Ova-FITC for 30 minutes and the intensities of Ova-FITC were measured over time. FACS plots overlay show the percentage of FITC⁺ cells (text box) and the mean fluorescence intensities of FITC below the respective histograms in control (regular letters) and YOD1-C160S expressing cells (bold letters). (B) Fifty μg of Ova-FITC was injected into footpads of control and YOD1-C160S mice and the percentages of Ova-FITC⁺ CD8⁺CD11c⁺ cells were measured at 6 and 24 hours in the draining popliteal LNs by flow cytometry. Overlay FACS plots are shown. Thin and thick lines represent the Ova-FITC staining in cells isolated from control and YOD1-C160S mice, respectively. C. BMDCs were pulsed with 200 μg/mL of Ova for 30 minutes and surface displayed SIINFEKL-peptide/H-2K^b complexes on control and YOD1-C160S BMDCs are shown after 12 hours (IC stands for isotype control). (D-G) Influence of chemical/pharmacologic inhibitors or TAP1-deficiency on antigen cross-presentation. Control and YOD1-C160S BMDCs were pulsed with 50, 100, or 500 μg/mL of ovalbumin for 5 hours. Different chemical inhibitors were added during the pulse period as described in supplemental Figure 5A. Treated BMDCs were then cocultured with CFSE labeled OT-I cells and their proliferation and cytokine production were measured. Proliferation (D) and IL-2 production (E) by OT-I cells under indicated conditions is shown. Experiments were repeated 4 times with similar results and data are shown as mean values ± SEM. (F-G) Control and YOD1-C160S BMDCs pulsed with 500 μg/mL of ovalbumin were treated with indicated doses of proteasome inhibitor (zL₃VS) and cocultured with CFSE labeled OT-I cells. Proliferation (F) and IL-2 levels (G) under indicated conditions are shown. Experiments were repeated 3 times and mean values ± SEM are shown. (H-J) BMDCs from TAP1^−/−YOD1-C160S and YOD1-C160S mice were generated in the presence or absence of doxycycline. These cells were pulsed with different doses of Ova and cocultured with CFSE labeled OT-I cells. After 72 hours of coculture, proliferation of OT-I cells (H) IL-2 production (I) in culture supernatants were measured. (J) Bar diagrams show the fold changes in the levels of IL-2 from OT-I cells cocultured with control and YOD1-C160S BMDCs pulsed with 50 μg/mL of Ova. (K-L) TAP1^−/−YOD1-C160S mice fed with regular or doxycycline supplemented water for 7 days were transferred with 5 × 10⁵ OT-I cells. These mice were then immunized subcutaneously in the base of tail with 200 μg of Ova emulsified in incomplete Freund adjuvant (IFA) and the frequencies of K^b-SIINFEKL-Tet⁺ cells were measured at 7 days after immunization in the draining iliac LNs. K. Representative FACS plots show the frequencies of K^b-SIINFEKL-Tet⁺ cells. (L) Bar diagram show the cumulative frequencies of SIINFEKL-Tet⁺ cells in 2 groups of mice. Three animals were included in each group and experiments were repeated twice.