Figure 6. A role for LBs in cross-presentation.

(A–D) Restoration of LB accumulation and cross-presentation by Irgm3 complementation in Irgm3−/−mDCs

Differentiated Irgm3−/− mDCs were infected with pMx control retrovirus or pMx-Irgm3.

(A) IB from cell lysate after 24 hour culture.

(B) FACS analysis after overnight culture of infected DCs using CD11c and Bodipy staining.

(C) IF with anti- Irgm3 staining and the LB specific Bodipy probe.

(D) Cross-presentation assay using Irgm3−/− mDCs infected after OVA-beads uptake with pMx or pMx-Irgm3. mDCs were extensively washed and cultured with anti-OVA OT-I CD8+ T cells. Cross-presentation was monitored by CD69 upregulation. Results are plotted ± SEM.

(E, F) Efficient cross-presentation is retained within the lipid body high fraction of WT mDCs

WT DCs having phagocytosed one bead were sorted according to their Bodipy signal, in order to obtain two DC populations, the Bodipy “high” (black line) and the Bodipy “low” (gray line), which present distinct LB accumulation.

(F) In vitro cross-presentation of OVA or BSA coated beads in Bodipy high and low DC populations was measured for different DC:T cells ratio. Cross-presentation was monitored by CD69 upregulation. The average of three independent experiments, each one normalized to maximal OT-I response is shown (average ± SEM).

(G– I) Pharmacological inhibition of lipid bodies inhibit cross-presentation in WT cells

(G) Bodipy staining after gating on CD11c+ cells of mDCs treated during four hours with 85 µM Xanthohumol or the control concentration of DMSO.

(H) CD69 plot after gating on Vα2+CD8+ of OT-I cells cultured overnight with fixed mDCs exposed to DMSO or 85 µM Xanthohumol during four hours after Ag uptake.

(I) Quantification of OT-I T cell activation (average ± SEM).