Figure 4.

Autophagy is required for efficient cross-presentation of soluble antigen, but not cell-associated antigen or antigen delivered via DEC205. Purified Atg7-WT or Atg7-DC CKO splenic CD8⁺ DC were incubated with (A) OVA protein (+ 0.5 μM CpG) for 45 min or (B) irradiated H-2^bm1 splenocytes coated with OVA protein (OVA-coated spleen). (C) For DEC205 targeting, purified CD8⁺ DC were activated by culture at 37°C overnight. Activated CD8⁺ DC were incubated with biotinylated anti-DEC205 antibodies followed by anti-biotin antibodies conjugated to OVA. Presentation of OVA by MHC I (cross-presentation) was assessed by monitoring the division of Cell Trace Violet-labeled OT-I T cells, while presentation of OVA by MHC II was assessed by monitoring the division of Cell Trace Violet-labeled OT-II T cells. In all cases, experiment was performed in triplicate (mean ± 1 SD) with data normalized and pooled from 2 independent experiments. Data were analyzed by 2-way ANOVA followed by Bonferroni multiple comparison test ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01.