Figure 2.

Antigen cross-presentation assay

(A) Representative experiment of Gal3-GFP-expressing DC2.4 cells treated with nOVA (upper image) versus aOVA (lower image). White arrows point to some damaged endosomes.

(B) Puncti quantification per cell in transfected cultures treated with OVA (black circles) or Accum-OVA (green circles). For this experiment, n = 12 fields of view from three different cultures with ∗∗p < 0.01.

(C) A representative flow cytometry experiment to assess fluorescent OVA-AF647 or Accum-OVA-AF647 uptake by mature DCs.

(D) A representative flow cytometry experiment investigating OVA-DQ versus aOVA-DQ processing by mature DCs.

(E) Quantification of the mean fluorescent intensity of the OVA-DQ/aOVA-DQ signals shown in (D). For this experiment, n = 5/group with ∗∗∗p < 0.001.

(F) A representative flow cytometry assessment of OVA-DQ processing following mature DC treatment with proteasome inhibitors.

(G) Schematic diagram showing the set-up of the antigen cross-presentation used to assess OVA-responding OT-I (CD8) and OT-II (CD4) T cells.

(H) IFN-gamma quantification using OT-I-derived CD8 T cells co-cultured with pulsed mature DCs.

(I) IL-2 quantification using OT-II-derived CD4 T cells co-cultured with pulsed mature DCs. For both (H and I), n = 4/group with ∗∗∗p < 0.001. The nOVA concentration is 1 mg/mL whereas the aOVA concentrations used were 1, 0.1, 0.05, 0.25, and 0.0125 mg/mL.

(J) IFN-gamma quantification using OT-I-derived CD8 T cells co-cultured with pulsed mature DCs pre-treated with proteasome inhibitors.

(K) Antigen cross-presentation assay using iDCs pulsed with nOVA or aOVA. For (J and K), n = 5/group with ∗∗∗p < 0.001. See also Figures S1–S3. All experiments presented in (C–K) were repeated at least three times.