Fig. 4. Intestinal DCs after oral administration of OVA plus CT cross-presenting OVA antigens captured by the DEC-205 receptor.

a B6 mice were orally administered PBS, 10 mg of OVA alone, 10 μg of CT alone, or OVA plus CT. After 16 h, CD11c⁺ DCs were purified from LP and MLNs of the mice using the MACS system and co-cultured with CFSE-labeled OT-I T cells for 4 days. The cultured OT-I T cells were stained with APC-labeled anti-CD8 Ab. The CFSE-dilution profiles of CD8⁺ OT-I T cells were analyzed by flow cytometry. b The cultured OT-I T cells as described in (a) were re-stimulated by plate-bound anti-CD3 Ab for 16 h. OVA-specific cytotoxic activity of the re-stimulated OT-I T cells was measured by the ⁵¹Cr-release assay using EL4 cells pulsed with the OVA-peptide SIINFEKL and using E.G7-OVA cells as target cells. The E:T ratio was 10:1. IFN-γ release of the re-stimulated OT-I T cells was also assessed. The results are shown as the mean + SE in duplicate. c, d B6 mice were orally administered 10 mg of OVA alone or plus 10 μg of CTA subunit, CTB subunit or CT. After 24 h, CD11c⁺ DCs were purified from MLNs and co-cultured with CFSE-labeled OT-I T cells. The CFSE dilution profiles (c) and OVA-specific cytotoxic activity (d) were assessed as described in a and b, respectively. e, f B6 mice were orally administered PBS or 10 mg of OVA plus 10 μg of CT. After 24 h, CD11c⁺ DCs were purified from spleens and MLNs and co-cultured with OT-I T cells. After 4 days of culture, the CFSE dilution profiles (e) and OVA-specific cytotoxic activity (f) were assessed as described in a and b, respectively. g B6 mice were orally administered 10 mg of OVA plus 10 μg of CT. After 16 h, CD11c⁺ DCs, CD8⁺CD11c⁺ DCs, or CD8⁻CD11c⁺ DCs were purified from MLN cells using the MACS system and co-cultured with CFSE-labeled OT-I T cells for 4 days. The CFSE-dilution profiles were assessed as described in (a. h, i) CD11c⁺ DCs were purified from LP, MLNs, or spleens of B6 mice using the MACS system and labeled using biotinylated anti-DEC-205, anti-DCIR2, or subtype control Ab. Subsequently, the cells were loaded with anti-biotin Ab conjugated to OVA antigens and co-cultured with CFSE-labeled OT-I T cells for 4 days. The CFSE-dilution profiles (h) and OVA-specific cytotoxic activity i were assessed as described in a and b, respectively. The E:T ratio is 4:1 and the results are shown as the mean + SE in duplicate. The results are representative of two independent experiments