Fig. 7. HMGB1 released through oral CT administration contributes to activation of intestinal DCs and cross-priming of CTLs in vivo.

a B6 mice were orally administered PBS or 10 μg of CT and simultaneously treated with i.v. injection or oral administration of 500 μg of GL. Six hours later, some mice were re-treated with GL in the same manner. Feces and blood were collected 16 h after oral administration of CT and GL treatment, and HMGB1 levels in the fecal extracts and plasma were measured by ELISA assay. The results are shown as the mean + SE in four to five mice per group. *P < 0.05; Welch’s t-test. b B6 mice were orally administered PBS or 10 μg of CT and treated with i.v. injection or oral administration of 500 μg of GL at the same time and 6 h after CT administration. MLN cells were isolated from the mice 24 h after CT administration and double-stained with FITC-labeled anti-CD11c Ab and APC-labeled anti-CD80, anti-CD86 (gray fill for PBS-treated mice and bold solid line for mice treated with CT and GL), or subtype control Ab (solid line). CD11c⁺ cells were gated and the expression of CD80 or CD86 was analyzed. Each value represents the MFI of CD80 or CD86. c MFI data as described in (b) are shown as the mean + SE in four to six mice. *P < 0.05, **P < 0.001; Welch’s t-test. d MLN cells were isolated from mice treated as described in (b) and triple-stained with FITC-labeled anti-CD11c Ab, PE-labeled anti-DEC-205 or anti-DCIR2 Ab, and APC-labeled anti-CD80, anti-CD86, or subtype control Ab. DEC-205⁺CD11c⁺ cells and DCIR2⁺CD11c⁺ cells were gated and the expression of CD80 or CD86 was analyzed. The results are representative of two independent experiments. e B6 mice were orally administered 10 μg of CT alone or plus 10 mg of OVA and treated with i.v. injection or oral administration of 500 μg of GL at the same time and 6 h after the CT administration. MLN cells were isolated from mice 24 h after CT administration and CD11c⁺ DCs were purified from MLN cells using the MACS system and co-cultured with CFSE-labeled OT-I or OT-II T cells for 4 days. The cultured OT-I or OT-II T cells were stained with APC-labeled anti-CD8 Ab or anti-CD4 Ab, respectively. CD8⁺ OT-I or CD4⁺ OT-II T cells were gated and the CFSE dilution profiles were analyzed by flow cytometry. f Data for the proliferation of CD8⁺ OT-I T cells (%) as described in (e) are shown as the mean + SE in three mice. *P < 0.05, **P < 0.005, ***P < 0.001; Welch’s t-test. g The cultured OT-I T cells as described in (e) were re-stimulated by plate-bound anti-CD3 Ab for 16 h and OVA-specific cytotoxic activity of the re-stimulated OT-I T cells was assessed. The E:T ratio is 10:1 and the results are shown as the mean + SE in triplicate of pooled cells from two mice. *P < 0.01, **P < 0.005, ***P < 0.0005; Welch’s t-test. h B6 mice were orally administered 100 mg of OVA alone or plus 10 μg of CTA subunit, CTB subunit, or CT every week. Some of the mice administered with OVA plus CT were treated by i.v. injection or oral administration of 500 μg of GL concurrently with, and 6 h after, the administration of OVA plus CT every week. Feces and blood were collected 3 weeks after the first oral administration and levels of anti-OVA IgA in the fecal extracts and anti-OVA IgG1 in the plasma were measured by ELISA assay. The results are shown as the mean + SE (four to five mice per group). *P < 0.05, **P < 0.001, not significant (ns); Welch’s t-test