Fig 1.
Oral administration of live LG2809 enhanced oral tolerance induction in DO11.10 mice via non-apoptotic pathways.
DO11.10 mice were fed live LG2809 (1 mg/day; treated) or 0.85% NaCl (saline; untreated) for 12 days. For the last 5 days, the mice did (oral tolerance group) or did not (control group) have OVA added to the drinking water. According to feeding regimes, the groups were as follows: saline/water (untreated control group), saline/OVA (untreated oral tolerance group), LG2809/water (treated control group), and LG2809/OVA (treated oral tolerance group). SPL CD4+ T cells from each group were cultured with APCs and 1 μM OVA323–339. (A) The cultures were pulsed with [3H]-thymidine for the last 24 hrs of the 96 hrs culture period and [3H]-thymidine incorporation was measured. (B) IL-2 in the supernatant after 48 hrs in culture was measured by ELISA. (C, D and E) Whole SPLs from DO11.10 mice of each group were stained with anti-CD4-PerCP, anti- KJ-1.26-FITC, and annexin V-PE. The ratio of apoptotic cells was determined by flow cytometric analysis. The ratio of CD4+ KJ-1.26+ cells (C), of live (CD4+ KJ-1.26+ annexin V- 7-AAD-) cells (D), of CD4+ KJ-1.26+ annexin V+ apoptotic cells (E) and CD4+ KJ-1.26+ Foxp3+ cells (F) is shown for each group. Data are shown as the means ± SD (n = 6). Data are representative of two independent experiments. Statistical differences were analyzed by Student’s t-test and were considered significant (*) when p was <0.05. n.s., not significant.
Fig 2.
LG2809 enhanced IL-10 secretion and T-cell suppressive activity in splenocytes from orally tolerized mice.
DO11.10 mice were treated as described in the legend to Fig 1. (A) SPL CD4+ T cells from DO11.10 mice of each group were cultured with APCs and 1 μM OVA323–339. IL-10 in the supernatant after 48 hrs in culture was measured by ELISA. (B) SPL CD4+ T cells from DO11.10 mice of the saline/OVA or LG2809/OVA groups (effector cells) were cultured with APCs, 1 μM OVA323–339, and responder DO11.10 CD4+ CD25- T cells at the ratio indicated in the figure. IL-2 in the supernatant was measured by ELISA. Data are shown as means ± SD. (n = 6). Data are representative of two independent experiments. Statistical differences were analyzed by Student’s t-test. *p<0.05 and **p<0.01, significantly different vs. saline/OVA group; ##p<0.01 and ###p<0.001, significantly different vs. responder DO11.10 CD4+ CD25- T cells alone.
Fig 3.
LG2809 increased the ratio of CD62Llow CD44high CD4+ T cells with IL-10-producing and suppressive activities.
DO11.10 mice were treated as described in the legend to Fig 1. (A and B) Whole SPLs from DO11.10 mice of each group were stained with anti-CD4-PerCP, KJ-1.26-FITC, anti-CD44-PE, and anti-CD62L-APC. The expression of CD44 and CD62L on CD4+ KJ-1.26+ T cells was analyzed by flow cytometric analysis. The mean ratio of CD62Llow CD44high cells in CD4+ KJ-1.26+ T cells is shown (B). Data are shown as means ± SD (n = 6). Data are representative of two independent experiments. Statistical differences were analyzed by Student’s t-test. *p<0.05. (C) The correlation between the concentration of IL-10 in the culture supernatant of CD4+ T cells shown in Fig 1C and the ratio of KJ-1.26+ CD62Llow CD44high cells among CD4+ T cells. Closed circles, LG2809/OVA group; open circles, LG2809/water group; closed triangles, saline/OVA group; open triangles, saline/water group. The continuous line is a linear regression for LG2809/OVA group and the dashed line is a linear regression for saline/OVA group. R, Pearson correlation coefficient. Data are representative of two independent experiments. (D) CD62Lhigh CD44high and CD62Llow CD44high cells sorted from CD4+ T cells isolated from DO11.10 mice of the LG2809/OVA group were cultured with APCs and 0.3 μM OVA323–339. After 48 hrs, IL-10 in the supernatant was measured by ELISA. (E) The sorted CD62Lhigh CD44high and CD62Llow CD44high CD4+ T cells from DO11.10 mice of the LG2809/OVA group were incubated with responder CD4+ T cells from DO11.10 SPL (at a ratio of 1:1), plus APCs and 0.3 μM OVA323–339. IL-2 in the supernatant was measured by ELISA. (F) The sorted CD62Lhigh CD44high and CD62Llow CD44high CD4+ T cells from DO11.10 mice of the saline/OVA, LG2809/OVA group, and CD4+ CD25- T cells were incubated with APCs and 0.3 μM OVA323-339. The cultures were pulsed with [3H]-thymidine for the last 24 hrs of the 96 hrs culture periods and [3H]-thymidine incorporation was measured. Data are representative of two independent experiments.
Fig 4.
LG2809 enhanced plasmacytoid dendritic cells in the lamina propria.
BALB/c mice were fed live LG2809 (1 mg/day) or saline for 7 days. The ratio of CD11c+ B220+ (A), CD11c+ Ly-6G+ (B), CD11c+ CD11b+ (C), and CD11c+ CD103+ (D) cells among CD3- LP cells was determined by flow cytometric analysis. Data are shown as the means ± SD. (n = 4). Data are representative of two independent experiments. Statistical differences were analyzed by Student’s t-test. *p<0.05. n.s., not significant.
Fig 5.
CD4+ T cells differentiated with lamina propria APCs from mice fed LG2809 had stronger suppressive activity.
(A and B) BALB/c mice were fed live LG2809 (1 mg/day) or saline for 7 days. CD4+ CD25- T cells from DO11.10 mice were cultured with 1 μM OVA323–339 and CD3- LP cells from BALB/c mice fed LG2809 or saline as APCs. After 72 hrs, the CD4+ T cells were purified by MACS. The activated CD4+ T cells were incubated with responder CD4+ CD25- T cells from DO11.10 SPL cells (at ratios of 0.4:1 and 1:1), plus APCs and 1 μM OVA323–339. After 48 hrs, IL-2 in the supernatant was measured by ELISA (A). The activated CD4+ T cells were incubated with APCs and 1 μM OVA323–339. After 48 hrs, IL-10 in the supernatant was measured by ELISA (B). Data are shown as means ± SD (n = 4). Data are representative of two independent experiments. Statistical differences were analyzed by Student’s t-test. *p<0.05, significantly different vs. saline group; n.s., not significant.; ###p<0.001, significantly different vs. responder DO11.10 CD4+ CD25- T cells alone.