Fig 1.
IL-35 increased the number of Treg in CD4+ T cells.
(A)The ratio of Treg in CD4+ T cells. (B) Representative diagram showing in comparison with the control group, IL-35 markedly enhanced the proliferation of Tregs (50.7% in IL-35 group vs. 9.5% in control group on day 5, P<0.01) under these conditions with time. Results are expressed as means ± SEM, from two experiments (n = 3 times/group/experiment). Unpaired t-tests were performed for comparisons between control- and IL-35- groups on corresponding days. *, ** and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively.
Fig 2.
IL-35 decreased the prevalence of Th17 in CD4+ T cells.
(A)The ratio of Th17 in CD4+ T cells. (B) Representative diagram showing Th17’s ratio in CD4+ T cells was significantly decreased in the IL-35 group compared to that in the control group (0.3% vs. 7.2% on day 5, P<0.01). Results are expressed as means ± SEM, from two experiments (n = 3 times/group/experiment). Unpaired t-tests were performed for comparisons between control- and IL-35- groups on corresponding days. *, ** and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively.
Fig 3.
IL-35 down-regulated the ratio of Th17/Treg in CD4+ T cells.
The ratio of Th17/Treg in CD4+ T cells in IL-35 group or control group on day 1, 3, 5 after co-culturing. A remarkable difference in the ratio of Th17/Treg in CD4+ T cells was observed under these conditions, particularly on day 5 after co-culture (1.4% in control group vs. 0.1% in IL-35 group, P<0.01). Results are expressed as means ± SEM, from two experiments (n = 3 times/group/experiment). Unpaired t-tests were performed for comparisons between control- and IL-35- groups on corresponding days. *, ** and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively.
Fig 4.
IL-35 ameliorated the function of islets in co-culture system.
The amount of insulin release of 15 co-cultured islets in low glucose level (a) and in high glucose level (b). Results are expressed as means ± SEM, from two experiments (n = 3 times/group/experiment). Unpaired t-tests were performed for comparisons between control- and IL-35- groups on corresponding days. *, ** and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively.
Fig 5.
IL-35 delayed the survival of co-cultured islets.
The islet cells were stained using AO/EB staining kit. The dead islet cells were labeled with yellow fluorescence, while the living islet cells were labeled with green fluorescence. In control group, most of islet cells were dead (a); however, the islet cells in IL-35 group were still living (b).
Fig 6.
Treg inhibited the proliferation of Th17 and IL-35 enhanced the suppressive function of Treg.
Representative histograms showing different Th17/Treg ratio (1:0, 1:0.5, 1:1, 1:2) in the co-culture system. With an increase of Treg ratio, on day 5, the proliferation of the CFSE-labeled Th17 cells was suppressed markedly (suppression ratio rising from 7.82% in 1:0 group to 48.2% in 1:2 group); besides, stronger suppression was observed when IL-35 was added, compared to that in the control group (suppression ratio rising from 20.5% in 1:0 group to 76.7% in 1:2 group).
Fig 7.
Up-regulating the prevalence of Treg ameliorated the function of islets.
The function of co-cultured islet cells in Th17:Treg = 1:0 group and Th17:Treg = 1:2 group in low glucose level (2.5mM) or high glucose level (16.7mM). The results showed that regardless of the glucose level, islet cells in the Th17/Treg = 1:2 group had better insulin secretory function than that in the Th17/Treg = 1:0 group. Results are expressed as means ± SEM, from two experiments (n = 3 times/group/experiment). Unpaired t-tests were performed for comparisons between control- and IL-35- groups on corresponding days. *, ** and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively.
Fig 8.
Model of IL-35 regulating Th17/Treg differentiation.