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. 2015 May 12;6:7086. doi: 10.1038/ncomms8086

Figure 8. Reduced Tregs in CD11c-Flip-KO mice contribute to the pathogenesis of arthritis.

Figure 8

(a) The percent and number of Tregs in the thymus (n=15 per group), defined as CD4+CD25+Foxp3+ in the CD11c-Flip-KO (KO) and control mice. (b) Tregs in spleens at 4 weeks (n=11) and ≥20 weeks (n=6–9), defined as CD4+CD25+Foxp3+. (c) Correlation (Pearson's) between the number of spleen Tregs and the inflammation score of arthritis in CD11c-Flip-KO mice (≥20 week) determined just before killing. (d) The in vitro suppressive function of Tregs. The CD45.2+CD4+CD25+ T cells from CD11c-Flip-KO or control mice were co-cultured with CFSE-labelled CD45.1+CD4+D25− Tresp cells at indicated ratios on anti-CD3 mAb-coated plates for 3 days, and examined for the CFSE dilution of CD4+ T cells. The results are representative of three experiments. Cell number was calculated by flow counting beads. (e) The in vitro suppressor function by Tregs for autoreactive CD4+T-cell proliferation, determined by in vitro syngeneic mixed lymphocyte response same as in Fig. 4. Results from three independent experiments, n=3 without Tregs and n=5 with Tregs. (f)The in vivo Treg function. CD11c-Flip-KO mice (n=5) were adoptively transferred with two dosages (∼ 2 × 106 per dose) of CD4+CD25+ T cells isolated from the spleens of CD45.1 mice at 0 and 3 weeks. Six age-matched CD11c-Flip-KO mice served as the no-treatment controls. The values in the left panel represent the mean ±1 s.e of the inflammation scores, ##P<0.01 (paired t test) compared with the score at week 0. The data in right panel reflect the change in the inflammation score (Δ). (g) In vitro syngeneic mixed lymphocyte response. The T cells from the pLNs and brachial LNs of the Treg-treated mice (in f) were CFSE-labelled and incubated with syngeneic APCs to identify autoreactive T cells. The values for the autoreactive T cells of CD11c-Flip-KO mice without Treg treatment are same as presented in Fig. 4c, which are used here for comparison (*P<0.05, **P<0.01 and ***P<0.001, unpaired two-sided t-test, except(e) ANOVA plus Tukey).