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. 2021 Feb 22;6(4):e142376. doi: 10.1172/jci.insight.142376

Figure 5. LLL12b restores Th17.

Figure 5

Treg balance of CD4+ T cells from MS patients. (A–L) PBMCs from treatment-naive MS patients (n = 22) were activated with αCD3/CD28 plus TGF-β/IL-6 for 3 days with LLL12b. DMSO was used as vehicle control. (A–D) IL-17 in supernatant of 1 patient sample was determined by ELISA and compared with 1-way ANOVA (A). IL-17 in LLL12b (0.125 μM) group was compared with control group of the same patient using Wilcoxon matched-pairs signed rank test (B). The percentage of decrease of IL-17 was calculated (C), and the patients in different ranges were shown in a pie chart (D). (E–H) Tregs (FoxP3+CD25+) were determined by intracellular staining, gating on CD45RA+CD4+ cells (E), and compared between LLL12b- (0.125 μM) and control-treated groups of the same patient using Wilcoxon matched-pairs signed rank test (F). The percentage of increase of Tregs was calculated (G), and the patients in different ranges were shown in a pie chart (H). (I–K) IL-17/Treg ratio of each patient was calculated and compared between LLL12b and control groups with Wilcoxon matched-pairs signed rank test (I). The percentage of decrease of IL-17/Treg ratio was calculated (J) and shown in a pie chart (K). (L) A nonparametric Pearson correlation test was used to analyze the degree of relatedness between percent increase of Treg and percent decrease of IL-17. (M) PBMCs from MS patients (n = 3) were activated with αCD3/CD28 under iTreg differentiation condition for 3 days. CFSE-labeled PBMCs from the same 3 patients were cultured with LLL12b (0.25 μM) or vehicle control DMSO for 2 hours, washed and mixed with iTregs cultured cells (Teff:Treg = 16:1), followed by activation with αCD3/CD28 for 5 days. CFSE in CD4+ T cells was determined by flow cytometry. The percentage of suppression was calculated and compared with a paired Student’s t test. **P < 0.01; ****P < 0.0001.