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. 2020 Jan 21;10:3047. doi: 10.3389/fimmu.2019.03047

Figure 2.

Figure 2

TNFα-TNFR2 signaling reduces IL-17A expression in activated effTreg, conceivably via the anti-inflammatory regulator TNFAIP3/A20. (A) Flow cytometry of intracellular IL-17A expression in FOXP3high effTreg that were stimulated with αCD3/CD28/rhIL-2 for 5 days in the absence or presence of rhTNFα or anti-TNF (n = 15). (B) RT-qPCR gene expression of IL-17A, IL-17F, and RORC, (C) NFκB target genes NFKB1, NFKB1A, NFKB2 (n = 5), and (D) TNFAIP3 (n = 8) at day 4 of culture. (E,F) TNFAIP3 and IL-17A gene expression of non-targeting-gene control (NTC) and siTNFAIP3 effTreg after 6 days under αCD3/CD28/rhIL-2 stimulation (n = 3). (G) Histogram depicting the expression of TNFR1 and TNFR2 on effTreg directly after FACS sorting (n = 9). (H) Flow cytometry of IL-17A expression in FOXP3high effTreg that were stimulated with αCD3/CD28 beads plus rhIL-2 with or without TNFR2 agonist for 5 days (n = 9). All data are shown as mean ± SEM. For statistical analysis, a Friedman test followed by Dunn's multiple comparison test (A), a two-way ANOVA followed by a Bonferroni posttest (B,C), and a Wilcoxon matched-pairs signed-rank test (D,G,H) were used. *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001, ns, not significant.