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. Author manuscript; available in PMC: 2020 Dec 3.
Published in final edited form as: Cytotherapy. 2019 Dec 3;21(12):1216–1233. doi: 10.1016/j.jcyt.2019.10.011

Figure 3: Function of thymic Tregs expanded with cell-free activation reagents.

Figure 3:

Isolated thymic Tregs were expanded with the indicated type of activation reagent and analyzed by flow cytometry after 12 or 15 days. Expression of (A) intracellular cytokines in Treg (CD4+FOXP3+) and Tconv after 4 hours of activation with PMA, ionomycin and brefeldin A, (B) CTLA-4 or (C) LAP and GARP after 24 hours of activation with anti-CD3/CD28 beads at a 1:16 bead to cell ratio. (D) After 15 days of expansion, thymic Tregs were cocultured with cell proliferation dye (CPD)-labeled PBMC at the indicated ratios and stimulated with a 1:16 ratio of anti-CD3/CD28 beads for 4 days. Suppression of CD8+ T cells within PBMC was determined by division index. For A-C, within each group, each symbol represents cells from a different subject and bars indicate median ± interquartile range. For D, median ± interquartile range is shown. n=4–6 for L cell aAPCs + anti-CD3 mAbs, n=4–6 for CD3/CD28 T Cell Activator, n=4–14 for CD3/CD28/CD2 T Cell Activator, n=2–6 for Dynabeads Treg Xpander, and n=3–4 for Tconv all tested in 4–9 individual experiments. *P < 0.05, **P < 0.01 as determined by a (A) Kruskal-Wallis test with Dunn’s multiple comparisons test to compare expression for each cytokine among activation reagents or (B) two-way ANOVA with Tukey’s multiple comparisons test to compare CTLA-4 expression between conditions on each day.