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. 2019 Apr 18;4(8):e126194. doi: 10.1172/jci.insight.126194

Figure 2. Foxp3 expression is stable after transduction, bead expansion, and restimulation.

Figure 2

(A) Intracellular staining of Foxp3 and CD25 as a percentage of total CD3+CD4+mCherry+ after sorting (day 0), bead expansion, and rest (day 14) and on day 23, 9 days after the addition of irradiated anti-CD3 K562 (TCR stim) or CD19-K562 (CAR stim) (n = 6 human donors). Methylation status using direct bisulfite modification and pyrosequencing of (B) the TSDR (n = 2 human female donors) and (C) the CTLA4 promotor (n = 3 human donors) on day 0 after sort, day 14, and day 23 with CAR stimulation. Surface expression of (D) CTLA4, (E) LAP, and (F) CD39, 9 days after TCR or CAR stimulation with irradiated K562 cells (n ≥ 3 human donors). All data are represented as box-and-whisker plots. *Adj-P < 0.05 by paired t test for CAR stimulation versus TCR stimulation (D) and between Tr 28ζ and Tr BBζ (E) with Holm-Bonferroni method adjustment for 3 and 2 tests, respectively. Blue bars for D0 represent UT Tregs and UT Tconvs immediately after sort. Tc, Tconvs; Tr, Treg.