Foxp3 induction dependent on cell cycle progression in vitro.

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Foxp3 induction dependent on cell cycle progression in vitro. Foxp3 induction dependent on cell cycle progression in vitro. (A) Naive CD4 T cells were isolated from Foxp3gfp mice, labeled with cell proliferation dye, and stimulated in vitro in Treg-inducing conditions prior to FACS analysis. Representative time course of Foxp3 expression versus cell division from day 0 to day 4 of culture. (B and C) Cells stimulated for 1.5 d were left untreated; treated initially with either Mimosine (2.5 mM) or Nocodazole (5 or 10 μg/ml); or treated for the final 16 h with Cytochalasin B (10 μM). (B) Representative FACS plots of cell proliferation dye versus indicated protein, gated on live CD4+ lymphocytes. Foxp3 signal is the sum of GFP-Foxp3 emission plus anti-Foxp3 Ab staining. (C) Division, Foxp3 expression, cell size (forward light scatter [FSC-H]), and viability among CD44hi-gated, CD4+ lymphocytes. (D) Frequency of live CD4+ lymphocytes expressing Foxp3 at day 1.5 of in vitro Treg induction (n = 4). ***p = 0.0002 compared with untreated. (E) Frequency of live CD44hiCD4+ lymphocytes expressing Foxp3 at day 1.5 of in vitro Treg induction (n = 4). ***p < 0.0001 compared with untreated. Bonnie Yen et al. ImmunoHorizons 2018;2:119-128 Copyright © 2018 The Authors