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. 2021 Jun 3;99(8):833–847. doi: 10.1111/imcb.12475

Figure 4.

Figure 4

Hp‐TGM‐induced FOXP3+ cells acquire Treg‐defining functions. (a) In vitro suppression assays were performed by culturing cell proliferation dye (CPD) eFluor450‐labeled PBMCs (responder cells) with CPDeFluor670‐labelled cells from control, TGF‐β or Hp‐TGM cultures 5–7 days after second restimulation at the cell ratios indicated. After 4 days of anti‐CD3/CD28 stimulation, proliferation of responder cells was assessed by calculating division index (DI) and calculating the percentage suppression based on responder only wells. (b) The percentage of suppression of responder cells that were gated as live CD4+ or CD8+ T cells (n = 3). Cells from control, TGF‐β or Hp‐TGM cultures 5–7 days after second restimulation were cultured with PMA and Ionomycin with or without brefeldin A for 5 h and (c) percentages of IFN‐γ+ (n = 7) and IL‐2+ (n = 3) cells were measured by flow cytometry (from cultures with brefeldin A added) and (d) levels of secreted IFN‐γ, IL‐2, TNF, IL‐4 and IL‐13 were measured in supernatants (from cultures without brefeldin A) by cytometric bead array (n = 6; IL‐13 is n = 5). A two‐way repeated measures ANOVA was used in (b and a) Friedman one‐way ANOVA with Dunn’s post‐test were used in c and d, error bars represent median ± interquartile range, *P ≤ 0.05, **P ≤ 0.01 and ns, not significant.