Figure 3

Tregs expanded at 33 °C are more stable and express higher amounts of Treg associated markers, than the cells cultured at 37 °C despite the same proportions of naive and memory subsets. (A) Frequency of FoxP3^High cells within CD4⁺FoxP3⁺ population on day 7 and 14 of Treg expansion at 37 and 33 °C (gray and red symbols, respectively; n = 11). (B) Intensity of CTLA-4 and CD39 expression (MFI) by CD4⁺FoxP3⁺CTLA-4⁺ and CD4⁺FoxP3⁺CD39⁺ cells, respectively after 7 and 14-day expansion at 37 and 33 °C (gray and red symbols, respectively; n = 7). MFI is presented as value to power of 4 (e.g. 1E4 = 1 × 10⁴). (C) Proportions of naive (Tn), central memory (Tcm) and effector memory (Tem) cells within CD4⁺FoxP3⁺ population at 37 and 33 °C on day 7 and 14 of the culture (n = 11). (D) Frequency of cells with demethylated FOXP3 TSDR on day 7 and 14 of Treg expansion at 37 and 33 °C (gray and red symbols, respectively; n = 5). CD4⁺ Teffs derived from the same donors (black symbols, n = 5) and expanded at 37 °C are shown as a control. The statistical differences (A–D) were calculated with Mann-Whitney U test and are presented as medians (min.-max.). *p < 0.05.