Figure 2

Temperature of 33 °C enhances expression of FoxP3, CD25 and Helios in Tregs. (A) Representative dot plots depicting FoxP3 expression in Tregs cultured at 37 and 33 °C for 14 days. CD4⁺ Teffs derived from the same donor and expanded at 37 °C are shown as a control. FoxP3⁺ cells are gated and their frequency within CD4⁺ population is shown in the right upper corner of each dot plot. Red line divides FoxP3⁺ population into FoxP3^High and FoxP3^Low cells which proportions are depicted with red and blue font, respectively. (B) Frequency of FoxP3⁺ cells within CD4⁺ population on day 7 and 14 of Treg expansion at 37 and 33 °C (gray and red symbols, respectively; n = 11). (C) Intensity of FoxP3 expression (MFI) in CD4⁺FoxP3⁺ cells after 14-day expansion at 37 and 33 °C (gray and red symbols, respectively; n = 11). (D) Representative dot plots depicting CD25 expression in Tregs cultured at 37 and 33 °C for 14 days. CD4⁺ Teffs derived from the same donor and expanded at 37 °C are shown as a control. CD25^High cells are gated and their frequency is shown in the right upper corner of each dot plot. (E) Frequency of CD25^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). (F) Intensity of CD25 expression (MFI) by CD4⁺FoxP3⁺CD25^High cells after 7 and 14-day expansion at 37 and 33 °C (gray and red symbols, respectively; n = 11). (G) Representative dot plots depicting Helios expression in Tregs cultured at 37 and 33 °C for 14 days. CD4⁺ Teffs derived from the same donor and expanded at 37 °C are shown as a control. Helios⁺ cells are gated and their frequency is shown in the right upper corner of each dot plot. (H) Percentage of Helios⁺ cells within CD4⁺FoxP3⁺ population at day 7 and 14 of Treg expansion at 37 and 33 °C (gray and red symbols, respectively; n = 5). MFI is presented as value to power of 4 or 5 (e.g. 1E5 = 1 × 10⁵). Statistical differences (B-C,E-F,H) were calculated with Mann- Whitney U test and are presented as medians (min.-max.). *p < 0.05.