Surface Phenotype Changes and Increased Response to Oxidative Stress in CD4+CD25high T Cells

Abstract

Conversion of CD4⁺CD25⁺FOXP3⁺ T regulatory cells (T_regs) from the immature (CD45RA⁺) to mature (CD45RO⁺) phenotype has been shown during development and allergic reactions. The relative frequencies of these T_reg phenotypes and their responses to oxidative stress during development and allergic inflammation were analysed in samples from paediatric and adult subjects. The FOXP3^lowCD45RA⁺ population was dominant in early childhood, while the percentage of FOXP3^highCD45RO⁺ cells began increasing in the first year of life. These phenotypic changes were observed in subjects with and without asthma. Further, there was a significant increase in phosphorylated ERK1/2 (pERK1/2) protein in hydrogen peroxide (H₂O₂)-treated CD4⁺CD25^high cells in adults with asthma compared with those without asthma. Increased pERK1/2 levels corresponded with increased Ca²⁺ response to T cell receptor stimulation. mRNA expression of peroxiredoxins declined in T_regs from adults with asthma. Finally, CD4⁺CD25^high cells from paediatric subjects were more sensitive to oxidative stress than those from adults in vitro. The differential T_reg sensitivity to oxidative stress observed in children and adults was likely dependent on phenotypic CD45 isoform switching. Increased sensitivity of T_reg cells from adults with asthma to H₂O₂ resulted from a reduction of peroxiredoxin-2, -3, -4 and increased pERK1/2 via impaired Ca²⁺ response in these cells.

Keywords: CD45 isoform; allergy; extracellular signal-regulated kinases 1/2; oxidative stress; regulatory T cell.

Figure 1

Flow cytometric surface marker analysis of CD4⁺ T cells isolated from paediatric PBMCs are shown from representative cases. In total, 1 × 10⁶ CD4⁺ cells were stained with Alexa 488-FOXP3, PE-CD25, and PC5-CD127 (a), or with Alexa 488-FOXP3, PC5-CD25, and PE-CD45RO (b). Representative flow histograms are shown. (c) The cells were stained with Alexa 488-FOXP3, PC5-CD25, and PE-CD45RO or CD45RA. Representative flow histograms are shown. (d) Results from individual paediatric subjects are plotted by bar graph. (e) Data from (d) were combined into 4 groups by age, and the difference was statistically analysed. All histograms were gated with CD25 because of the analysis of purified CD4⁺ T cells.

Figure 2

Flow cytometric measurement of p38 MAPK phosphorylation in response to H₂O₂ exposure in CD4⁺CD25^high T cells from paediatric and adult subjects. 3 × 10⁵ PBMCs were incubated for 10 min and stained with PC5-CD4, PE-CD25, and Alexa 488-pp38 MAPK. Representative flow histograms from paediatric subjects without allergy and adult subjects without asthma are shown (a). Phosphorylation of p38 MAPK in cells exposed to 50 μM H₂O₂ or vehicle were compared between paediatric subjects with and without allergy and adult subjects with or without asthma. Representative flow histograms (b) and average percentage of pp38 MAPK-positive cells are plotted by bar graphs (c). Gating was performed on cells highly expressing CD25 antigen.

Figure 3

Flow cytometric measurement of ERK1/2 phosphorylation by H₂O₂ exposure in CD4⁺CD25^high T cells from paediatric subjects without allergy and adult subjects without asthma that were exposed to 10 mM H₂O₂ were stained with PC5-CD4, PE-CD25, and Alexa 488-pERK1/2. The representative flow histograms are shown in (a). The typical patterns of pERK1/2 expression among all child subject groups are shown in (b). The average percentage of pERK1/2- positive cells in all adult subject groups were plotted by bar graphs, and asthma subjects were further divided into two groups by Ca²⁺ response (c). Typical patterns (poor response and robust response) of Ca²⁺ response are depicted in (d). Gating was performed on cells highly expressing CD25 antigen.

Figure 4

Assessment of the sensitivity of FOXP3⁺ cells to oxidative stress. The measurement of antioxidant levels and caspase activity in response to H₂O₂ were compared with those in control cells. Glutathione content in FOXP3 2-day and FOXP3 5-day cells were compared with control cells (a). Caspase activity in response in the abovementioned cells exposed to 100 μM H₂O₂ for 4 h were measured (b). mRNA expression of peroxiredoxins in CD4⁺CD25⁺CD127^−/low T cells were quantified by real-time PCR (c).