Figure 1.

Representative gating strategy – determination of the proportion of peripheral blood Treg cells expressing Helios. Peripheral blood mononuclear cells were obtained and processed as in the Patients and Methods section. During flow cytometry data analysis, lymphocytes were first identified based on their forward (FSC-H) and side scatter (SSC-H) (A). Contents of the lymphocyte gate were then analyzed for the presence of CD3 and CD4 markers and CD3⁺CD4⁺ T cells were gated (B). Then the contents of the CD3⁺CD4⁺ T cell gate were analyzed for the expression of CD25 and FoxP3 antigens and the cells displaying the CD3⁺CD4⁺CD25^highFoxP3⁺ phenotype (Tregs) were delineated (C). Finally, the cells displaying the Treg phenotype were analyzed for the proportion of Helios⁺ cells ((D) sample stained with isotype control, (E) sample stained with anti-Helios antibody). Rounded rectangles mirror the actual gates used for analysis.