Regulatory T-cell Immunophenotyping and Cytokine Analysis

Fresh peripheral blood mononuclear cells (PBMNCs) from patients 2 and 5 (Supplementary Table 3) and six healthy controls were used. For regulatory T-cell immunophenotyping, cells were stained using monoclonal antibodies (mAb) against the antigens CD3, CD4 and CD25 (BD Biosciences) and FOXP3 (clone259D, eBioscience) and data collected with 4- or 6-color flow cytometry. Regulatory T-cells were defined as CD3⁺CD4⁺CD25^highFOXP3⁺. Evaluation of T-cell cytokine production is described in detail elsewhere^{23 (link)}. Briefly, fresh mononuclear cells were stimulated for 6 hours with anti-CD3, anti-CD28 and anti-CD49d (BD Biosciences). The cells were analyzed using a 6-color flow cytometry panel with mAbs against the antigens CD3, CD4, CD8, IFN-γ and TNF-α (BD Biosciences). The data were analyzed with FACSAria II flow cytometer and FACSDiva software (BD Biosciences).