FIGURE 1.

Gating strategies for regulatory T cells (Treg) in freshly collected cord blood samples. Umbilical cord blood (up to 30ml) was collected by syringe and immediately added to 10ml of RPMI 1640 (Gibco, Life Technologies) containing preservative‐free sodium heparin (Pfizer, final concentration of 10 IU/ml). Venous peripheral blood was collected at 6 and 12 months of age and added to a 15‐ml tube containing 100Ul preservative‐free sodium heparin (Pfizer, final concentration 10 IU/ml). Mononuclear cells (MNC) were isolated by density gradient centrifugation (Lymphoprep, Axis‐Shield). The MNC were stained with fluorochrome‐labelled monoclonal antibodies to CD4, CD45RA, and FoxP3. Events were gated to the lymphocytes (FSC and SSC plot) and then to the CD4⁺ T cells (FL‐2 and SSC plot). FoxP3⁺ subsets were then selected on the basis of CD45RA and FoxP3 expression (FL‐1 and FL‐3 plot). CD45RA⁺ FoxP3^low populations were classified as naïve Treg (nTreg); the CD45RA⁻ FoxP3^high as activated Treg (aTreg); and CD45RA⁻FoxP3^low as non‐suppressive FoxP3⁺ T‐cell; respectively