4.2. Immunolabeling of Tregs in peripheral blood and flow cytometry

The samples used for the cytometric analysis of Treg populations in the whole blood of patients were prepared using the FoxP3 Staining Kit (Becton Dickinson, Franklin Lakes, NY, USA), according to the manufacturer's instructions and analyzed using a BD FACS Canto II flow cytometer and the BD FACS Diva Software (Becton Dickinson, Franklin Lakes, NY, USA). For each sample, 3 × 10⁴ lymphocytes were collected and gated on an SSC × CD45 dot plot. Subsequently, the populations of CD4 FITC, CD25 APC, and double-positive CD4+/CD25+ cells were distinguished among the lymphocytes, and the gate of FoxP3+ cells was established on the CD4+/CD25^high+ subpopulation, which are the only cells which exhibit regulatory function in humans [42]. Recently published data identified new population of Treg cells, expressing C-C chemokine receptor 4 (CCR4) and considered CCR+ Tregs as effector Tregs cells [58]. Our preliminary, unpublished results showed about 98% convergence of Treg frequency in CD4+/CD25^high+ subpopulation and CCR4+ Tregs.

The frequency of Tregs was defined as the percentage of cells with the CD25^high+/FoxP3+ phenotype in the subpopulation of CD4+ T lymphocytes. In addition to the specific-staining cell analysis, a negative control was used for each sample to determine the frequency of autofluorescence, and an isotypic control, performed for each blood sample, was used to exclude nonspecific staining of the antibodies.