*2.2. Flow Cytometry and Immunological Assessment*

None of the patients had acute infections at the time of sample collection for the immunological evaluation. Lymphocyte counts, serum immunoglobulin concentration, and serum immunoglobulin subclasses were evaluated through standard methods and compared with age-related normal values. An extended immunological phenotype was performed in all patients and the data were compared with age-matched normal values [17–20]. Eight-color flow cytometric analysis was performed on fresh peripheral whole blood anticoagulated with ethylenediaminetetracetic acid (EDTA), according to standard protocols, to determine the following cell subpopulations: T lymphocytes (CD3+), helper T lymphocytes (CD3+CD4+), cytotoxic T lymphocytes (CD3+CD8+), B lymphocytes (CD19+), and natural killer (NK) cells (CD16+/56+). Helper and cytotoxic T lymphocytes were also analyzed for the expressions of CD45RA, CD62L, and CD31 to identify naïve (CD45RA+CD62L+), central memory (CM: CD45RA−CD62L+), effector memory (EM: CD45RA−CD62L−), terminal effector memory re-expressing CD45RA (TEMRA: CD45RA+CD62L−), and recent thymic emigrants (RTEs: CD45RA+CD62L+CD31+) [21]. Circulating Treg cells were identified as a CD4+CD25+CD127<sup>−</sup> cell population, as previously described [22]. The expression

of CD45RA was evaluated to estimate the amount of naïve Treg cells. The expression of CD185 (CXCR5) was analyzed on memory T helper cells (CD3+CD4+CD45RO+) to identify follicular T cells. We defined naïve B cells as CD19+CD27−IgD+ and switched memory B cells as CD19+CD27+IgD−IgM−. Circulating dendritic cells (DCs) were enumerated and phenotypically characterized directly into the two major subsets, namely, myeloid (mDCs) and plasmacytoid (pDCs), as previously described [22]. Due to the lack of a specific marker to detect DCs, we used a mixture of monoclonal antibodies specifically established to identify DCs, purchased from Immunotech (Beckman Coulter Inc., Brea, CA, USA). Cells were stained with the following antibodies: CD14, CD16, CD85k, CD33, or CD123 for the mDC and pDC subsets, respectively. Dendritic cells were identified as CD14low/<sup>−</sup>CD16low/−CD85k<sup>+</sup> and CD33+ or CD123+. The absolute numbers of DCs were estimated by multiplying the percentage of DCs in the mononuclear cell (MNC) gate by the absolute peripheral blood MNC count determined using a standard hemocytometer (Abbott Laboratories, Abbott Park, IL, USA). DC data were compared with our laboratory age-related normal values [22]. Data acquisition and analysis were performed on a dual laser BD FACSCanto (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) using the FACSDiva software (San Jose, CA, USA).
