Infants younger than 12 months with a positive serology in whom a urine or blood PCR test could not be performed were excluded from the study, since it was not possible to ascertain their HCMV infection status. Detection of anti-HCMV antibodies was carried out by the clinical laboratory using standard diagnostic tests. Detection of HCMV genome was performed by using Q-CMV Real Time Complete

Kit (Nanogen Advanced Diagnostics, Torino, Italy), a nucleic acid amplification assay based on TaqMan®-MGB (Minor Groove Binder) technology for detection and quantification of CMV DNA. The amplification reaction targets the gene region that encodes the Major Immediate Early Antigen (MIEA) of HCMV as well as a region of the human beta globin gene, PLX4032 mw which is amplified simultaneously C59 wnt chemical structure with the target sequence to verify successful DNA isolation in order to exclude false-negative results. Anti-NKG2C was from R&D Systems (Minneapolis, MN). Anti-NKG2A (clone Z199, kindly provided by Dr. A. Moretta, University of Genova), anti-LILRB1 (clone HP-F1), anti-CD161 (clone HP-3G10), and the anti-Myc (clone 9E10) negative control, were directly produced in our

laboratory. Indirect immunofluorescence staining with these reagents was carried out with a phycoerythrin (PE)-labeled F(ab′)2 rabbit anti-mouse Ig (Dako, Glostrup, Denmark). Anti-CD3-peridin-chlorophyll-protein (PerCP) and anti-CD56-allophycocyanin were from BD Biosciences (San Diego, CA); anti-CD45-allophycocyanin-Cy7 was from BioLegend (San Diego, CA). The expression of NKG2C, NKG2A, LILRB1, and CD161 by NK and T cells was analyzed by multicolor flow cytometry in fresh peripheral blood samples, obtained by venous

puncture in EDTA tubes. Whole blood out samples were pretreated with human aggregated Ig (30 μg/mL) to block Fc receptors, incubated with individual NKR-specific mAbs, washed and further incubated with a PE-tagged F(ab′)2 rabbit anti-mouse Ig. Washed samples were incubated with anti-CD3-PerCP, anti-CD56-allophycocyanin, and anti-CD45-allophycocyanin-Cy7. Erythrocytes were lysed using BD PharmLyse lysing buffer (BD Biosciences). Samples were analyzed in a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). BD FACSDiva software (BD Biosciences) was used for data analysis and calculation of the MFI values. Results from hemograms, obtained in parallel to the samples used for immunophenotypic analysis, were used to calculate the absolute numbers of NK and T-cell populations.