Cd4 clone 13b8.2

An analysis of the cell purity of the isolated T_mem cells was performed via flow cytometry, using the following fluorescent labeled antibodies directed against CD45RA (clone HI10), CD45RO (clone UCHL1), CD3 (clone SK7) (all purchased from BD Biosciences, Heidelberg, Germany), and CD4 (clone 13B8.2) (purchased from Beckmann Coulter, Krefeld, Germany), using a FACS Calibur device (BD Biosciences) as described earlier [65 (link)]. Data processing and analysis were performed using associated CellQuest Pro software (version 4.0.2).

The phenotype of pDCs was determined with the following primary mAbs and the appropriate isotype controls (from BD Biosciences, San Jose, CA): CCR5 (clone 2D7), CXCR4 (clone 12G5), CD40-APC (clone 5C3); HLA-DR-APC (clone L243); CCR7-FITC (clone 3D12), CD83-APC (clone HB15e), CD86-APC (clone 2331) and TRAIL-PE (clone RIK-2). CD4 (clone 13B8.2) was purchased from Beckman Coulter. Cells were stained for 30 minutes at 4°C, washed twice in PBS/BSA/NaN3 (0.5% BSA, 0.01% NaN3) and fixed with 1% PFA. For intracellular staining, cells were fixed with 4% PFA, permeabilized using 0.5% BSA, 0.01% NaN3, 0.5% Saponin buffer, stained for 20 minutes at room temperature with FITC-labeled anti-HMGB1 pAbs (ABCAM). At least 5,000 events were acquired using a FACScalibur flow cytometer (BD Biosciences), and stained cells were analysed using FlowJo software (Tree Star, Inc., Ashland, OR). DC survival was assessed using the 7-AAD assay, as described previously [99 (link)]. When phenotypic characterization of pDCs was performed in NK-DC cocultures, NK cells were excluded through the gating of CD56^neg cells. Surviving pDCs were identified as CD56^neg 7-AAD^neg cells. When phenotypic characterization of NK cells was performed in NK-DC cocultures, NK cells were gated through their expression of CD56 marker.

T-cell clonal expansion was detected using TCR Vβ repertoire analysis by means of an IOTest Beta Mark TCR V Kit (Beckman Coulter, Brea, CA, USA). Relative frequencies of 24 Vβ T cell receptor families were analyzed in CD4+ and CD8+ T cells by flow cytometry in the peripheral blood of 17 recipients of ASCT from the LTR cohort. TCR Vβ panels included antibodies against CD3 (clone UCHT1), CD4 (clone 13B8.2), and CD8 (clone T8) (Beckman Coulter). Cells were acquired on a BD FACSCanto II cytometer and data were analyzed with FlowJo Software v.10 (BD Biosciences, San Jose, CA, USA).