Cd107a pe cf594

T cells were magnetically purified from PBMC using a PAN T-cell isolation kit (Miltenyi Biotec) from healthy donors. Healthy donor T cells (1 × 10⁵) were then cultured with bead-purified (Miltenyi Biotec) CD19+ B cells from CLL patients (1 × 10⁶) at an optimal 1:10 ratio per well in the presence or absence of CXCL12 (250 ng/ml), lenalidomide (10 µM), CXCR4, or mouse anti-human IL-10 blocking antibodies (2 µg/ml, R&D). Each experiment was performed in duplicate. After 48 h of culture, T cells were magnetically purified again as described above (purity >95%) and activated with anti-CD3/CD28 magnetic Dynabeads (Invitrogen) for 6 h. A negative control (no stimulation) was included in every experiment. Brefeldin A (10 µg/mL) and CD107a PE-CF594 (BD Biosciences) were added to the culture. Cells were stained with a Live/Dead-Aqua (Invitrogen), CD3-BV650, CD8-FITC, CD4-APC-Cy7, fixed/permeabilized (BD Biosciences) followed by intracellular staining with IFN-γ-V450, TNF-α-Alexa 700, and IL-2-PE (BD Biosciences).

For intracellular cytokine staining, PBMCs were stimulated with the corresponding pooled peptide at a final concentration of 8 μg/mL for 1 h at 37 °C. Then, the cells were incubated for an additional 4 h with 1 μg/mL GolgiPlug (Brefeldin A, BD) and 0.7 μg/mL GolgiStop (Monensin, BD) and surface stained with CD107a-PE-CF594 (BD Bioscience). Unstimulated cells were used as negative controls. A combination of 50 ng/mL phorbol-12-myristate-13-acetate and 1 μg/mL ionomycin (both Sigma-Aldrich, Seelze, Germany) was used as a positive control. Dead cells were stained with LIVE/DEADR Fixable Aqua dye (Invitrogen). Surface markers, including CD3-AF700 (BioLegend), CD4-FITC (BD Bioscience), and CD8-APC-H7 (BD Bioscience), were stained. Cells were then washed and permeabilized using Cytofix/Cytoperm (BD Bioscience). Subsequently, the cells were washed with Perm/Wash buffer (BD Bioscience), stained intracellularly with Tumour Necrosis Factor (TNF)-α-APC (BioLegend), IFN-γ-BV786 (BD Bioscience), and IL-2-PE (BioLegend), fixed with 1 × CellFix solution (BD Biosciences) and acquired immediately on a BD LSR Fortessa. The data were analysed using FlowJo (Tree Star Inc., Ashland OR).

For immune-monitoring studies, patient PBMC samples were stained with cocktail fluorescent-conjugated antibody mix diluted in PBS containing 2% FBS + 0.01% azide for 30 min at 4 °C in the dark. After a 2-step washing with PBS, cells were fixed with 2% PFA for 20 min at 4 °C. Samples were analyzed on a BD Fortessa Flow Cytometer (UV-Violet-Blue-Yellow/Green-Red 5-Laser configuration). For the first cohort (aviremic patients) the following antibodies were used: CCR5-FITC, CD158a-PE, CD57-PE-CF594, CD14-Alexa Fluor 700, CD19-Alexa Fluor 700, CXCR4-BV421, CD62L-BV605, NKG2D-BV650, CD16-BV711, CD3-BV786, CD56-BUV395 and CD4-BUV737 (BD Biosciences), CD158b-PE, CD158e/k-PE, NKp46-PE-Vio770, NKG2C-APC, CD45RA-APC-Vio770, and CD45RO-VioGreen (Miltenyi). For the second cohort (viremic patients) the similar panel of antibodies were re-used, except for additional antibodies for CXCR4-BV421 and CD107a-PE-CF594 (BD Biosciences). Cell death and viability were determined by DAPI (BD Biosciences). Data were analyzed using FlowJo software (v10.8) (BD Biosciences) with appropriate plugins for high-dimensional analysis and visualization.