Cd20 pe

The CLL cells from all the blood samples were stained with optimal concentrations of antibody combinations and directed against the following surface antigens: CD183(CXCR3)-FITC, CD20-PE, CD5-PerCP-Cy5.5, CD38-Pe-Cy7, CD49d-APC, CD19-APC-Cy7, CD184 (CXCR4)-BV421 and HLA-DR-BV510 (all procured from BioLegend), as previously reported [5 (link),15 (link)]. Isotype-matched antibodies (BioLegend) were used as negative controls.
The determination of s-CLL and l-CLL cells was conducted using FSC data and a back-gating strategy. The analysis was performed using a BD FACSCanto II (Becton Dickinson) instrument, and data acquisition was performed using BD FACSDiva software (v.8.0.2; Becton Dickinson). Flow cytometry data were analysed using FlowJo v.X0.7 software (Tree Star, Inc., San Carlos, CA, USA). In all the experiments, a minimum of 10,000 events was counted. The results were expressed as a percentage and mean fluorescence intensity (MFI).

To determine the size of peripheral blood leukocyte and BM nucleated cell populations, cells were labeled with the following antibodies against human cell surface markers: anti-human cluster of differentiation (CD)235a-phycoerythrin (PE), CD14-PE-cyanin 5, CD16-PE, CD20-PE, CD203c-PE, CD3-PE-cyanin 5, CD4-PE-cyanin 5, CD4-PE-cyanin 7, CD8-PE-cyanin 5, and CD56-PE (all from BioLegend, San Diego, CA, USA); CD71-PE-cyanin 5, CD3-PE, and CD11c-PE (all from BD Biosciences); and CD141-allophycocyanin (Miltenyi Biotec, Bergisch Gladbach, Germany). The total lymphocyte population was determined from forward and side scatter, and CD4+ and CD8+ T cells, natural killer (NK) cells, and B cells were separated into CD3+/CD4+, CD3+/CD8+, CD56+/CD3−, and CD20+ populations, respectively. The monocyte population was determined from forward and side scatter and by CD14 expression. The granulocyte population was gated by forward and side scatter, and neutrophils and eosinophils were separated according to CD16 expression (CD16+ and CD16−, respectively). Basophils were isolated as the CD203c+ population. Plasmacytoid DCs, CD1c+ myeloid DCs, and CD141^high myeloid DCs were isolated from peripheral blood mononuclear cells on a FACSAriaII cell sorter as previously described [39 (link)]. Erythroid cells present in the BM cell population were gated according to CD235 and CD71 (TFR1) expression.

Thawed PBMCs samples were taken from five patients (two in cured group and three in uncured group). A total of 15 blood samples were collected at 3 time points: day 0 (the day before), week 24, and week 48 of treatment. Samples were washed 3 times by 1x PBS with centrifugation at 400 g for 6 min and counted for cell numbers. 500,000 cells per tube were then stained for 30 min on ice with antibodies. For flow cytometric analyses, the following antibodies were used: CD3 (CD3-FITC; BioLegend, cat#300305), CD4 (CD4-APC-Cy7; BioLegend, cat#317418), CD8 (CD8-PerCP-Cy5.5; BioLegend, cat#344710), CD19 (CD19-APC; BioLegend, cat#302212), CD20 (CD20-PE; BioLegend, cat#302306), CD25 (CD25-APC; BioLegend, cat#302610), CD127 (CD127-PE; BioLegend, cat#351304), and PD-1 (PD-1-BV421; BioLegend, cat#329920). Cells stained by antibodies were resuspended in 1x PBS, and analyzed by flow cytometry using BD LSR Fortessa cell analyzer. Flow cytometry data were analyzed using FlowJo (version 10.4.0). Accordingly, B cells and Treg cells were selected as CD19⁺ CD20⁺ and CD3⁺ CD4 ⁺ CD25⁺ CD127⁻ subsets, independent CD3⁺ clustering of either CD4⁺ or CD8⁺ T cells with PD-1⁺ used for identifying PD-1 expression respectively from lymphocytes. Flow cytometry gating strategies for CD3+CD4+/CD3+CD8+ T cell populations see Supplementary Figure 2.