Anti cd19 apc

To determine immune cell populations, the following antibodies were employed: anti-CD19-APC, anti-CD3-APC, anti-CD4-PerCP, and anti-CD8-APC (all from Immunostep); anti-CD3-FITC and anti-CD56-APC (Cytognos); and anti-CD3-PE/Cy7 and anti-CD5-APC/Cy7 (Biolegend). The subsets of immune cells were identified as follows: T cells were defined as CD3⁺CD56⁻, NK cells were identified as CD3⁻CD56⁺ and healthy B cells as CD19⁺. Leukemia cells were defined as CD19⁺, as the percentage of healthy B cells (CD19⁺CD5⁻) detected in PBMCs from patients with CLL was <2% (data not shown). Additionally, antibodies from Biolegend were used to detect surface expression of CD79A (clone: HM47), CD79B (clone: CB3-1), and IgM (clone: MHM-88). Cells were analyzed in a BD FACS Canto II flow cytometer with FACS Diva software (Beckton Dickinson).

Diagnosis of CLL for each patients was confirmed by flow cytometry, which revealed a typical CD19+, CD20+, CD5+, CD23+ and Ig light chain (κ or λ) restricted phenotype. Absolute counts of the main PBMCs subsets, including CD4 and CD8 T cells, B cells and NK cells were carried out by flow cytometry upon diagnosis. The distribution of these subsets of lymphocytic cells and the membrane expression of NKG2D were also analyzed at the time of patient enrollment. The protocol for B-cell chronic lymphoproliferative diseases used in all cases included the following antibody conjugates: anti-CD3-FITC, anti-CD4-PerCP, anti-CD8-CF-Blue, anti-CD56-APC, anti-CD19-APC (all from Immunostep), anti-CD3-PECy7 (eBioscience) and anti-NKG2D-PE (Miltenyi Biotec). The populations of cells were defined as follows: CD4 T cells were defined as CD3+CD4+, CD8 T cells were defined as CD3+CD8+, B cells were defined as CD19+ and NK cells were defined as CD3-CD56+. Cells were analyzed on a BD Biosciences FACSCanto II cytometer and data were analyzed by FACSDiva software (Beckton Dickinson).

Leukemic cells, B lymphocytes and NK cells from patients and HD were identified by flow cytometry. For this purpose, the following antibodies were employed: anti-CD19-APC and anti-CD3-PE (Immunostep, Salamanca, Spain), anti-CD3-FITC and anti-CD56-APC (both from Cytognos, Salamanca, Spain). Leukemic cells were identified as CD19+. NK cells were defined as CD56+CD3−. BTLA and HVEM expression was evaluated using anti-BTLA-PE (clone MIH26, Biolegend, San Diego, CA, USA) and anti-HVEM-PE (clone 122, Biolegend). Cells were analyzed in a Cytoflex S flow cytometer and CytExpert 2.3 software (Beckman Coulter, Brea, CA, USA).