Anti nkg2d pe

The following mouse monoclonal antibodies were used: CD56-APC (clone N901, Beckman Coulter, Miami, FL, USA), CD56-Brilliant Violet 421 (clone HCD56, Sony Biotechnology, San Jose, CA, USA), CD56-PE (clone N901 (HLDA6), Beckman Coulter, Miami, FL, USA), CD57-PE (clone TB01, eBioscience, San Diego, CA, USA), CD57-FITC (clone TB03, Miltenyi Biotec, Bergisch Gladbach, Germany), CD57-APC (clone TB03, Miltenyi Biotec, Bergisch Gladbach, Germany), CD16-PE (Sorbent, RF), CD2-PE-Cy7 (clone TS1/8, Sony Biotechnology, San Jose, CA, USA), anti-NKG2A-PE (clone 131411, R&D Systems, Minneapolis, MN, USA), anti-KIR2DL2/DL3-PE (clone DX27, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-HLA-DR-FITC (clone B8.12.2, Beckman Coulter, Miami, FL, CA, USA), NKG2A-PE (clone 131411), NKG2C-AlexaFluor488 (clone 108724), NKG2C-PE (clone 134591; R&D Systems, Minneapolis, MN, USA), NKp46-FITC (clone 9E2; Sony Biotechnology, San Jose, CA, USA), anti-NKG2D-PE (clone REA1175, Miltenyi Biotec, Bergisch Gladbach, Germany). Staining was performed according to the manufacturer’s instructions.

Peripheral blood mononuclear cells (PBMCs) obtained from CLL patients were cultured in the presence of anti-ILT2 blocking antibody (HP-F1; 10 μg/mL) or irrelevant mouse IgG1, and lenalidomide (1 μM) and IL-2 (50 U/ml) for 7 days. The expression of CD69, CD25, CD137, NKG2D, and DNAM-I on NK cells (defined as CD3⁻CD56⁺) was evaluated by flow cytometry analysis using specific antibodies; anti-CD69-FITC (Immunostep), anti-CD25-PE (Biolegend), anti-CD137-PE (Biolegend), anti-NKG2D-PE (Miltenyi), and anti-DNAM-I-PE (Biolegend).

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).