Nkp44 pe

HSPC-NK cell phenotype was determined by staining for CD56-PE-Cy7 (Biolegend, #318318), NKG2A-APC (Beckman Coulter, #A60797), CD16-FITC (Biolegend, #302006), pan-KIRs-PE (Biolegend, #339506, 312606 and 312708), DNAM-1-FITC (BD, #559788), NKp46-PE (Biolegend, #331908) and NKG2D-APC (Biolegend, #320808) (CellGro vs NK MACS) or CD56-BV510 (Biolegend, #318340), CD45-BV421 (Biolegend, #368522), NKG2A-PE-Cy7 (Beckman Coulter, #B10246), DNAM-1-FITC (Biolegend, #337104), NKp46-PE (Biolegend, #311908), NKp44-PE (Biolegend, #325108), NKp30-APC (Biolegend, #325210) and NKG2D-APC (Biolegend, #320808) (GMP validations runs). Briefly, 200.000 cells were washed using PBS/0.5% BSA and incubated with antibodies in PBS/0.5% BSA at 4 °C for 30 min. Cells were then washed twice with PBS/0.5% BSA and resuspended in PBS/0.5% BSA containing Sytox Blue (1:5000 diluted, invitrogen, #S34857) for CellGro vs NK MACS experiments or 7-AAD (1:1000 diluted, Sigma, #A9400) for GMP validation runs. Cells were acquired on the Gallios (CellGro vs NK MACS) or Navios (GMP validation runs) flowcytometers and analyzed using Kaluza V2.1.3.

All samples were stained with anti-CD56-APC, anti-CD3-FITC (Biolegend) and 7AAD (BD Biosciences). Transgene expression was detected by flow cytometry on 7AAD⁻ CD56(-APC)⁺ CD3(-PE)⁻ cells (Biolegend). For NK-cell receptor detection, samples were stained with DAPI, CD56-BV711, CD16-BV786, NKp30-AF647, NKp44-PE, NKp46-BV421 (Biolegend), NKG2D-APC (BD Biosciences), and NKG2A-PE (Miltenyi Biotec). CD3-BV650 and CD19-APC-Cy7 (Biolegend) markers were used as a gating exclusion strategy for the NK cell staining. Receptor expression was assessed on DAPI⁻ CD56(-BV711)⁺ CD3(-BV650)⁻ cells. To detect CAR-expression, cells were incubated with 2 μl Siglec2(CD22)-Fc chimera (50 mg/ml, R&D) for 30 min at 4°C, washed and stained with anti-Fc-PE (Jackson Immune).