Cd19 percp cy5.5 clone hib19

Peripheral blood mononuclear cells (PBMC) were isolated immediately after blood collection into heparinized collection tubes (within 30 min) by density gradient centrifugation using Biocoll Separating Solution (Merck). They were analyzed immediately by flow cytometry (BDFortessa) using the following antibodies (CD45RA-BUV737, clone HI100; CD4-BV785, clone RPA-T4; CD25-BV650, clone BC96; CD8-BV510, clone RPA-T8; CD14-PacificBlue, clone HCD14; CD19-PerCP-Cy5.5, clone HIB19; CD56-FITC, clone MEM-188; CD197-PE-Cy7, clone G043H7; CD127-PE/Dazzle594, clone A019D5; CD3-PE, clone SK7; γδTCR-APC, clone B1), all from Biolegend. Dead cells were excluded by staining with Hoechst (Sigma-Aldrich). For assessment of cellular markers of acute infection such as plasmablasts, activated CD8 T cells and cTfh cells, frozen PBMC from the same patient cohort were analyzed. Cells were stained with fluorescently labeled antibodies and samples were acquired using the spectral flow cytometer Cytec Aurora (Cytek^® Biosciences). The antibodies are indicated in Figure S8B and were all purchased from Biolegend. Unmixing was performed using the software provided by the Cytec Aurora. FlowJo v10 was used for further analysis of the data.

NSG adult female mice (6–8 weeks old) were purchased from Charles River and were sub-lethally irradiated (3 Gy) 24 h prior to transplantation. CD34+ HSPCs were thawed and cultured. At day 4 of culture (2 days after editing), 0.5 × 10⁶ viable cells/mouse were injected via tail vein into NSG mice with a 27-gauge × 0.5-inch needle. The same cell number per mouse was used for all experimental conditions tested. After 8 and 14 weeks post-transplantation, mice PB from the tail vein was lysed with 1× RBC lysis buffer (ThermoFisher Scientific) and stained with anti-human CD45 APC antibody (clone HI30, BioLegend) to evaluate human CD45+ engraftment by flow cytometry. At 14–16 weeks post-transplantation, level of human engraftment and lineage composition were determined in the BM and PB. Briefly, BM cells were harvested by flushing tibiae and femurs with 1× PBS and passing through a 40-μm strainer. Mononuclear cells were blocked with Fc blocking solution (BioLegend) and stained with the following antibody panels; CD45 BV421, CD19 PerCp Cy5.5 (clone HIB19, BioLegend), CD33 FITC (clone P67.6, BD Bioscience), and CD3 PE (clone OKT3, BioLegend) for flow cytometry analysis. To determine human stem cell composition within BM, cells were stained with the following antibody panels: CD45 BV421, CD34 FITC, CD38 APC-Cy7, CD90 PE-Cy7, and CD45RA PerCP Cy5.5.

Freshly and IL-2/IL-15-activated NK cells (10⁶ cells) collected from HDs were co-cultured with target cells at an effector-to-target (E:T) ratio of 3:1 in U-bottomed 96-well plates for 4 h in a 5 % CO₂ incubator with PE-conjugated anti-CD107a (H4A3, BioLegend) and Brefeldin A (Golgistop, BD). Stimulus with 25 ng/mL PMA plus 250 ng/mL ionomycin was used as a positive control and NK cells alone were used as a negative control. Cultured cells were then stained with fluorochrome-conjugated monoclonal antibodies against human blood surface antigens: CD3 PerCP/Cy5.5 (clone HIT3a), CD14 PerCP/Cy5.5 (clone M5E2), CD19 PerCP/Cy5.5 (clone HIB19), CD16 FITC (clone 3G8), and CD56 APC (clone HCD56), all purchased from BioLegend. The percentage of CD3⁻CD56⁺ NK cells positive for CD107a was calculated. All analyses were performed in duplicate using BD FACSCanto II and FlowJo analysis software.
Cytokines produced by 48 h IL-2/IL-15-activated NK cells co-cultured with tumor cells at an E:T ratio of 10:1 were measured using ELISA kits according to the manufacturer’s instructions (granzyme B and IFN-γ: Abcam, Cambridge, UK; and TNF-α: R&D Systems, MN, USA).