Rosettesep human cd3 depletion cocktail

The specific approach for human NKC expansion was carried out as previously outlined [29 (link)]. In brief, frozen CBMCs were sourced from RIKEN BRC derived from three volunteers (0 years old, 1 male, and 2 females). The CD3 fraction of the CBMCs was depleted using a RosetteSep™ Human CD3 Depletion Cocktail (STEMCELL Technologies, Vancouver, Canada). A total of 2 × 10⁶ or 1 × 10⁶ of CD3-depleted cells were placed in 6- or 12-well anti-NKp46 and antiCD16 antibody immobilization plates containing AIM-V medium (Thermo Fisher Scientific) supplemented with 10% autologous plasma, 50 ng/mL recombinant human IL-18 (rhIL-18, Medical & Biological Laboratories, Nagoya, Japan), and 3000 IU/mL rhIL-2 (COREFRONT, Tokyo, Japan) at 37 °C in a humidified incubator containing 5% CO₂. AIM-V medium supplemented with 3000 IU/mL rhIL-2 was replenished as needed for 28 days. The expanded cells were frozen in CELLBANKER 2 (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan). To assess the inhibitory effects on tumor cell growth, frozen cells were revived in AIM-V medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; MP Biomedicals, Tokyo, Japan) and 3000 IU/mL rIL-2, followed by a two-day incubation.

To isolate monocytes from RRMS patients, peripheral mononuclear cells (PBMC) from 40 mL whole blood samples were obtained by ficoll-hypaque gradient (Rafer, Zaragoza, Spain) and positive selection of CD14+ cells was performed using the EasySep uman CD14 Positive Selection kit (StemCell Technologies, Vancouver, BC, Canada) following the manufacturer’s instructions. In the case of buffy coats, a depletion of T lymphocytes (CD3+ cells) was performed incubating samples with RosetteSep Human CD3 Depletion Cocktail (StemCell Technologies) before ficoll-hypaque gradient separation due to the high number of cells present in the buffy coat samples.
Samples were incubated with 7-amino-actinomycin D (7-AAD) (BD Biosciences, San José, CA, USA) and annexin V-PE (Immunotools, Friesoythe; Germany) for 20 min at 4 °C in darkness to analyze the viability of monocytes. In addition, perfect count microspheres (Cytognos S.L, Salamanca, Spain) were added to each sample and acquired in a flow cytometer (FACS Canto II, BD Biosciences) to determine the number of viable monocytes obtained.

Peripheral blood mononuclear cells were obtained from leukocyte residues from healthy donors from the Blood and Tissue Bank (Barcelona, Spain) (n = 22) by Ficoll Hypaque Pus™ density gradient centrifugation (GE Healthcare Biosciences) at 1,800 rpm for 30 min, and CD3⁺ cells were depleted using the RosetteSep™ Human CD3 Depletion Cocktail (StemCell Technologies). Monocytes were then isolated using the EasySep™ Human anti-CD14 Positive Selection Kit (StemCell Technologies) or the MagniSort Human CD14 Positive Selection kit (eBioscience) following manufacturers’ instructions. Recovered cells were counted using PerfectCount Microspheres (Cytognos) and assessed for purity (>90% CD14⁺) and viability [≥93% by FSC/SSC and 7AAD⁻ (BD) gating] in a Canto II flow cytometer (BD).

NSG mice were bred and maintained under specific pathogen free conditions with acidified water (pH 5.3) at the animal facility of Gustave Roussy Institute. Animal experiments were performed in accordance with guidelines established by the Institutional Animal Committee. Peripheral blood mononuclear cells from AML patients were depleted in CD3⁺ cells by RosetteSep human CD3⁺ depletion cocktail (StemCell Technologies) and 5 × 10⁶ cells were intravenously injected to female mice (6–8 weeks old) 24 h after irradiation at 2.5 Gy from a ¹³⁷Cs source33 (link). Mice were analysed at 8–43 weeks post-injection. Cells from mouse BM were stained with rat anti-mouse CD45 (Biolegend) and mouse anti-human CD45, anti-human CD19, anti-human CD33 and anti-human CD3 antibodies (all from BD Pharmingen; clones and fluorochromes are indicated in Supplementary Table 12). Stained cells were analysed on FACSort or FACSCanto II cytometers (BD Biosciences). The presence of <0.5% of human CD45⁺ (hCD45⁺⁾ population was considered as non-engraftment. The presence of >5% of hCD45⁺ cells with major CD33⁺ population (>75% of the hCD45⁺ cells) was considered as overt AML engraftment. The presence of 0.5 to 5% of hCD45⁺ or the presence of >5% of hCD45⁺ with <75% of CD33⁺ in the hCD45⁺ population was considered as non-overt or non-leukaemic engraftment.

Highly purified human NK cell expansion method was performed as previously described [34 (link)]. Briefly, PBMCs were obtained from 16 mL heparinized peripheral blood obtained from a healthy volunteer (a 41-year-old man). The CD3 fraction of the PBMCs was depleted by the RosetteSep^TM Human CD3 Depletion Cocktail (STEMCELL Technologies, Vancouver, Canada). The CD3-depleted PBMCs were placed in a T25 culture flask (Corning, Steuben, NY, USA) containing AIM V medium (Life Technologies) supplemented with 10% autologous plasma, 50 ng/mL recombinant human IL-18 (rhIL-18, Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), and 3000 IU/mL rhIL-2 (Novartis, Basel, Switzerland) at 37 °C in a humidified atmosphere containing 5% CO₂ for 7 days. The AIM V medium containing 3000 IU/mL rhIL-2 was replenished as necessary. Then, 3 × 10⁶ of the expanded NK cells were electroporated to RNPs complexes targeting TIM3 using an Amaxa Human NK cell Nucleofector Kit (VPA-1005; Lonza, Basel, Switzerland) and electroporation program X-001. Subsequently, the cells were resuspended in AIM V medium containing 10% autologous plasma and 3000 IU/mL rhIL-2 and placed in a 12-well plate (Corning) at 37 °C in a humidified atmosphere containing 5% CO₂ for 7 days.

CD3-depleted peripheral mononuclear cells (PBMCs) were isolated using a RosetteSepTM Human CD3 Depletion Cocktail (STEMCELL Technologies, Vancouver, BC, Canada). CD3-depleted PBMCs were placed in a T25 culture flask (Corning, Steuben, NY, USA) containing AIM-V medium (Life Technologies, New York, NY, USA) supplemented with 5% autologous plasma, 50 ng/mL recombinant human (rh) interleukin (rhIL)-18 (Medical and Biological Laboratories Co., Ltd.; MBL, Nagoya, Japan), and 3000 IU/mL rhIL-2 (Novartis, Basel, Switzerland) at 37 °C in a humidified 5% CO₂-containing atmosphere for 14 days. AIM-V supplemented with 3000 IU/mL IL-2 was replenished as necessary.