Anti human cd16

The single-cell secretome analysis was performed using the IsoCode chip from IsoPlexis using the human NK cytokine panel. The assay was performed using the manufacturer’s kit and following the manufacturer’s instructions (IsoPlexis). In brief, NT-NK cells were stimulated using purified anti-human CD16 (BD Pharmingen, 555404; 1 μg ml⁻¹) and CAR-NK cells were stimulated using human CD19 antigen (ACRO, CD9-H5259; 10 μg ml⁻¹) for 4 h at 37 °C. NK cells were washed and labeled with a fluorescent dye (IsoPlexis stain cell membrane 405), and 30,000 cells were loaded onto the IsoCode chips. The IsoLight device was used to scan the chips, and IsoPlexis’s proprietary IsoSpeak software was used to analyze the data. The PSI, as computed by the software, was used for data representation. Stimulatory cytokines comprised GM-CSF, IL-12, IL-15, IL-2, IL-21, IL-5, IL-7, IL-8 and IL-9. Effector cytokines comprised GZMB, IFN-γ, MIP-1α, perforin, TNF-α and TNF-β. Chemokines comprised CCL-11, IP-10, MIP-1β and RANTES^{55 (link)}.

The KBM502 (cat no. 16025020; KOHJIN Bio, Sakado city, Saitama, Japan)-based culture medium used to expand and activate the isolated PBMC was supplemented with the following ingredients; 0.5% or 10% autologous human plasma depending on the step, each 2.5 μg of 3 different agonistic antibodies (anti-human CD56 (cat. no. 555513 and clone B159 (RUO); BD Biosciences, San Jose, CA, USA), anti-human CD16 (cat. no. 555403 and clone 3G8 (RUO); BD Biosciences) and anti-human CD355 (cat. no. MAB1850–500 and clone #195314; R&D SYSTEMS, Minneapolis, MN, USA)), and 3 different cytokines (200 ng/mL of IL-2 (cat. no. 653601261; Norvatis, Whippany, USA), 10 ng/mL of IL-12 (cat. no. 200–12; PEPROTECH, Rocky Hill, NJ, USA) and 100 ng/mL of IL-18 (cat. no. B003–2; R&D SYSTEMS)). All the cytokines were from PEPROTECH (Rocky Hill, NJ, USA).

A house protocol describing the preparation of ex vivo expanded NKLs has been recently published [6 (link)]. Briefly, blood samples were collected from healthy donors, and PBMCs and plasma were collected from buffy coats and the upper aqueous phase of blood, respectively, by density gravity centrifugation (Ficoll-Paque, GE Healthcare, Piscataway, NJ, USA). For expansion and activation of NKLs, isolated PBMCs were supplemented with the following bioactive components: 0.5% or 10% autologous human plasma depending on the step, 3 different agonistic antibodies, and 3 different cytokines. The cells were cultured for 2 weeks to produce a sufficient number of NKLs. The following antibodies and cytokines were used: anti-human CD56 (555513, BD Biosciences, San Jose, CA, USA), anti-human CD16 (555403, BD Biosciences), anti-human CD355 (MAB1850-500, R&D SYSTEMS, Minneapolis, MN, USA), IL-2 (653601261, Novartis, Whippany, NJ, USA), IL-12 (200-12, PeproTech, Rocky Hill, NJ, USA), and IL-18 (B003-2, R&D Systems).