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. 2021 Jul 8;9(7):e002267. doi: 10.1136/jitc-2020-002267

Figure 1.

Figure 1

N-803 increased the viability and proliferation of exPBNK with enhanced p-Stat3, p-Stat5, pAKT, p-p38MAPK and NK activating receptors. PBMNCs were stimulated with irradiated genetically modified K562-mbIL21-41BBL cells for 2–3 weeks. (A) Purified exPBNK cells were cultured in complete medium with 0.35 ng/mL (low) or 3.5 ng/mL (high) N-803 or molar equivalent dose of IgG for 3 days. NK viability and proliferation were monitored by MTS assays. The amount of 490 nm absorbance is directly proportional to the number of living exPBNK cells in the culture. The exPBNK cells with N-803 at 0.35 ng/mL or 3.5 ng/mL have significantly higher viability as compared with IgG or medium controls (p<0.001) and N-803 at 3.5 ng/mL significantly stimulated the proliferation of exPBNK cells as compared with N-803 at 0.35 ng/mL (p<0.001). (B) ExPBNK cell phenotypic changes cultured in medium with IgG or N-803 under light microscopy (Axiovert 200M; Carl Zeiss) are shown at day 6 (original magnification 200 x). (C) Purified exPBNK cells were cultured in medium with 3.5 ng/mL N-803 or molar equivalent dose of IL-15 or IgG for 3 days. Intracellular phosphorylated STAT3 (p-Stat3), phosphorylated Akt1 (p-AKT1), phosphorylated p38MAPK (p-p38MAPK), and phosphorylated STAT5 (p-Stat5) were monitored by flow cytometry analysis. N-803 at 3.5 ng/mL significantly enhanced the phosphorylation of STAT 3, Akt1 and p38MAPK as compared with IgG (p<0.001) at day 3. (D) Purified exPBNK cells were cultured in medium with 3.5 ng/mL N-803 or molar equivalent dose of IL-15 or IgG for 3 days or 10 days. ExPBNK cells were stained with indicated monoclonal antibodies. The expression of receptors on viable exPBNK cells were compared by flow cytometry analysis. Purified non-expanded NK (PBNK) cells were used as controls. At day 10, the expression levels of NKG2D, NKp30, NKp44, NKp46 and CD16 were significantly enhanced in exPBNK with N-803 as compared with exPBNK with IgG. ***p<0.001, Data were presented as mean±SEM from three independent experiments. ExPBNK, expanded peripheral blood natural killer cell; IL-15, interleukin-15; ns, not significant; PBMNCs, peripheral blood mononuclear cells.