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. 2024 Jul 3;73(9):179. doi: 10.1007/s00262-024-03765-8

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Characterization of NK cells incubated ex vivo with multifunctional fusion protein complexes. A Model of “Prime” of NK cells utilizing HCW9201 and “Expansion” of “Prime” NK cells with HCW9206/HCW9101 complex. B Bar graph showing expression of IL-7R and IL-21R on NK cells after overnight stimulation by either low-dose (LD) IL-15, mixed cytokines of IL-15, IL-12, and IL-18 or HCW9201 (n = 3 donors). C Purified NK cells were labeled with CellTrace Violet dye and incubated for 7 days with rhIL-2 (10ng/ml) or HCW9206/HCW9101 (“Expand”) or HCW9201 followed by HCW9206/HCW9101 (“Prime and Expand”) Cells were then harvested and examined for CellTrace Violet dye dilution by flow cytometry for cell proliferation. Percentage of cells divided was determined by FlowJo Proliferation Modeling (v. 10.3). Data are representative of 2 independent experiments. D Left—Expansion of NK cells purified from PBMC of 15 donors with 3 independent replicates each. NK cells were primed overnight in the presence of HCW9201 on day 0 followed by HCW9206/HCW9101 expansion from day 1 to day 14 of culture. Individual expansion data are indicated by grey lines. Purple line is the mean ± SD of all expansions shown. Right—Total fold expansion of “Expand” and “Prime and Expand” treated NK cells after 14 days of culture (Paired t-test, *< 0.05). E Surface protein expression of CD25, CD69, and NKG2A measured by flow cytometry of isolated NK, “Expand” NK cells, “Prime and Expand” NK cells. Cells were thawed from cryopreservation and then stained with antibodies to CD45, CD56, CD3, Viability, CD25, CD69, and NKG2A. NK cells were gated as non-Debris, single cell, CD45⁺, CD56⁺CD3⁻, Live cells and shown as mean ± SD of the MFI. (ANOVA, post-hoc Tukey, *< 0.05, **< 0.01, ****< 0.0001). F Metabolic parameters were examined for resting, IL-15 (control), “Prime” alone, “Prime” and supported by rhIL-15 and “Prime and Expand” NK cells using XFe96 Extracellular Flux Analyzer measurements of extracellular acidification rate (ECAR). Left—complete ECAR analysis consisted of four stages: basal (without drugs), glycolysis induction (10 mM glucose), maximal glycolysis induction (2 μM oligomycin), and glycolysis inhibition (100 mM 2-DG). Right—summary data (mean ± SD) showing glycolysis, glycolytic reserve, and glycolytic capacity. Data shown is from 6 donors analyzed in 3 independent experiments (n = 5). (ANOVA, *< 0.05, **< 0.01)