Journal: iScience

Article Title: From byproduct to biotherapeutic: Comparative study of buffy coats and leukoreduction system chambers for NK cell-based immunotherapies

doi: 10.1016/j.isci.2026.114907

Figure Lengend Snippet: Source-dependent differences in PBMC yield, NK cell isolation, and phenotype (A) Graphical overview of the PBMC isolation, NK cell enrichment, and phenotypic characterization workflow for LRSCs, BC, and PB. (B) Viability of isolated PBMCs as determined by trypan blue exclusion ( n = 10 for PB; n = 11 for BC and LRSCs). (C) Concentration of PBMCs (cells/mL starting material) obtained from each source ( n = 10 for PB; n = 11 for BC and LRSCs). Here, all comparisons were not significant and are summarized as such (n.s.: p > 0.05). (D) NK cell concentration (cells/10 6 PBMCs) from LRSCs and BCs. PB was excluded due to insufficient PBMC yield. ( n = 11 for LRSCs; n = 9 for BC); two outliers were identified post hoc in the BC roup. (E) Expansion of NK cells from LRSCs and BCs over 16 days. Cell count was assessed every 4 days using precision counting beads ( n = 6). (F) Correlation between NK cell frequencies in donor peripheral blood (measured via TBNK analysis) and isolated NK cell yield from LRSCs ( n = 10). Correlation was assessed using Pearson’s correlation analysis. Data are represented as mean ± SEM. Unless otherwise stated, n refers to biologically independent donor samples and is indicated per group. p -values were determined by Student’s t test with Welsh correction (D and E), by one-way ANOVA with Tukey posttest (B and C) or Pearson’s correlation analysis (F). All graphics were created with BioRender.com .

Article Snippet: According to the manufacturer’s instructions, NK cells were isolated from PBMCs using a magnetic-activated cell sorting (MACS) untouched NK cell isolation kit (Catalog no. 130-092-657; Miltenyi, Bergisch Gladbach, Germany).

Techniques: Cell Isolation, Isolation, Concentration Assay, Cell Characterization

Journal: iScience

Article Title: From byproduct to biotherapeutic: Comparative study of buffy coats and leukoreduction system chambers for NK cell-based immunotherapies

doi: 10.1016/j.isci.2026.114907

Figure Lengend Snippet: Functional characterization of NK cells (A) Degranulation capacity of NK cells following a 2 h co-culture with K562 leukemia cells, measured by surface CD107a expression ( n = 5). (B and C) Europium-based cytotoxicity assay assessing the specific lysis of K562 target cells at E:T ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 on day 7 (B) and day 14 (C) of culture to monitor functional decline over time ( n = 6 for both time points). Here, all comparisons were not significant and are summarized as such (n.s.: p > 0.05). (D) IFN-γ release following 16 h of co-culture of NK cells with K562 cells, quantified via Luminex assay ( n = 5). (E) Kinetic live-cell killing assay using the Incucyte monitoring of Nuclight Red signal loss in K562 cells over 72 h at an E:T ratio of 1:1 ( n = 6). (F) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis thresholds during Incucyte co-culture with K562 cells. (G) CD107a degranulation assay of NK cells with or without pre-incubation with trastuzumab (anti-HER2 antibody) during a 2 h co-culture with MDA-MB-453 cells ( n = 5). (H) Incucyte-based cytotoxicity assay shows the real-time lysis of MDA-MB-453 cells by trastuzumab-loaded NK cells at an E:T ratio of 1:1 over 72 h ( n = 6). (I) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the ADCC assay. (n varies as not all samples reached the benchmark values within the observation period). All ADCC experiments have been performed after 14 days of in vitro NK cell expansion. N/A indicates that statistical analysis was not possible for this group. 9J) CD107a degranulation of unmodified NK cells and ErbB2-CAR-NK cells after 2 h co-culture with MDA-MB-453 cells ( n = 5). (K) Incucyte-based cytotoxicity assay showing killing dynamics of ErbB2-CAR-NK cells against MDA-MB-453 cells at an E:T ratio of 1:1 over 72 h ( n = 6). (L) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the CAR-mediated cytotoxicity assay. (n varies as not all samples reached the benchmark values within the observation period). All CAR-killing experiments have been performed after 14 days of in vitro NK cell expansion. Unless otherwise stated, n refers to biologically independent donor samples and is indicated per group. Data are shown as mean ± SEM. p-values were determined by Student’s t test with Welsh correction (A, D, F, G, I, J, and L) or by one-way ANOVA (B and C) with Tukey post-test (K and L).

Article Snippet: According to the manufacturer’s instructions, NK cells were isolated from PBMCs using a magnetic-activated cell sorting (MACS) untouched NK cell isolation kit (Catalog no. 130-092-657; Miltenyi, Bergisch Gladbach, Germany).

Techniques: Functional Assay, Co-Culture Assay, Expressing, Cytotoxicity Assay, Lysis, Luminex, Degranulation Assay, Incubation, ADCC Assay, In Vitro

Journal: Cells

Article Title: Empowerment of CAR-T Cells by IL-7 and IL-15 Boosts Their Efficacy Against HER2-Positive Tumors with Enhanced Expansion and Persistence

doi: 10.3390/cells15060547

Figure Lengend Snippet: Treatment with IL-15 induced a population of CD57 + CAR-T cells, whose CD57 expression was downregulated upon tumor challenge. ( A , B ) CD57 expression level of CD4 + and CD8 + CAR-T cells with different interleukin treatments during CAR-T cell expansion measured by flow cytometry. ( A ) Representative histogram of CD57 expression on day 15 after initial anti-CD3/CD28 activation of CD4 + and CD8 + CAR-T cells. ( B ) Statistical evaluation of CD57 expression on day 8 and day 15 by matched ANOVA by donors. *: p < 0.05. ( C ) Nine days after the initial anti-CD3/28 activation (start), CAR-T cells were used for the repetitive tumor killing assay. Before each round of tumor killing, the CD57 level of CD4+ and CD8 + CAR-T cells was measured by flow cytometry. Group comparison of CD57 expressions was conducted between HER2-CAR-T cells with different treatments. ( D ) IL-2+7+15 HER2-CAR-T cells were sorted by anti-CD57 magnetic beads on day 15 after activation. On day 16, the positively sorted CD57 hi CAR-T cells, the remaining CD57 lo cells, and bulk CAR-T cells were proceeded to a repetitive tumor killing assay for three rounds. Before each round of tumor killing, the CD57 level of CD8 + cells was measured. ( E ) On day 16, CD57 hi , CD57 lo and bulk CAR-T cells were used to kill tumor cells at different effector–target (E:T) ratios. CAR-T cells were co-incubated with tumor cells for 24 h. Remaining live tumor cells and HER2 expression were identified by flow cytometry.

Article Snippet: The magnetic separation of CD57 hi and CD57 lo cells was conducted using anti-CD57 magnetic beads (Miltenyi Biotec, 130-092-073) and a QuadroMACSTM separator (Miltenyi Biotec, 130-090-976) according to the manufacturer’s instruction.

Techniques: Expressing, Flow Cytometry, Activation Assay, Comparison, Magnetic Beads, Incubation