Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Optimal genetic feeder cell-expanded and engineered NK cell products are composed of CD56 bright and CD56 dim NK cells

doi: 10.1007/s00262-026-04306-1

Figure Lengend Snippet: Both CD56 bright and CD56 dim NK cells are most optimally activated by the combination of K562 F cells and cytokines . A Experimental setup to assess the short-term (16 h) effects of individual or combined K562 F and/or cytokine (IL-2 and IL-15) stimulation on NK cell activation by flow cytometry. B Live, single NK cells were gated (Suppl Fig. ) and pooled from all samples and donors ( n = 5). Data were visualized using UMAP dimensionality reduction . The UMAP was generated using all arcsinh-normalized markers listed in C and shows the distribution of cells derived from each stimulation condition. C UMAP visualization of single-cell marker expression, with each cell colored according to expression level; respective markers are indicated above each plot. D–F Density plots of CD69, Granzyme B, and perforin expression in unstimulated and stimulated NK cells of one representative donor. G–I Mean fluorescence intensity (MFI) of CD69, Granzyme B, and perforin expression in CD56 bright and CD56 dim NK cells, determined by manual gating on CD56 expression. J–L The percentage of CD137, IFN-γ, and TNF-α expression in CD56 bright and CD56 dim NK cells, determined by manual gating for each activation marker. Each symbol represents an individual donor ( n = 5). Error bars indicate mean ± SD. Statistical significance was determined using two-way ANOVA followed by Sidak’s multiple comparison test; p values of relevant comparisons are shown (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001)

Article Snippet: CD56 + CD3 − NK cells were isolated from cryopreserved PBMCs using an untouched NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Flow Cytometry, Generated, Derivative Assay, Marker, Expressing, Fluorescence, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Optimal genetic feeder cell-expanded and engineered NK cell products are composed of CD56 bright and CD56 dim NK cells

doi: 10.1007/s00262-026-04306-1

Figure Lengend Snippet: Stimulation and expansion of CD56 bright and CD56 dim NK cells result in a homogenous NK cell phenotype that could be distinguished by CD16 expression. A NK cells within the PBMCs ( d = 0) and the NK cell subsets after sorting and expansion with K562 F cells and cytokines ( d = 7) were analyzed by flow cytometry. The viable NK cells (gating in Suppl Fig. from both conditions ( d = 0 and d = 7) of all donors ( n = 3) were combined and visualized using UMAP dimensionality reduction, based on the markers that are shown. The distribution of the cells derived from each NK cell subset on day 0 and day 7 is shown on the UMAP, followed by the normalized arcsinh-transformed marker expression of each cell surface marker, with the respective markers indicated above each plot. BCD Density plots of CD16, NKG2A, and CD94 expression of the NK cell subsets at day 0 and expanded CD56 bright and CD56 dim NK cells at day 7 of one representative donor. E–M The expression levels (MFI) of CD56, NKp46, NKG2D, and CD38, or the percentage of cells expressing CD16, NKG2A/CD94, CD62L, CD27, and CD2 of both CD56 bright and CD56 dim NK cells at day 0 and day 7, determined by manual gating. Each symbol represents a different donor ( n = 3). Error bars represent mean and SD of different donors. Statistical test used was a two-way ANOVA, followed by a Sidak’s multiple comparison test; the p value of each significant comparison is plotted (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0,0001). F Overlay of CD56 bulk NK cells over UMAP dimensional reduction plot. The same living single NK cells as described in panel A were in addition to a bulk NK cell population from the same 3 donors used as input for the UMAP. The bulk NK cells were also cultured with K562 F cells and cytokines for 7 days

Article Snippet: CD56 + CD3 − NK cells were isolated from cryopreserved PBMCs using an untouched NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Transformation Assay, Marker, Comparison, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Optimal genetic feeder cell-expanded and engineered NK cell products are composed of CD56 bright and CD56 dim NK cells

doi: 10.1007/s00262-026-04306-1

Figure Lengend Snippet: CD56 bright NK cells form clusters with K562 F cells more rapidly, leading to accelerated feeder cells clearance. A Representative images from longitudinal live-cell IncuCyte analysis at the indicated timepoints (in hours) showing CD56 bright and CD56 dim NK cells stimulated with GFP+ K562 F cells (green) and cytokines. B K562 F clearance over 7 days ( t = 168 h), presented as the fold change in green calibrated unit (GCU) integrated intensity of the GFP signal (GFP+ K562 F cells) relative to t = 0 ( n = 4 donors). C Fold expansion of CD56 bright and CD56 dim NK cells over 7 days ( t = 168 h), shown as the percentage of area confluence of NK cells (GFP−) relative to K562 F cells (GFP+) ( n = 4 donors). D Fold expansion of CD56 bright , CD56 dim , and CD56 bulk NK cells isolated from healthy donors ( n = 7) after initial stimulation with K562 F cells and cytokines, calculated as the fold change in counted cell numbers at day 7 compared to day 0. E Frequency of Ki-67+ CD56 bright and CD56 dim NK cells in unstimulated NK cells present in cryopreserved PBMC from healthy donors ( n = 7), measured by flow cytometry at day 0. F Fold expansion of CD56 bright and CD56 dim NK cells ( n = 3) following restimulation at day 7 with K562 F cells and cytokines, calculated as the fold change in counted cell numbers at day 14 compared to day 7. Each symbol represents an individual donor ( n = 3). Error bars show mean ± SD. Statistical significance was determined using a paired t test; p values of relevant comparison are indicated (ns = nonsignificant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001)

Article Snippet: CD56 + CD3 − NK cells were isolated from cryopreserved PBMCs using an untouched NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Isolation, Flow Cytometry, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Optimal genetic feeder cell-expanded and engineered NK cell products are composed of CD56 bright and CD56 dim NK cells

doi: 10.1007/s00262-026-04306-1

Figure Lengend Snippet: CD56 bright and CD56 dim NK cells exhibit similar NK-mediated cytotoxicity, but CD56 dim NK cells remain the principal effector cell for antibody-dependent cellular cytotoxicity. A 16-h flow cytometry-based cytotoxicity assay of 7-day K562 F and cytokine-stimulated sorted CD56 bright and CD56 dim NK cells against the dTomato + , NK-sensitive, HLA-negative cell line K562 ( n = 3 donors) at different E/T ratios. Cytotoxicity was calculated as: Cytotoxicity (%) = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left(1-\frac{\#\text{ dTomato}+\text{ living target cells after co}-\text{culture }}{\#\text{ dTomato}+\text{ living untreated target cells}}\right)$$\end{document} 1 - # dTomato + living target cells after co - culture # dTomato + living untreated target cells × 100%. B ADCC-mediated flow cytometry-based cytotoxicity assay of 7-day K562 F and cytokine-stimulated CD56 bright and CD56 dim cells against the CD20+ dTomato + B-ALL cell line ALL BV. C Burkitt’s lymphoma cell line Raji at two E/T ratios (1:1 and 1:3) in the presence of 10 or 100 µg/mL rituximab, respectively. Cytotoxicity was calculated as above. Rituximab-mediated cellular cytotoxicity was determined as: % cytotoxicity (tumor cells + RTX)—% cytotoxicity (tumor cells alone). Each symbol represents an individual donor. Error bars represent mean and SD of the donors. Statistical significance was determined using a paired t test, with p values indicated (ns = nonsignificant, * = p < 0.05)

Article Snippet: CD56 + CD3 − NK cells were isolated from cryopreserved PBMCs using an untouched NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Cytotoxicity Assay, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Optimal genetic feeder cell-expanded and engineered NK cell products are composed of CD56 bright and CD56 dim NK cells

doi: 10.1007/s00262-026-04306-1

Figure Lengend Snippet: CD56 bright NK cells are more susceptible to retroviral transduction than CD56 dim NK cells. A Schematic overview of the 14-day production protocol for engineered NK:TCR and NK:CAR cell products derived from FACS-sorted CD56 bright , CD56 dim , and CD56 bulk NK cells. B Transduction efficiency of the indicated retroviral constructs (Suppl Fig. ) measured by flow cytometry at day 7 post-isolation. Percentages of TCR/CD3 + were calculated of total CD8β+ -transduced NK cells, and percentages of CAR+ and tNGFR+ were calculated of total NK cells. Each symbol represents an individual donor ( n = 3–5). Error bars indicate mean ± SD across donors. Statistical significance was assessed using a one-way repeated measurement ANOVA, with p values of each comparison plotted (ns = nonsignificant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). C Representative flow cytometry plots of transduced and subsequently MACS-purified final NK:TCR and NK:CAR products at day 14, derived from FACS-sorted CD56 bright , CD56 dim , and CD56 bulk NK cells

Article Snippet: CD56 + CD3 − NK cells were isolated from cryopreserved PBMCs using an untouched NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Retroviral, Transduction, Derivative Assay, Construct, Flow Cytometry, Isolation, Comparison, Purification

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Optimal genetic feeder cell-expanded and engineered NK cell products are composed of CD56 bright and CD56 dim NK cells

doi: 10.1007/s00262-026-04306-1

Figure Lengend Snippet: Both CD56 dim and CD56 bright NK cells mediate CAR- and TCR-specific antitumor cytotoxicity. Sixteen-hour flow cytometry-based cytotoxicity assays using NK:BOB1-TCR, NK:CD19-CAR, and NK:MOCK cell products derived from FACS-sorted CD56 bright and CD56 dim NK cells. A–C NK:BOB1-TCR and NK:MOCK cell subsets were tested against A dTomato + LCL JY (TCR Ag+ ; BOB1 + /HLA-B7 +), LCL HHC (TCR Ag+ ; BOB1 + /HLA-B7+), B LCL IZA (TCR Ag−; BOB1 + /HLA-B7−), and C MM cell line UM9 (TCR Ag+ ; BOB1 + /HLA-B7+) using NK:BOB1-TCR and NK:MOCK cells derived from CD56 bright , CD56 dim , and CD56 bulk NK cells. DE NK:CD19-CAR and NK:MOCK cells derived from CD56 bright , CD56 dim , and CD56 bulk NK cells against D dTomato + LCL IZA (CAR Ag + ; CD19+) and E dTomato + B-ALL cell line ALL BV (CAR Ag+ ; CD19+). Cytotoxicity was calculated as: Cytotoxicity (%) = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left(1-\frac{\#\text{ dTomato}+\text{ living target cells after co}-\text{culture }}{\#\text{ dTomato}+\text{ living untreated target cells}}\right)$$\end{document} 1 - # dTomato + living target cells after co - culture # dTomato + living untreated target cells × 100%. Each symbol represents an individual donor. Error bars show mean ± SD. Statistical significance was assessed using multiple paired t tests, with p values indicated (ns = nonsignificant, * = p < 0.05)

Article Snippet: CD56 + CD3 − NK cells were isolated from cryopreserved PBMCs using an untouched NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Derivative Assay, Co-Culture Assay

Journal: Science Advances

Article Title: Resident CD49a + CD103 + NKG2C + NK cells restrict HIV infection in human lymphoid tissue explants

doi: 10.1126/sciadv.adz1565

Figure Lengend Snippet: Expression of CD56, CD16, NKp44, resident (CD69, CD49a, and CD103), and memory-like (KIRs and NKG2C) markers was measured by flow cytometry in different tonsillar tissue specimens. ( A ) Frequencies of CD45 + CD14 − CD19 − lymphocytes (left), total CD56 + NK cells (middle), and classic CD16 +/− NK cell subsets (right). ( B ) Frequency of the expression of resident markers within main CD56 + CD16 +/− subsets. ( C ) Frequency of the expression of memory-like markers within main CD56 + CD16 +/− subsets. ( D ) Frequency of the expression of memory-like markers within CD56 + CD16 +/− CD69 + CD49a + and CD49a − NK cells. ( E ) Coexpression frequencies of resident and memory-like markers within CD56 + CD16 +/− NK cell subsets. ( F ) Frequency of ieILC1s (NKp44 + CD103 + within CD127 − CD56 + CD16 − cells). ( G ) Frequency of NKp44 expression within CD127 − CD56 + CD16 − CD69 + CD49a + CD103 + NK cells. ( H and I ) Frequencies of CD127 + CD16 − ILCs within (H) CD45 + CD14 − CD19 − lymphocytes and (I) CD56 + cells. ( J ) Frequency of resident CD127 + CD16 − ILCs. ( K ) Frequency of memory-like CD127 + CD16 − ILCs. ( L ) Coexpression frequencies of resident markers within CD127 + CD16 − ILCs. For [(A) to (D)] and [(F) to (K)], the median with interquartile range is represented. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test and the Skillings-Mack test with Bonferroni’s correction for multiple comparisons, as appropriate. Significant comparisons P < 0.05. A minimum of 30 cells for (A) to (L) was analyzed per NK cell subpopulation. N = 18 biological replicates for (A) (left), N = 55 biological replicates for (A) (middle and right) and (B) and (E), and N = 17 biological replicates for (F) to (L). Green circles: CD16 + ; blue squares: CD16 − .

Article Snippet: The tonsillar cell suspension containing resident NK cells was depleted from CD19 + cells using a commercial kit (EasySep Human CD19 Positive Selection Kit II; StemCell) and NK cells were purified using the CD56 + Microbeads (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry

Journal: Science Advances

Article Title: Resident CD49a + CD103 + NKG2C + NK cells restrict HIV infection in human lymphoid tissue explants

doi: 10.1126/sciadv.adz1565

Figure Lengend Snippet: Expression levels of the inhibitory ICs NKG2A, LAIR-1, TIGIT, PD-1, and KLRG1 were analyzed by flow cytometry in tonsillar tissue samples. ( A ) Frequencies of IC expression within CD56 + CD16 +/− CD69 + subsets. ( B and C ) Frequencies of IC expression within resident (B) CD16 + and (C) CD16 − NK cells. ( D ) Frequencies of IC expression within resident CD16 − KIR + NKG2C − NK cells. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test or the Friedman test with Dunn’s correction for multiple comparisons, as appropriate. Significant comparisons P < 0.05. A minimum of 30 cells was analyzed per NK cell subpopulation. N = 11 biological replicates. Green circles: CD16 + ; blue squares: CD16 − .

Article Snippet: The tonsillar cell suspension containing resident NK cells was depleted from CD19 + cells using a commercial kit (EasySep Human CD19 Positive Selection Kit II; StemCell) and NK cells were purified using the CD56 + Microbeads (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry

Journal: Science Advances

Article Title: Resident CD49a + CD103 + NKG2C + NK cells restrict HIV infection in human lymphoid tissue explants

doi: 10.1126/sciadv.adz1565

Figure Lengend Snippet: Tonsillar explants derived from two samples were infected by HIV BaL for 5 days. The CD14 − CD19 − CD3 − CD56 + population from HIV-uninfected and HIV-infected explants was isolated and analyzed using scRNA-seq. ( A ) UMAP visualization of clusters identified by unsupervised hierarchical clustering and proportion of cells per cluster and sample (right). ( B ) UMAP representation of diffusion pseudotime trajectories, indicating maturation states from dark blue (least mature) to yellow (most mature). ( C ) Bee swarm plot illustrating the distribution of clusters across pseudotime. ( D ) Volcano plots of clusters showing DEGs between uninfected and infected tissue cells. ( E ) GO pathway analysis in selected clusters in response to HIV exposure. ( F ) Frequency of ieILC1s in the infected culture. ( G ) Expression of PD-1 on CD16 + CD69 + CD49a + NKG2C + NK cells upon infection. ( H ) Degranulation potential (FC of CD107a + IFN-γ − cells) of NK cells in uninfected and infected explants upon K562 stimulation. ( I ) Expression of KIRs on NK cells in the infected culture upon infection. ( J ) Cytokine production potential (FC of CD107a + IFN-γ + cells) of NK cells in uninfected and infected explants upon K562 stimulation. ( K ) Expression of LAIR-1 upon infection on NK cells. ( L ) Expression of TIGIT on NK cells upon infection. ( M ) Degranulation potential (FC of CD107a + IFN-γ − cells) of NK cells in uninfected and infected explants upon K562 stimulation. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. Significant comparisons P < 0.05. For (F) to (M), a minimum of 30 cells was analyzed. N = 2 biological replicates for (A) to (E), N = 7 biological replicates for (F) and (G) and (K) and (L), N = 7 biological replicates for (H), (J), and (M), and N = 24 biological replicates for (I). Green circles: CD16 + ; blue squares: CD16 − .

Article Snippet: The tonsillar cell suspension containing resident NK cells was depleted from CD19 + cells using a commercial kit (EasySep Human CD19 Positive Selection Kit II; StemCell) and NK cells were purified using the CD56 + Microbeads (Miltenyi Biotec).

Techniques: Derivative Assay, Infection, Isolation, Diffusion-based Assay, Expressing