Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Flow cytometric analysis of cell populations during the process. Panel A. Number of cells and proportion of populations obtained at the different steps of the process starting from buffy coat (BC) or leukapheresis (LA). Panels B–D. One representative experiment using LA (#3) as staring material is shown. Panel B. CD14 + monocytes (a), CD19 + B cells (b) CD3 + T cells (c), CD3 − CD56 + NK cells (d), γδ and αβ T lymphocytes (e) as well as Vδ1 and Vδ2 subsets (f), present after Ficoll-Paque gradient in gated lympho-mononuclear cells. Panels C and D. CD14 + monocytes (a), CD19 + B cells (b) CD3 − CD56 + NK cells (c), γδ and αβ T lymphocytes (d) as well as Vδ1 and Vδ2 subsets (e), gated mononuclear cells at the end of expansion (day 14) (Panel C) and harvested after αβ T-cell depletion (Panel D). Panel E shows γδ T cell differentiation, by APC-CD27 and PEVio770-CD45RA staining.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Cell Differentiation, Staining

Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Expression of CD16, natural cytotoxic receptors and exhaustion markers. Panel a. Expression of CD16, NKG2A, NKG2D, NKp30, NKp44 and NKp46 was tested by flow cytometry and analyzed in gated lympho-mononuclear cells. Experiments performed using batch from LA#4 is reported. NK and γδ T cells were discriminated by the different expression of CD3 (negative in NK and positive in γδ T lymphocytes). Results from all batches produced are reported in Panel b. Expression of PD-1 and TIM-3 in γδ (gated in CD3 + cells) and NK cells (gated in CD3 − ) present in batch #4 is shown in Panel c. Percentages of the two exhaustion markers expression in all batches produced are detailed in Panel d.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Expressing, Flow Cytometry, Produced

Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Cytotoxic activity of the GADEKILL against target tumor cells. CD107a surface expression on γδ T lymphocytes (Panel a), Vδ2 cells (Panel b), and NK cells (Panel c) in the absence (ctr) or presence of target cells (K562, SK-N-AS and SK-N-SH) have been analyzed on the four batches. γδ T cells and Vδ2 cytotoxic subsets were analyzed in gated CD45 + CD3 + cells, by staining with APC-TCRγδ, Vioblue anti-Vδ2. NK were analyzed on gated CD45 + CD3 − cells, by staining with APC-CD56 mAb. CD107a degranulation assay was performed using a 1:1 E:T ratio. Horizontal bars indicated medians. Asterisks indicated statistically significant differences. One representative experiment from batch #2 is shown in panels d, e and f. Degranulation of effector cells in the absence of the targets (Panel d) and against the myeloid leukemia K562 cells (Panel e) and the neuroblastoma SK-N-AS cell line (Panel f) is reported.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Activity Assay, Expressing, Staining, Degranulation 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: 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: iScience

Article Title: High-grade serous ovarian cancer induced in different sites of origin in mice exemplifies diverse features of the human disease

doi: 10.1016/j.isci.2025.114238

Figure Lengend Snippet: FTE-HGSCs have an increased number of tumor-infiltrating T cells and macrophages (A) Examples of IHC staining for CD3 (T lymphocytes), CD4 (T helper lymphocytes), and CD8a (cytotoxic T lymphocytes) in tumors of the four ovarian cancer models. (B) Quantification of IHC staining for T cell and macrophage markers in tumors of the four models. Data presented as means ± SD, N = 5–7 tumors/group. Statistical analysis performed by one-way ANOVA. ∗∗ indicates p < 0.01.

Article Snippet: Rat Anti Human CD3 , BioRad , Cat# MCA1477; RRID: AB_321245.

Techniques: Immunohistochemistry