easysep human cd3 t cell isolation kit  (Miltenyi Biotec)

Journal: Science Advances

Article Title: Armored human CAR T reg cells with PD1 promoter-driven IL-10 have enhanced suppressive function

doi: 10.1126/sciadv.adx7845

Figure Lengend Snippet: ( A ) Schematic diagram of alloantigen-based suppression assay shown in (B) and (C). HLA-A2 + DCs were cocultured with HLA-A3 − T reg cells for 72 hours. Allogeneic CPD eFluor 450–labeled HLA-A3 + CD3 + responder T cells were then added, and cocultures were maintained for an additional 96 hours. ( B ) Representative CD4 + responder T cell proliferation, gated on live HLA-A3 + CPD eFluor 670 − CPD eFluor 450 + CD4 + cells. ( C ) % Suppression of CD4 + responder T cell proliferation, relative to responder T cells cultured without T reg cells. ( D ) Schematic diagram of islet antigen–based suppression assay shown in (F) to (H). HLA-A2 + HLA-DR4 + DCs were cocultured with T reg cells for 48 hours. CD4 + 4.13-TCR + responder T cells and 1 nM GAD65 peptide were then added, and cocultures were maintained for an additional 48 hours. ( E ) CD4 + 4.13-TCR + T cell proliferation following 96-hour coculture with immature HLA-DR4 + DCs and varying GAD65 peptide concentrations (without T reg cells). Control cells were pulsed with an irrelevant hemagglutinin peptide (100 nM). Division indices provided. ( F ) Representative CD4 + responder T cell proliferation, gated on live CD4 + CPD eFluor 670 − mTCRβ + cells. ( G ) % Suppression of responder T cell proliferation, relative to responder T cells stimulated without T reg cells. ( H ) Relative cytokine analysis from 1:128 T reg cell:responder T cell (T resp cell) ratio. TNFα, IL-17A & IL-17F were not detected. Averaged data are means ± SEM with non-linear regression lines ( n = 8 to 10). Statistical significance was determined using mixed-effects analysis with P values shown. n.s., not significant.

Article Snippet: After 3 days, CD3 + responder T cells (previously enriched from an HLA-A3 + individual using the EasySep Human CD3 Positive Selection Kit II; STEMCELL Technologies) were labeled with CPD eFluor 450 and added to the cocultures at a 5:1 responder T cell:DC ratio.

Techniques: Suppression Assay, Labeling, Cell Culture, Control

Journal: Experimental Hematology & Oncology

Article Title: Engineered circular RNA-based DLL3-targeted CAR-T therapy for small cell lung cancer

doi: 10.1186/s40164-025-00625-8

Figure Lengend Snippet: Optimization of electroporation conditions for circRNA expression in Human CD3 + T cells. A Comparison of EGFP expression in T cells electroporated with circRNA at different voltages (1400V, 1600V, and 1800V). B Flow cytometry analysis of anti-DLL3 CAR expression at varying electroporation voltages. C Evaluation of CAR expression and apoptosis rates in T cells electroporated with various concentrations of circRNA (1–6 µg/million cells). D Assessment of immunogenicity in circRNA and mRNA-transfected T cells under optimized electroporation conditions. E Assessment of CAR expression duration in circRNA and mRNA-transfected T cells. F Evaluation of cell proliferation in circRNA and mRNA-transfected T cells using the CCK-8 assay. G Evaluation of cell proliferation in circRNA and mRNA-transfected T cells using the CFSE assay. circRNA circular RNA, mRNA messenger RNA, EGFP enhanced green fluorescent protein, CAR-T chimeric antigen receptor T cell, DLL3 Delta-like Ligand 3, CCK-8 Cell Counting Kit-8, CFSE Carboxyfluorescein Succinimidyl Ester

Article Snippet: CD3 + T cells were then purified employing the EasySep TM Human CD3 Positive Selection Kit II (Stemcell, 17851).

Techniques: Electroporation, Expressing, Comparison, Flow Cytometry, Immunopeptidomics, Transfection, CCK-8 Assay, CFSE Assay, Cell Counting

Journal: Cell Reports Medicine

Article Title: CAR T cells, CAR NK cells, and CAR macrophages exhibit distinct traits in glioma models but are similarly enhanced when combined with cytokines

doi: 10.1016/j.xcrm.2025.101931

Figure Lengend Snippet: Manufacturing of mRNA-based mouse CAR immune effector cells and functional assessment thereof (A) Schematic overview for rapid and high number expansion protocols of mouse immune cells. Spleen-derived T cells are activated using αCD3 and αCD28 antibodies and expanded with IL-2. Spleen-derived NK cells are isolated using negative selection and expanded with high doses of IL-15. Macrophages are expanded from bone marrow (BM) cells using conditioned medium from L929 cells. (B–D) Mouse T cells (B), NK cells (C), or macrophages (D) were transfected with ZsGreen mRNA, and fluorescence was analyzed using flow cytometry in the following days. Quantifications for ZsGreen + cells are shown. (E) Representative microcopy images of ZsGreen mRNA-transfected mouse T cells, NK cells, and macrophages one day post-transfection. Scale bars, 100 μm. (F) Schematic representation of CAR mRNA and CARΔ(CD3ζ) mRNA lacking the intracellular CD3ζ domain. Both mRNAs co-express RQR8 and were generated using in vitro transcription with optimized 3′-UTRs, 5′-UTRs, and pseudouridine (Ψ). (G–I) Quantifications of tdTomato + GL-261 glioma cell confluence after co-cultures with mouse (G) T cells, (H) NK cells, or (I) macrophages that were mock transfected or transfected with mRNA coding for CAR or CARΔ(CD3ζ) and seeded at an E:T ratio of 2:1. Data are represented as mean + SD based on n = 3 FOVs. (J and K) Flow cytometry quantifications of GL-261, CT-2A, or SB-28 glioma cells that were co-cultured at an E:T ratio of 8:1 for 24 h with mouse immune cells that were mock transfected or transfected with mRNA coding for CAR or CARΔ(CD3ζ). (J) Glioma cell lysis and (K) total remaining glioma cells after co-culture are shown (mean + SD of n = 3, one-way ANOVA with ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). FOV, field of view; E:T ratio, effector to target cell ratio; MFI, mean fluorescence intensity.

Article Snippet: Human T cells and NK cells were expanded from CD3 + and CD3 − fractions separated from PBMCs using the EasySep Release Human CD3 Positive Selection Kit (Stemcell Technologies, #17751).

Techniques: Functional Assay, Derivative Assay, Isolation, Selection, Transfection, Fluorescence, Flow Cytometry, Generated, In Vitro, Cell Culture, Lysis, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: CAR T cells, CAR NK cells, and CAR macrophages exhibit distinct traits in glioma models but are similarly enhanced when combined with cytokines

doi: 10.1016/j.xcrm.2025.101931

Figure Lengend Snippet: 3D confocal microscopy and flow cytometry unveil the tumor-infiltrative capacity of CAR T cells, CAR NK cells, and CAR macrophages (A–F) GL-261 iRFP720 glioma-bearing C57BL/6 mice received intravenous injections of 5 × 10 6 CAR immune effector cells co-expressing ZsGreen on day 11 after glioma cell implantation. Mice were perfused two days later, and brain sections stained for DAPI and the endothelial cell marker endomucin (Emcn). (A) Representative 3D images of tumor-bearing hemispheres with Emcn + endothelium (turquoise) and ZsGreen + CAR immune effector cells (yellow). Scale bars are 200 μm (left) and 50 μm (right). (B–D) CDF plots of distances to the endothelium are shown in red compared to the simulated CSR distributions in gray for CAR T cells (B), CAR NK cells (C), and CAR macrophages (D). Two-sample Kolmogorov-Smirnov test was used to analyze significance (CDF versus CSR plots: ∗ p < 0.05). (E) Representative 2D tissue maps display the spatial distribution of CAR T cells, CAR NK cells, and CAR macrophage as single cells or clusters within whole tumor sections. (F) Quantification of tumor-infiltrating CAR T cells, CAR NK cells, and CAR macrophages in brain slices as cell numbers per tumor volume ( n = 6, one-way ANOVA with ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (G and H) GL-261 iRFP720 glioma-bearing CD45.2 + C57BL/6 received either intravenous injections of 5 × 10 6 CD45.1 + CAR immune effector cells or local injections of 2 × 10 6 CD45.1 + CAR immune effector cells on day 11 after glioma cell implantation. Mice were perfused two days later, and tumor-bearing hemispheres dissociated. (G) Absolute cell counts per tumor and (H) the viability of CD45.1 + CD11b − CD3 + CAR T cells, CD45.1 + CD11b − CD335 + NK cells, and CD45.1 + CD11b + CAR macrophages were quantified using flow cytometry (boxplot with median +/− quartiles and min to max of n = 4, one-way ANOVA with ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). CDF, cumulative distribution function; CSR, complete spatial random.

Article Snippet: Human T cells and NK cells were expanded from CD3 + and CD3 − fractions separated from PBMCs using the EasySep Release Human CD3 Positive Selection Kit (Stemcell Technologies, #17751).

Techniques: Confocal Microscopy, Flow Cytometry, Expressing, Staining, Marker

Journal: Cell Reports Medicine

Article Title: CAR T cells, CAR NK cells, and CAR macrophages exhibit distinct traits in glioma models but are similarly enhanced when combined with cytokines

doi: 10.1016/j.xcrm.2025.101931

Figure Lengend Snippet: Human CAR and multifunctional CAR lymphocytes kill glioma cell lines and glioblastoma cells from patient samples with an intact microenvironment (A) Human LN-229 and ZH-161 glioma cells were co-cultured with human T cells or NK cells that were mock transfected or transfected with mRNA coding for CAR or CARΔ(CD3ζ) at different E:T ratios for 24 h. Glioma cell lysis was assessed using flow cytometry (mean ± SD of n = 3, one-way ANOVA with ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (B) Same setup as in (A) but with human macrophages that were polarized using M-CSF or GM-CSF/IFNγ and co-cultured at an E:T ratio of 8:1. Total remaining glioma cells after co-culture are shown (mean + SD of n = 3, one-way ANOVA with ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (C and D) Human immune cells transfected with ZsGreen mRNA and mRNA coding for CAR or cytokines or both were ex vivo co-cultured with surgically derived patient glioblastoma samples, and images of co-cultures were quantified using pharmacoscopy. (C) Remaining fractions of glioblastoma cells from n = 10 glioblastoma patient samples are represented as boxplot with median +/− quartiles and min to max. Significances were quantified by comparing co-cultures with mock-transfected immune cells and co-cultures with mRNA-transfected immune cells using paired nonparametric t test (∗ p < 0.05; ∗∗ p < 0.01). (D) Representative immunofluorescence images of co-cultures are shown with CD45 + immune cells in red, tumor cells in orange, and CAR immune cells in green. White arrows denote immune cell clusters. Scale bar, 40 μm.

Article Snippet: Human T cells and NK cells were expanded from CD3 + and CD3 − fractions separated from PBMCs using the EasySep Release Human CD3 Positive Selection Kit (Stemcell Technologies, #17751).

Techniques: Cell Culture, Transfection, Lysis, Flow Cytometry, Co-Culture Assay, Ex Vivo, Derivative Assay, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: CAR T cells, CAR NK cells, and CAR macrophages exhibit distinct traits in glioma models but are similarly enhanced when combined with cytokines

doi: 10.1016/j.xcrm.2025.101931

Figure Lengend Snippet:

Article Snippet: Human T cells and NK cells were expanded from CD3 + and CD3 − fractions separated from PBMCs using the EasySep Release Human CD3 Positive Selection Kit (Stemcell Technologies, #17751).

Techniques: Virus, Recombinant, Derivative Assay, Activation Assay, Electron Microscopy, Lysis, Transfection, Cell Isolation, Selection, Enzyme-linked Immunosorbent Assay, Retroviral, Software, Pore Size