cd3ε antibody, anti-mouse, reafinity  (Miltenyi Biotec)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Lipid nanoparticle mRNA delivery preserves CAR T cell cytotoxicity and limits exhaustion compared to electroporation

doi: 10.1016/j.omtn.2026.102929

Figure Lengend Snippet: Transfection efficacy of CD19-CAR mRNA and co-expression of multiple IVT mRNAs in polyclonally activated T cells (A) Experimental workflow: PBMCs from healthy donors were isolated, CD3 + T cells enriched by MACS, polyclonally activated with plate-bound anti-CD3/CD28 antibodies for 48 h, and electroporated with IVT mRNA on day 3. Created with BioRender.com . (B and C) Flow cytometric analysis of CD19-CAR + frequency (B) and CD19-CAR molecules per cell (C) in CD3 + , CD4 + , and CD8 + T cell subsets at 8 h and 24 h post-electroporation. (D) Schematic of single and co-transfection approach using CD19-CAR ± CCR7 mRNA. Created with BioRender.com . (E) Representative plots of mock-transfected control, CD19-CAR-only, CCR7-only, and CD19-CAR + CCR7 co-transfected T cells, showing CD19-CAR and CCR7 expression 8 h post-transfection. (F) Viability of T cells following single and dual mRNA transfection, assessed by live/dead staining 8 h post-transfection. (G–I) Quantification of CD19-CAR-only versus CD19-CAR + CCR7 co-expression: CD19-CAR + frequency (G), CD19-CAR geometric mean fluorescence intensity (H), and CCR7 geometric mean fluorescence intensity (I) in CD3 + T cells, 8 h after mRNA transfection ( n = 8 donors). (J) Schematic of multiplexing approach using four IVT mRNAs (CD19-CAR, CCR7, CXCR3, and GFP). Created with BioRender.com . (K–N) Flow cytometric analysis of four-mRNA multiplexing showing frequency of CD19-CAR + GFP + double-positive cells (K and L) and geometric mean fluorescence intensity of CXCR3 (M) and CCR7 (N), 8 h post-transfection ( n = 3 donors). Statistical analysis between subpopulations was performed by one-way ANOVA with Tukey’s post hoc test or paired t test. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data represent mean ± SD from the indicated number of independent donors.

Article Snippet: PBMCs were isolated from healthy donors using Biocoll (Biochrom) gradient centrifugation, and CD3 + T cells were enriched using magnetic cell separation with CD3 Microbeads (Miltenyi Biotec).

Techniques: Transfection, Expressing, Isolation, Electroporation, Cotransfection, Control, Staining, Fluorescence, Multiplexing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Lipid nanoparticle mRNA delivery preserves CAR T cell cytotoxicity and limits exhaustion compared to electroporation

doi: 10.1016/j.omtn.2026.102929

Figure Lengend Snippet: Memory phenotype characterization of IVT mRNA-transfected CD19-CAR T cells (A) Representative flow cytometry plots showing the gating strategy for CD8 + memory T cell subsets in primary human CD3 + -enriched T cells. Subsets were defined by CD3 + , CD4 – , CD8 + , and CCR7/CD45RA expression as follows: naive like (T naive like ; CCR7 + CD45RA + ), central memory (T CM ; CCR7 + CD45RA – ), effector memory (T EM ; CCR7 – CD45RA – ), and terminally differentiated effector (T EMRA ; CCR7 – CD45RA + ). (B) Frequencies of CD4 + and CD8 + T cells within CD19-CAR + populations compared with mock-transfected controls after electroporation or LNP transfection. (C and D) Geometric mean fluorescence intensity (gMFI) of CD19-CAR expression in bulk CD4 + (C) and CD8 + (D) T cell populations following electroporation or LNP-mediated transfection; representative experiment. (E and F) CD19-CAR surface expression across memory T cell subsets within the CD8 + population following electroporation (E) or LNP-mediated (F) mRNA delivery over 108 h post-transfection. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test (B) or by two-way repeated-measures ANOVA with Šidák’s multiple-comparison test (E and F). ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data represent mean ± SD from n = 4 independent donors, unless stated otherwise.

Article Snippet: PBMCs were isolated from healthy donors using Biocoll (Biochrom) gradient centrifugation, and CD3 + T cells were enriched using magnetic cell separation with CD3 Microbeads (Miltenyi Biotec).

Techniques: Transfection, Flow Cytometry, Expressing, Electroporation, Fluorescence, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Lipid nanoparticle mRNA delivery preserves CAR T cell cytotoxicity and limits exhaustion compared to electroporation

doi: 10.1016/j.omtn.2026.102929

Figure Lengend Snippet: Functional characterization of CD19-CAR mRNA transfected T cells following antigen encounter (A) Experimental workflow of the overnight stimulation assay: mock-transfected and mRNA-transfected CD19-CAR T cells were stimulated with NALM6 (CD19 + ) cells for 16 h in the presence of brefeldin A to capture intracellular cytokine production by flow cytometry. Created with BioRender.com . (B) Representative flow cytometry plots showing activation marker expression (CD137 and CD154) in CD3 + T cells with (stimulated) and without (unstimulated) target cell stimulation. Comparisons are shown for electroporated (left) and LNP-transfected (right) CD19-CAR T cells. (C) Frequencies of activated (CD137 + and/or CD154 + ) CD3 + , CD4 + , and CD8 + T cell populations, comparing electroporation and LNP delivery methods. Data are background-subtracted. (D) Intracellular effector cytokine production (IFN-γ and TNF-α) within activated CD3 + , CD4 + , and CD8 + T cell populations. Data showing the relative increase in cytokine-producing T cells for LNP transfection normalized to electroporation. Statistical analysis was performed by paired t test. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data represent mean ± SD from n = 4 independent donors.

Article Snippet: PBMCs were isolated from healthy donors using Biocoll (Biochrom) gradient centrifugation, and CD3 + T cells were enriched using magnetic cell separation with CD3 Microbeads (Miltenyi Biotec).

Techniques: Functional Assay, Transfection, Flow Cytometry, Activation Assay, Marker, Expressing, Cell Stimulation, Electroporation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Lipid nanoparticle mRNA delivery preserves CAR T cell cytotoxicity and limits exhaustion compared to electroporation

doi: 10.1016/j.omtn.2026.102929

Figure Lengend Snippet: Functional characterization of CD19-CAR T cells produced by LNP-mediated versus electroporation-based mRNA delivery (A) Experimental workflow: electroporated and LNP-transfected T cells were assessed in an in vitro transwell assay, first migrating toward CCL21 for 3 h, followed by co-culture with NALM6 target cells (CD19 + GFP + ). Killing efficiency was monitored over 28 h with 4-h intervals using live-cell imaging. Created with BioRender.com . (B) Killing efficacy of NALM6 (CD19 + GFP + ) target cells using untouched, mock-transfected and CD19-CAR mRNA transfected T cells (EP and LNP) over 28 h assessed using live-cell imaging every 4 h. (C) Area under the curve (AUC) analysis of total killing capacity over the 28-h observation period for the same groups relative to untouched T cells. (D) Fold increase of migrated CD3 + T cells toward CCL21 chemokine normalized to untouched T cells comparing LNP and electroporation delivery methods for mock-transfected and CD19-CAR mRNA transfected T cells analyzed using flow cytometry. (E) Flow cytometric analysis of exhaustion marker expression (TIM-3 and LAG-3) in CD3 + , CD4 + and CD8 + T cell populations following cytotoxic activity of LNP-transfected T cells normalized to electroporated T cells. (F) Mean percentage of CAR T cells expressing zero, one, two, or three exhaustion markers in electroporated T cells (upper) and LNP-transfected CD19-CAR T cells (lower) following cytotoxic activity. (G) Supernatants from migrated T cell killing assays were collected and analyzed for pro-inflammatory cytokines. Mean levels of granzyme B, IFN-γ, TNF-α, IL-8, and IL-10 are shown. Normalized to CD19-CAR frequency for each sample. Statistical analysis for differences between subpopulations was performed by Friedman test followed by Dunn’s multiple comparison test (C, D, and G) or paired t test (E). ∗ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Data represent mean ± SEM from n = 4 independent donors.

Article Snippet: PBMCs were isolated from healthy donors using Biocoll (Biochrom) gradient centrifugation, and CD3 + T cells were enriched using magnetic cell separation with CD3 Microbeads (Miltenyi Biotec).

Techniques: Functional Assay, Produced, Electroporation, Transfection, In Vitro, Transwell Assay, Co-Culture Assay, Live Cell Imaging, Flow Cytometry, Marker, Expressing, Activity Assay, Comparison