Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CAR T cell engineering impacts antigen-independent activation and co-inhibition

doi: 10.1016/j.omtm.2025.101586

Figure Lengend Snippet: Expression patterns of activation and co-inhibition markers in healthy-donor- and CLL-patient-derived CAR T cells by flow cytometry (A) CD69 surface expression in either scFv + ,CD4 + or scFv + ,CD8 + CAR T cells over the course of 4 weeks. Bars represent the mean ± SD; each data point represents an individual donor. Statistics: two-sided t test between LV or SB samples from days 9 and 30. (B) Representative flow cytometry image of target epitope expression in K562 and NALM-6-R110 cells. (C) Pooled results of in vitro co-culture assays indicating the killing of CD19 + , IGLV3-21 R110+ NALM-6-R110 target cells in relation to CD19 − ,IGLV3-21 R110 K562 non-target cells. Bars represent the mean ± SD of pooled experiments. E:T ratios are indicated as symbol color and source donor as symbol shape. (D) Histogram of the PD-1, TIM-3, and LAG-3 expression in CD3 + T cells (HD175) on day 9. (E) Visualization of the CD4, CD8, PD-1, and LAG-3 distribution pattern of CD3 + T cells (HD175) on day 16. Technical duplicate measurements were concatenated and tSNE analysis performed with standard settings.

Article Snippet: Immediately after combining target and effector cells, half of each well was subjected to flow cytometric analysis and the other half incubated for 24 h. Killing of target cells was assessed by gating for viable (DAPI-negative, #130-111-570, Miltenyi Biotec) CD3 − ,CD19 + ,R110 + target NALM-6-R110 cells and by normalizing their percentage after 24-h incubation to 0-h incubation time.

Techniques: Expressing, Activation Assay, Inhibition, Derivative Assay, Flow Cytometry, In Vitro, Co-Culture Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CAR T cell engineering impacts antigen-independent activation and co-inhibition

doi: 10.1016/j.omtm.2025.101586

Figure Lengend Snippet: Single-cell RNA sequencing analysis of CD3 + T cells from SB and LV production pools (A) Uniform manifold and approximation projection (UMAP) plot of CD3 + T cells from the SB and LV production pools. Stacked bar plots display the cluster cell distribution based on CAR T production batches (including UTD control cells) or production mode (SB, sleeping beauty transposition; LV, lentiviral transduction) for all CD3 + T cells and CAR T cells only (scFv + cells). (B) Heatmap of average normalized expression for conventional T cell marker genes in each cluster. (C) UMAP plot showing CD4 , CD8A , and TRDC expression in CD3 + T cells with respect to CAR T production mode. (D) UMAP plot of CD4 + T cells after re-clustering. Expression of T cell markers and selected cluster-defining genes as dot plot grouped by production mode. Dotted lines indicate proliferating T cell clusters. (E) UMAP plot of CD8 + T cells after re-clustering. Expression of T cell markers and selected cluster-defining genes as dot plot grouped by production mode. Dotted lines indicate proliferating T cell clusters. (F) UMAP plot of CD4 + and CD8 + T cell subsets with marked scFv + T cells per production mode. (G) Proportion of scFv + cells per cluster in (F). (H) Violin plots showing expression of marker genes for stem cell-like T memory cells (T scm ) in scFv + cells from the SB and LV production. (I) UMAP plot showing enrichment of gene signature scores for the indicated T cell subpopulations depending on production mode. (J) Dot plot showing the expression of selected T cell exhaustion/activation markers in scFv +/− cells. (K) Dot plot showing the expression of selected T cell exhaustion/activation markers in scFv + CD4 + or CD8 + T cells depending on production mode.

Article Snippet: Immediately after combining target and effector cells, half of each well was subjected to flow cytometric analysis and the other half incubated for 24 h. Killing of target cells was assessed by gating for viable (DAPI-negative, #130-111-570, Miltenyi Biotec) CD3 − ,CD19 + ,R110 + target NALM-6-R110 cells and by normalizing their percentage after 24-h incubation to 0-h incubation time.

Techniques: RNA Sequencing, Control, Transduction, Expressing, Marker, Activation Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CAR T cell engineering impacts antigen-independent activation and co-inhibition

doi: 10.1016/j.omtm.2025.101586

Figure Lengend Snippet: Characterization of production-protocol-specific transcriptomic signatures in CD3 + T cells from SB and LV production pools (A) Volcano plot showing genes differentially expressed (adjusted p < 0.01) between SB and LV production modes for CD4 + scFv + (upper panel) and CD4 + scFv − T cells (lower panel). (B) Gene set enrichment of GSEA/KEGG (upper panel) and GSEA/Hallmark pathways (lower panel) in SB-manufactured scFv + CD4 + T cells as compared to the LV production. (C) Dot plot showing the average normalized expression of genes from the RIG-I receptor signaling gene set in SB- and LV-produced scFv + CD4 + T cells. (D) Volcano plot showing genes differentially expressed (adjusted p < 0.01) between SB and LV production modes for CD8 + scFv + (upper panel) and CD8 + scFv − T cells (lower panel). (E) Enrichment of GSEA/Hallmark pathway genes in SB-manufactured scFv + CD8 + T cells as compared to the LV production. (F) Heatmap displaying surface expression (MFI) of indicated markers in bulk CD3 + or CD4 + and CD8 + T cells as determined by flow cytometry on day 7 for mono-electroporation of SB mRNA, anti-CD19, or anti-R110 CAR DNA constructs. Control T cells underwent electroporation only.

Article Snippet: Immediately after combining target and effector cells, half of each well was subjected to flow cytometric analysis and the other half incubated for 24 h. Killing of target cells was assessed by gating for viable (DAPI-negative, #130-111-570, Miltenyi Biotec) CD3 − ,CD19 + ,R110 + target NALM-6-R110 cells and by normalizing their percentage after 24-h incubation to 0-h incubation time.

Techniques: Expressing, Produced, Flow Cytometry, Electroporation, Construct, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CAR T cell engineering impacts antigen-independent activation and co-inhibition

doi: 10.1016/j.omtm.2025.101586

Figure Lengend Snippet: Analysis of donor-dependent impact on SB- and LV-manufactured CAR T cell transcriptomes (A) UMAP plot of healthy donor (HD)- or CLL-patient-derived CD3 + T cells displaying expression of selected T cell markers. (B) Cell numbers in the indicated subpopulations depending on the donor. (C) Heatmap showing differential expression of genes between HD-/CLL-derived CD3 + T cells. (D) Radar plot displaying the contribution of HD-/CLL-derived CD4 + scFv ± and CD8 + scFv ± T cells to the single subsets in the bulk CD4 + and CD8 + populations from the SB and LV production pools. (E) Violin plot showing expression of indicated exhaustion/co-inhibition markers in HD-/CLL-derived CD4 + and CD8 + scFv + T cells.

Article Snippet: Immediately after combining target and effector cells, half of each well was subjected to flow cytometric analysis and the other half incubated for 24 h. Killing of target cells was assessed by gating for viable (DAPI-negative, #130-111-570, Miltenyi Biotec) CD3 − ,CD19 + ,R110 + target NALM-6-R110 cells and by normalizing their percentage after 24-h incubation to 0-h incubation time.

Techniques: Derivative Assay, Expressing, Quantitative Proteomics, Inhibition