Journal: Molecular Therapy Advances

Article Title: Epigenetic editing balances TCR suppression and persistence in CAR T cells

doi: 10.1016/j.omta.2026.201712

Figure Lengend Snippet: Optimization of epigenetic CD3ε silencing (A) Illustration of the epigenetic editing and analysis workflow in Jurkat cells. (B) Initial results of CRISPRoff v.2-meditated CD3ε silencing in Jurkat cells by antibody staining and flow cytometry at day 4 post transfection. (C) Schematic of the CD3ε genomic promoter region. Upper line represents the promoter region with gray box labeled ‘P’ with an arrow, indicating the promoter and the bigger box indicating the first CD3ε exon. The depiction below shows a zoomed in view on the narrower promoter region, in which functional sgRNAs were identified. Designed sgRNAs ( green ) and designer zinc fingers ( blue ) are indicated as arrows with their numbers labeled. (D) Protein domains and features of optimized epigenetic editor constructs and their names. (E) Barplot of flow cytometry data after CD3ε silencing with EpiE-3 and 20 pmol of different sgRNAs and silencing using sgCD3ε-9 with the optimized concentration of 50 pmol. All bar plots represent the mean of three biological replicates, measured at 4 days post electroporation in Jurkat cells and the error bars show their standard deviation ( p values compared to ‘no’ control sample: ∗∗∗< 0.001, ∗∗ <0.1, ∗ <0.5). (F) Comparison of CD3ε silencing with 50 pmol of sgCD3ε-9 and the optimized epigenetic editors with CRISPRoff-v.2 as benchmark. (G) CD3ε silencing with 1 pmol mRNA of different zinc finger-based epigenetic editors. (H) Cell viability determined by 4',6-diamidino-2-phenylindole (DAPI) staining after CD3ε silencing with all epigenetic editor constructs. (I) Time course of CD3ε silencing in Jurkat cells over 17 days. (J) Illustration of the experimental workflow to test silencing durability in primary T cells under resting (unsupplemented RPMI) or activated (TexMACS medium with TransAct CD3/CD28 and IL-2, 7, and 15) conditions. (K) Epigenetic silencing dynamics in activated and resting primary T cells over 17 days. T cells from two independent donors were edited by conventional CRISPR-Cas9 TRAC knockout or epigenetic CD3ε silencing with optimized conditions (EpiE-3/sg CD3ε-9) and the CD3ε silencing monitored under two distinct conditions. T cells from the ‘activated’ condition were activated 1:500 with TransAct right after electroporation and cultured with IL-2, IL-7, and IL-15 in G-Rex 24 well plates from day 3 post electroporation, allowing for optimal expansion, whereas T cells from the ‘resting’ condition were not TransAct-activated and cultured in RPMI without interleukins and standard 24 well plates from day 3 post electroporation.

Article Snippet: Isolated T cells were stained with fluorescently labeled mAbs against human CD3 (#130-113-138), CD4 (#130-113-225), CD8 (#130-110-683) (Miltenyi Biotec).

Techniques: Staining, Flow Cytometry, Transfection, Labeling, Functional Assay, Zinc-Fingers, Construct, Concentration Assay, Electroporation, Standard Deviation, Control, Comparison, CRISPR, Knock-Out, Cell Culture

Journal: Molecular Therapy Advances

Article Title: Epigenetic editing balances TCR suppression and persistence in CAR T cells

doi: 10.1016/j.omta.2026.201712

Figure Lengend Snippet: Application in primary RevCAR T cells (A) Schematic of CAR T production and editing workflow. After the isolation of CD3+ (mixed CD4+/CD8+) healthy donor-derived T cells on day 0, the cells are activated with TransAct and RevCAR-transduced with a lentivirus. Thereafter, CD3ε was epigenetically silenced by electroporation of sgCD3ε-9 and mRNA of EpiE-3 and expanded for 3 days in a G-Rex 24 Well plate in TexMACS supplemented with IL-2, IL-7, and IL-15. The cells were thereafter rested for 24 h in RPMI without interleukins and subsequently subjected to co-culture assays. (B) Expansion curves of RevCAR T cells after electroporation until start of experiments at day 4. RevCAR T Cells from four independent donors are represented as individual lines for each condition (untreated—no electroporation, mock-electroporation with mCherry mRNA, TCR-KO-electroporation with Cas9 mRNA and TRAC-targeting sgRNA, CD3-EpiE—electroporation with EpiE-3 and sgCD3ε-9). (C) Barplot of flow cytometry data of RevCAR T cells from the four independent donors. Viability was assessed by DAPI staining. In all barplots, the bars represent the mean of T cells from four independent donors ( n = 4), which are individually represented by colored dots and the error bars represent their standard deviation. (D) Barplot of flow cytometry data measuring the RevCAR+ T cell population by EGFP signal intensity. The lentiviral RevCAR construct expresses a RevCAR-T2A-EGFP construct, allowing for an indirect readout. (E) RevCAR+ T cells were determined via EGFP signal and CD3+ T cells by staining with anti-CD3-APC Ab. Representative flow cytometry plots of live RevCAR transduced T cells 4 days post electroporation. Efficiency of CD3ε silencing can be estimated from the x axis and CAR+ percentage from the y axis with the percentages in each quadrant indicated by numbers. One representative sample is displayed for each treatment and the barplot to the right summarizes the data from all four donors ( p values compared to ‘untreated’ control sample: ∗∗∗< 0.001). (F) Volcano plot of RNA-seq data after editing of primary T cells from two independent donors in technical triplicates for each treatment (untreated, TCR-KO, and CD3-EpiE). The data from both donors was analyzed separately. The x axis represents the average log2 fold change of transcript abundance between untreated and TCR-KO or CD3-EpiE samples. The y axis represents the significance (-log10 ( p value)) of these changes across the triplicates. Significantly misregulated genes are classified with cut-off values (log2 fold change >1.5; p value <0.05) as indicated by gray dotted lines. The on-target gene, is highlighted in green, other significantly misregulated genes are highlighted in blue and genes that were found to be significantly misregulated in both donors were highlighted in yellow. Top, shows all data points and bottom shows a zoomed-in view with the y axis capped at -log10 ( p value) = 35, for better resolution and with gene labels.

Article Snippet: Isolated T cells were stained with fluorescently labeled mAbs against human CD3 (#130-113-138), CD4 (#130-113-225), CD8 (#130-110-683) (Miltenyi Biotec).

Techniques: Isolation, Derivative Assay, Transduction, Electroporation, Co-Culture Assay, Flow Cytometry, Staining, Standard Deviation, Construct, Control, RNA Sequencing

Journal: Molecular Therapy Advances

Article Title: Epigenetic editing balances TCR suppression and persistence in CAR T cells

doi: 10.1016/j.omta.2026.201712

Figure Lengend Snippet: Functional validation upon epigenetic CD3ε silencing in vitro and in vivo (A) Schematic representation of the RevCAR construct and its interaction with the target cell. The RevCAR T cells co-expresses eGFP via a T2A. The RevCAR consists of the CD3z activation domain, the CD28 co-stimulatory, transmembrane, and hinge domains and the extracellular E5B9 peptide epitope. A soluble RevTM is required to redirect RevCAR T cells toward cancer cells as it binds to the target cell’s surface molecule, such as PD-L1, on cancer cells and simultaneously to the RevCAR-E5B9 T cells. (B) Cytotoxicity assays of RevCAR T cells toward luciferase-expressing MDA-MB-231 cancer cells with or without PD-L1 RevTM. After a co-culture time of 40 h at an E:T ratio of 5:1, the number of residual MDA cancer cells was determined by an luciferase assay. Experiments were conducted with RevCAR T cells from four independent donors ( n = 4) and three technical replicates. Bars represent the average specific killing, the error bars indicate the standard deviation between replicates donors and colored dots visualize the individual datapoints donors ( p values compared to unedited ‘mock’ sample: ∗∗∗< 0.001). (C) Barplots of investigated flow cytometry panel after 48 h of co-culture of RevCAR T cells with cancer cells with or without PD-L1 RevTM (gray and black bars, respectively). Experiments were conducted with RevCAR T cells from three independent donors ( n = 3) and three technical replicates. Bars represent the average percentage of T cells positive for a respective marker, the error bars indicate their standard deviation and colored dots visualize the individual data points from each replicate and donor. (D) Schematic representation of in vivo experiment timeline. (E) Kaplan-Meier analysis of survival of mice treated with edited T cells. (F) Persistence of T cells in vivo , quantified as chimerism ratio. The chimerism ratio was calculated as the ratio of mouse CD45 + cells to human CD45 + cells as analyzed by flow cytometry from peripheral blood samples. A Mann-Whitney U test was performed to determine statistical significance. (G) Expression of CD3 on human T cells in vivo , quantified on human CD45 + cells as analyzed by flow cytometry from peripheral blood samples. Since TCR-knockout T cells did not persist, no data points could be collected after week 2.

Article Snippet: Isolated T cells were stained with fluorescently labeled mAbs against human CD3 (#130-113-138), CD4 (#130-113-225), CD8 (#130-110-683) (Miltenyi Biotec).

Techniques: Functional Assay, Biomarker Discovery, In Vitro, In Vivo, Construct, Activation Assay, Luciferase, Expressing, Co-Culture Assay, Standard Deviation, Flow Cytometry, Marker, MANN-WHITNEY, Knock-Out

Journal: Respiratory Research

Article Title: HLA-DRA-mediated inhibition of T-Cell proliferation by differentiated airway epithelium and its disruption in smoking-associated airway inflammation

doi: 10.1186/s12931-026-03676-5

Figure Lengend Snippet: Differentiated HAECs suppress pre-activated T cells proliferation. A Schematic of Naïve T cell or Pre-activated T cell being co-cultured with differentiated (ALI day-21) or undifferentiated (ALI day-0) airway epithelium. B Morphology of T cells after 3‑day co‑culture with differentiated HAECs; Pre-activated T cell in ALI co-culture formed fewer and smaller clusters than in monoculture, whereas naïve T cells did not form clusters; scale, 10 µm. C Proliferation of T cells after 3‑day co‑culture with differentiated HAECs, assessed by EdU staining. Naïve T cell (−), Naive T cell + ALI (−), Pre-activated T cell (++), Pre-activated T cell+ALI (−); Red fluorescence represented EdU positive (EdU+) cells; EdU, a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis; Blue fluorescence indicated nuclei stained with DAPI; * proliferation levels are semi‑quantified as: (–), (+) <5%, (++) 5%~10%, (+++)>10%; scale, 10 µm. D Viable T cells number recovered after co‑culture. Data is represented as mean±SD from three independent experiments. *, p <0.05, ****, p <0.0001, ns, not significant. E Morphology of T cells after 3‑day co‑culture with undifferentiated HAECs. The number and size of T cells clusters were similar between Pre-activated T cells in ALI co-culture and monoculture; scale, 10 µm. F Proliferation of T cells after 3‑day co‑culture with undifferentiated HAECs, assessed by EdU staining. Naïve T cell (−), Naive T cell + ALI (−), Pre-activated T cell (+++), Pre-activated T cell+ ALI (+++); scale, 10 µm. G Viable T cells number recovered after co‑culture. Data is represented as mean±SD. *, p <0.05; **, p <0.01; ***, p <0.001, ****, p <0.0001, ns, not significant. Note: Pre-activated T cell: cells were stimulated with α-CD3/α-CD28-coupled beads for 3 days before seeding into ALI co-culture system

Article Snippet: First, the 6-well cell culture plates were precoated with 1 μg/ml anti-CD3 monoclonal antibody (mAb) and 1 μg/ml anti-CD28 mAb (#130-093-387, and #130-093-375, Miltenyi Biotec, Auburn, CA, USA) at 4 °C overnight.

Techniques: Cell Culture, Co-Culture Assay, Staining, Fluorescence, DNA Synthesis