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: Nature Communications

Article Title: NR2F6 deletion revives CAR-T cell function and induces antigen-agnostic immune memory in solid tumors

doi: 10.1038/s41467-026-69796-0

Figure Lengend Snippet: A Experimental setup and timeline. Created in BioRender. Klepsch, V. ( https://BioRender.com/772xola ). Average tumor growth curve ( B ) and survival curve ( C ) in immunodeficient Rag1 −/− recipient mice. Rag1 −/− mice injected subcutaneously with 5 × 10 5 Panc02-EpCAM (100% positive) cells and treated 2 days later with 1 × 10 6 genetically modified 28ζ CAR-T cells using CRISPR/Cas9 (NTC vs Nr2f6 crispr−/− . g04 or Nr2f6 crispr−/− . g03 ). Tumor growth and survival was compared to the no therapy group that received PBS. Tumor volume was measured by caliper. n = 6 mice for Nr2f6 crispr−/−g03 and PBS and n = 5 mice for NTC and Nr2f6 crispr−/− . g04 , representative of two independent experiments. B two-way ANOVA Nr2f6 crispr−/− . g04 vs. Nr2f6 crispr−/− . g03 : ns = p = 0.9889, C overall ns = p = 0.2295, PBS vs Nr2f6 crispr−/− . g04 ns = p = 0.1213. D Experimental setup and timeline for FACS and ELISpot analysis. Ly5.1 mice were used as recipients to track Ly5.2 NTC or Nr2f6 crispr−/−.g03 CAR-T cells. Created in BioRender. Klepsch, V. ( https://BioRender.com/oeq94aw ). E Flow cytometric analysis of splenocytes from three NTC and three Nr2f6 crispr−/−.g03 CAR-T cell-treated wild type tumor-bearing mice on d8 after tumor inoculation (1 × 10 6 Panc02-EpCAM mosaic ). Increased proportion of cDC1 in Nr2f6 crispr−/−.g03 CAR T cell treated animals compared to NTC. cDC1 were gated as exemplified in Supplementary Fig. , n = 3 mice per group. F IL12p40 concentration in serum of NTC and Nr2f6 crispr−/− CAR T treated tumor-bearing mice d6 after ACT determined by LUMINEX technology. Data was normalized to NTC CAR T receiving mice. n = 2 PBS, 3 NTC CAR, and 6 Nr2f6 crispr−/− CAR mice. G , H Granzyme B ELISpot assay. Immunocompetent wild type mice were injected subcutaneously with 1 × 10 6 Panc02-EpCAM mosaic cells and left untreated (PBS) or treated 2 days later with 3.6 × 10 6 genetically modified 28ζ NTC or Nr2f6 crispr−/− . g03 CAR-T cells. Assay was performed with isolated endogenous CD3 + T cells on day 21 after tumor inoculation. NTC versus Nr2f6 crispr−/− . g03 CAR-T. n = 5 mice per group from 2 independent experiments. E – G two-tailed unpaired Student’s t-test. Data shown as mean ± SEM.

Article Snippet: For antigen presentation, mouse bone-marrow-derived dendritic cells (BMDCs) were generated as described elsewhere and either left unstimulated or stimulated with an activating antibody against CD3 (Mabtech, Nacka Strand, Sweden, 3605-1-50) as positive control as well as a set of peptides and lysates.

Techniques: Injection, Genetically Modified, CRISPR, Enzyme-linked Immunospot, Concentration Assay, Luminex, Isolation, Two Tailed Test