Journal: Physiological Reports

Article Title: Sex differences in circulating platelet‐derived CD41 + extracellular vesicles in healthy adults

doi: 10.14814/phy2.70932

Figure Lengend Snippet: Nano flow cytometry measures of MV phenotype in males and females. (a) CD3+ lymphocyte‐derived MVs. (b) CD14+ monocyte‐derived MVs. (c) CD16+ neutrophil‐derived MVs. (d) CD45+ leukocyte‐derived MVs. (e) CD31+ endothelial cell‐derived MVs. (f) CD62E+ endothelial activation‐derived MVs. (g) CD41+ platelet‐derived MVs. (h) MVs expressing Annexin A5. Individual data points are presented as MV counts/μL. Clear circles represent females and dark filled circles represent males. Statistical comparisons between sexes were performed using linear models adjusted for age and BMI. Data are presented as untransformed values for visualization following outlier removal using a 3*SD criterion. Statistical analyses were performed on log‐transformed data where appropriate.

Article Snippet: Panel 2 received 2 μL each of CD3 PE (130‐114‐519, Miltenyi Biotec), CD14 PerCP‐Vio700 (130‐110‐523), CD16 PE‐Vio615 (130‐119‐995), CD31 FITC (130‐110‐668), CD41 APC (130‐123‐301), and 10 μL of CD62E PE (130‐104‐643).

Techniques: Flow Cytometry, Derivative Assay, Activation Assay, Expressing, Transformation Assay

Journal: Nature Medicine

Article Title: Multi-antigen-targeting T cells in pediatric central nervous system tumors: a phase 1 trial

doi: 10.1038/s41591-026-04449-9

Figure Lengend Snippet: Flow immunophenotyping release testing data are presented in A-G and IFNγ ELISpot data presented in H-K (Arm A: n = 12; Arms B/C: n = 27). Each data point represents a separate TAA-T cell product infused into patients (biological replicates). Lower whisker is defined as the minima, upper whisker is defined as the maxima, center is defined as median, lower bound of box is defined as Q1 (25th percentile), and upper bound of box is defined as Q3 (75th percentile). a ) % Total T cells (CD3 + ). Arm A: Minima=51, Maxima=99, Median=97, 25th Percentile=95, 75th Percentile=99; Arms B/C: Minima=31, Maxima=100, Median=97, 25th Percentile=94, 75th Percentile=98. b ) % CD4 + T cells (CD3 + CD4+ of CD45 + ). Arm A: Minima=0.50, Maxima=88, Median=16, 25th Percentile=2.6, 75th Percentile=38; Arms B/C: Minima=0.40, Maxima=55, Median=9.9, 25th Percentile=6.1, 75th Percentile=17. c ) %CD8 + T cells (% CD3 + CD8+ of CD45 + ). Arm A: Minima=4.3, Maxima=88, Median=37, 25th Percentile=22, 75th Percentile=47; Arms B/C: Minima=19, Maxima=91, Median=51, 25th Percentile=36, 75th Percentile=71. d ) % αβ T cells (% TCRαβ+ of CD3 + ). Arm A: Minima=8.2, Maxima=94, Median=69, 25th Percentile=28, 75th Percentile=86; Arms B/C: Minima=32, Maxima=98, Median=71, 25th Percentile=48, 75th Percentile=84. e ) % γδ T cells (% TCRγδ+ of CD3 + ). Arm A: Minima=5.1, Maxima=79, Median=28, 25th Percentile=8.4, 75th Percentile=67; Arms B/C: Minima=1.1, Maxima=83, Median=25, 25th Percentile=10, 75th Percentile=53. f ) % CD3 + CD16 + CD56+ of CD45 + . Arm A: Minima=2.8, Maxima=25, Median=11, 25th Percentile=7.2, 75th Percentile=17; Arms B/C: Minima=3.4, Maxima=64, Median=16, 25th Percentile=8.1, 75th Percentile=28. g ) % NK cells (CD16/CD56 + CD3- of CD45 + ). Arm A: Minima=0.40, Maxima=48, Median=1.0, 25th Percentile=0.48, 75th Percentile=4.6; Arms B/C: Minima=0.10, Maxima=62, Median=1.6, 25th Percentile=0.60, 75th Percentile=6.1. h - i ) IFNγ ELISpot with background correction for Arm A ( h ) and Arms B/C ( i ). Negative values after background normalization have been normalized to zero for visual representation.

Article Snippet: Antibodies for the T/NK panel included CD8 FITC (Miltenyi Biotec, cat. no. 130-110-677), CD16 PE (Miltenyi Biotec, cat. no. 130-113-393), CD56 PE (Miltenyi Biotec, cat. no. 130-113-312), CD3 Per CP Vio-770 (Miltenyi Biotec, cat. no. 130-113-141), CD4 PE-Vio-770 (Miltenyi Biotec, cat. no. 130-113-227) and CD45 APC (Miltenyi Biotec, cat. no. 130-110-633).

Techniques: Enzyme-linked Immunospot, Whisker Assay

Journal: Science Advances

Article Title: Harnessing skin-resident γδ T cells for immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1126/sciadv.aec7215

Figure Lengend Snippet: ( A ) Expression of indicated chemokine receptors by in vitro expanded, live gated CD3 + Vδ1 + γδ T cells. Blood-derived αβ T cells were used as staining control. ( B ) As (A), graphical summary of the percentage of Vδ1 + γδ T cell expressing the chemokine receptors ( n = 5 skin donors). Error bars represent mean ± SD. ( C ) 10 × 10 6 human skin–derived T cells, containing approximately 7% of Vδ1 + γδ T cells, were injected intravenously (i.v.) into NSG mice carrying a xSCC of a volume ranging from 100 to 200 mm 3 [reached approximately 60 to 80 days (d) post–i.d. injection of SCC-13 cells]. Each mouse was injected intraperitoneally (i.p.) with recombinant IL-2 and IL-15 daily until the harvest day. Figure created in BioRender. I. Gratz (2026) https://BioRender.com/zmnd28b . ( D ) Representative plot of the percentage of Vδ1 + γδ T cells engrafted in the spleen, blood, xSCC and murine skin 2, 7, or 14 days posttransfer. ( E to G ) Bar graphs show the absolute numbers of ingoing Vδ1 + γδ T cells, Vδ1 + γδ T cells engrafting spleen and blood normalized to mouse weight (grams), and xSCC and murine skin normalized to tissue weight (grams). (E) n = 7 mice per group; pool of two independent experiments. (F) n = 12 mice per group; pool of four independent experiments; (G) n = 12 mice per group; pool of two independent experiments. Each symbol represents one skin donor. Error bars represent mean ± SD. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons test. All data points, including extreme values, are shown. ( H ) Representative immunofluorescent staining of colocalized TCRδ/DAPI in HD skin and xSCC 7 days after γδ transfer. Scale bars, 100 μm. Staining controls are shown in fig. S4.

Article Snippet: Human skin–derived T lymphocytes were labeled with Cell Proliferation Dye eFluor 450 (10 μM, Thermo Fisher Scientific, catalog no. 65- 0842-85), washed with PBS, and subsequently stimulated for 6 days with rhuIL-2 (100 IU/ml; BioLegend, catalog no. 589108), rhuIL-15 (20 ng/ml; BioLegend, catalog no. 570603), in addition with mouse anti-human CD3 monoclonal antibody (1 μg/ml; Miltenyi Biotec, catalog no. 130-093-387, RRID:AB_1036144) alone or with rhuIL-1α (9 ng/ml; BioLegend, catalog no. 570004) and rhuIL-18 (9 ng/ml, BioLegend, catalog no. 592102) applied individually or in combination in cTexMacs medium.

Techniques: Expressing, In Vitro, Derivative Assay, Staining, Control, Injection, Recombinant

Journal: Science Advances

Article Title: Harnessing skin-resident γδ T cells for immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1126/sciadv.aec7215

Figure Lengend Snippet: ( A ) Levels of cytokines (pg/mg tissue) produced by HD skin, huSCC, ES, and xSCC. Heatmap bars represent the mean of n = 5 HD skin and huSCC donors, and mean n = 5 of xenograft mice. ( B ) Representative gating strategy and bar graphs of the human skin–derived and ex vivo expanded Vδ1 + γδ T cells expressing IL-1RAcP and IL-18Rα. Peripheral blood αβ T cells and ex vivo expanded skin-derived αβ T cells were used as staining controls. Mean of n = 5 skin donors. ( C ) eFluor450-labeled γδ T cells were cultured under basal conditions [unstimulated or with IL-2 (100 IU/ml) and IL-15 (20 ng/ml)] or stimulated with anti-CD3 (1 μg/ml) and/or IL-1α and IL-18 (9 ng/ml) for 6 days. Proliferation was assessed by the median fluorescence intensity (MFI) of eFluor450 in Vδ1 + by flow cytometry. The representative histograms show the eFluor450 dilution in Vδ1 + T cells in the different conditions. Cell counts were normalized to unit area. ( D ) Bar graphs show the fold change of efluor450 MFI of Vδ1 + treated with anti-CD3, IL-1α, and IL-18 relative to IL-2 and IL-15. Mean of n = 6 skin donors. Statistical analysis was performed using a Friedman test followed by Dunn’s multiple comparisons test. Data in bar graphs (B) and (D) are shown as mean ± SD.

Article Snippet: Human skin–derived T lymphocytes were labeled with Cell Proliferation Dye eFluor 450 (10 μM, Thermo Fisher Scientific, catalog no. 65- 0842-85), washed with PBS, and subsequently stimulated for 6 days with rhuIL-2 (100 IU/ml; BioLegend, catalog no. 589108), rhuIL-15 (20 ng/ml; BioLegend, catalog no. 570603), in addition with mouse anti-human CD3 monoclonal antibody (1 μg/ml; Miltenyi Biotec, catalog no. 130-093-387, RRID:AB_1036144) alone or with rhuIL-1α (9 ng/ml; BioLegend, catalog no. 570004) and rhuIL-18 (9 ng/ml, BioLegend, catalog no. 592102) applied individually or in combination in cTexMacs medium.

Techniques: Produced, Derivative Assay, Ex Vivo, Expressing, Staining, Labeling, Cell Culture, Fluorescence, Flow Cytometry

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