mabs against human cd3  (Miltenyi Biotec)

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: Emerging Microbes & Infections

Article Title: Predictive markers of SARS-CoV-2 spike-specific cytotoxic T cell activity following Omicron breakthrough infection

doi: 10.1080/22221751.2025.2602317

Figure Lengend Snippet: Schematic overview of PBMC stimulation and experimental design. Serial PBMC samples were recovered overnight and a fraction applied for 24 h to ELISpot plates with either vehicle (negative control), the full SARS-CoV-2 S peptide pool, anti-CD3 (positive control) or individual peptides to assess circulating S-specific T cell frequency by IFN-γ and/or IL-2 production. Remaining PBMC were stimulated with the full SARS-CoV-2 S peptide pool or individual peptides in the presence of IL-7 with IL-2 added on day 3. On day 7, an aliquot of the cultured cells was restimulated for 5 h with either vehicle, the full SARS-CoV-2 S peptide pool or individual peptides, followed by flow cytometric analysis of individual T cell subsets for IFN-γ and IL-2 production. On day 10, the remaining expanded cells were assessed for CTL activity using a 51 Cr-release assay against autologous BLCL pulsed with vehicle, the full SARS-CoV-2 S peptide pool, or individual peptides selected based on donor HLA typing. A matrix approach was used to deconvolute unknown CTL epitopes using BLCLs pulsed with matrix peptide pools, with confirmation using individual peptides from BEI on days 11-12.

Article Snippet: Effector T cells were stained with directly conjugated mAb against human CD3 (VioGreen, REA613, Miltenyi Biotec), CD4 (APC-Vio770, REA623, Miltenyi Biotec), CD8 (AlexaFluor® 700, HIT8a, BioLegend), CD57 (FITC, NK-1, BD Biosciences, Toronto, ON, Canada), and IFN-γ (APC, 4S.B3, Invitrogen) and IL-2 (PE, MQ1-17H12, Invitrogen) using the Inside Stain Kit (Miltenyi Biotec) as per manufacturer’s instructions and as previously described in [ ].

Techniques: Enzyme-linked Immunospot, Negative Control, Positive Control, Cell Culture, Activity Assay, Release Assay, Immunopeptidomics

Journal: Emerging Microbes & Infections

Article Title: Predictive markers of SARS-CoV-2 spike-specific cytotoxic T cell activity following Omicron breakthrough infection

doi: 10.1080/22221751.2025.2602317

Figure Lengend Snippet: Circulating S-specific SARS-CoV-2 T cell activity. Circulating T cell responses were measured over 24 h in ELISpot assays (in duplicate) using vehicle control, full SARS-CoV-2 S peptide pool, anti-CD3 or individual peptides. Persons with two (PV2; blue, right panel; n = 6) or three (PV3; pink, left panel; n = 22) vaccinations followed by Omicron breakthrough infection (POMI) were tested for (A) IFN-γ, (B) dual IFN-γ/IL-2, and (C) IL-2 SFC/10 6 PBMC. Data are shown as median with IQR (PV3/POMI) and as mean ± SD (PV2/POMI). Groups compared are spanned by lines above them with significant differences ( p value) or non significance (ns) indicated on the line. Wilcoxon matched-pairs signed rank test or Student’s paired t -test was used as appropriate for comparisons based on normality of data distribution.

Article Snippet: Effector T cells were stained with directly conjugated mAb against human CD3 (VioGreen, REA613, Miltenyi Biotec), CD4 (APC-Vio770, REA623, Miltenyi Biotec), CD8 (AlexaFluor® 700, HIT8a, BioLegend), CD57 (FITC, NK-1, BD Biosciences, Toronto, ON, Canada), and IFN-γ (APC, 4S.B3, Invitrogen) and IL-2 (PE, MQ1-17H12, Invitrogen) using the Inside Stain Kit (Miltenyi Biotec) as per manufacturer’s instructions and as previously described in [ ].

Techniques: Activity Assay, Enzyme-linked Immunospot, Control, Infection

Journal: Emerging Microbes & Infections

Article Title: Predictive markers of SARS-CoV-2 spike-specific cytotoxic T cell activity following Omicron breakthrough infection

doi: 10.1080/22221751.2025.2602317

Figure Lengend Snippet: IFN-γ and IL-2 production from expanded SARS-CoV-2 S-specific CD8 + T cells. (A) Representative gating strategy for identifying CD3 + CD8 + T cells. On day 7, expanded T cells were restimulated for 5 h with the full SARS-CoV-2 S peptide pool. Cytokine production was assessed for (B) IFN-γ, (C) dual IFN-γ/IL-2, and (D) IL-2 expression. Background signals from vehicle controls were subtracted before plotting. Data are shown as median with IQR (PV3/POMI n = 22; PV2/POMI n = 6). (E) Correlation between IL-2 production by CD8 + T cells on day 7 and IFN-γ spot-forming units per 10⁶ total T cells on day 1 was assessed. P values in (B-D) were calculated using Wilcoxon matched-pairs signed rank test and are shown above lines spanning the groups compared when significant or as ns when not significant. Significance of correlation in (E) was assessed using Spearman’s correlation.

Article Snippet: Effector T cells were stained with directly conjugated mAb against human CD3 (VioGreen, REA613, Miltenyi Biotec), CD4 (APC-Vio770, REA623, Miltenyi Biotec), CD8 (AlexaFluor® 700, HIT8a, BioLegend), CD57 (FITC, NK-1, BD Biosciences, Toronto, ON, Canada), and IFN-γ (APC, 4S.B3, Invitrogen) and IL-2 (PE, MQ1-17H12, Invitrogen) using the Inside Stain Kit (Miltenyi Biotec) as per manufacturer’s instructions and as previously described in [ ].

Techniques: Expressing

Journal: Immunologic Research

Article Title: Metformin suppresses gammadelta T17 cell differentiation alleviating DSS-induced colitis

doi: 10.1007/s12026-025-09601-0

Figure Lengend Snippet: Metformin suppresses human γδT17 differentiation. A Summarized data shows the UCAI of UC patients ( n = 10). B Summarized data shows the percentages of γδT17 cell in UC patients. PBMCs were freshly isolated with Ficoll reagent and were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) and monensin (2 µg/ml) for 5 h at 37 °C. CD3 + γδTCR + IL-17. + cells were analyzed by FCM. C – E Summarized data shows the correlations of γδT17 cell percentage with IL-17A, IL-23, and fecal calprotectin in UC patients. F Summarized data shows RORC level in γδ T cells. G Summarized data shows IL-17A level in culture medium of γδ T cell. In F and G , γδT cells from healthy donor were treated with Sup for 24 h. RORC was detected by qRT-PCR, and IL-17A level was detected by ELISA. Data represents the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, by one-tailed Student’s t test ( A ), and one-way ANOVA ( B , F , and G ), and by Pearson correlation analysis ( C – E )

Article Snippet: Cytofix/Cytoperm kit and fluorescein-conjugated mAbs against mouse CD3 (LT3), γδTCR (GL3), CD4 (L3T4), IFN-γ (XMG1.2), IL-17A (TC11-18H10), and TCR V4 (UC3-10A6) were from eBioscience.

Techniques: Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: Migratory DCs associate with tumor immune status, patient prognosis, and immunotherapeutic response in patients with cancer (A and B) Representative images of multiplex immunostaining of migratory DC and T cell markers on tumor sections derived from immune-“hot” and “cold” patients with PDAC/NSCLC are given. (C and D) Correlation analysis between migratory DC population and overall survival across our collected PDAC and NSCLC patient cohorts ( n = 66 patients with PDAC, n = 65 patients with NSCLC). (E and F) ROC curves represent the accuracy of migratory DCs and CD3 + CD8 + T cells in predicting overall survival in patients with PDAC and NSCLC. (G and H) Spatial analysis of multiplex IF images from each group. Magnified images are given. (I and J) Stack bar charts show the density of CD3 + CD8 + T cells at varying distances from migratory DCs within both hot and cold tumors from patients with PDAC/NSCLC. (K) Representative images of multiplex immunostaining of migratory DC and T cell markers on tumor sections from NSCLC immunotherapy non-responder and responder. (L) High population of migratory DCs correlated with better progression-free survival in patients with advanced NSCLC treated with anti-PD-1 ( n = 19 patients). (M) ROC curves illustrate the predictive accuracy of migratory DCs and CD3 + CD8 + T cells for progression-free survival in patients with advanced NSCLC treated with anti-PD-1. (N) Spatial proximity analysis of multiplex IF images from each group. Magnified image is given. (O) Stack bar chart illustrates the density of CD3 + CD8 + T cells at different distances from migratory DCs in each group. (C, D, and L) Log rank (Mantel-Cox). Scale bars in (A), (B), and (K) represent 50 μm. See also Figure S1 and Tables S1 , , and .

Article Snippet: rabbit mAb against mouse CD3 , CST , Cat# 78588S; RRID:AB_2889902.

Techniques: Multiplex Assay, Immunostaining, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: cNP cancer cell @MV DC treatment triggers DC maturation and CD8 + T and NK cell activation in vitro (A and B) FACS analysis measured CD86 expression in immature human/mouse DCs after treatments. Bar charts display the percentage of CD11c + CD86 + DCs per group ( n = 3 independent experiments). (C and D) FACS analysis examined human/murine CD3 + CD8 + T cell proliferation after treatments ( n = 3 independent experiments). (E–H) FACS analysis assessed human/murine CD3 + CD8 + T cell activation after treatments. Bar charts show the percentage of CD3 + CD8 + CD69 + T cells or CD3 + CD8 + IFN-γ + T cells per group ( n = 3 independent experiments). (I–L) FACS analysis assessed human/murine NK cell activation after treatments. Graphs show the percentage of CD3 − CD56 + CD69 + , CD3 − NK1.1 + CD69 + , CD3 − CD56 + IFN-γ + , or CD3 − NK1.1 + IFN-γ + relative to the total human/murine NK population in each group ( n = 3 independent experiments). (M and N) Schematic diagrams illustrate LDH release assays conducted on PDAC patient-derived tumor organoids (T, target), using varying ratios of CD3 + CD8 + T cells (E, effector) pre-treated with the indicated treatments (M), or co-cultured with hDCs pre-treated with the indicated treatments (N). Bar charts indicate the killing efficiency percentage at each E:T ratio among different groups ( n = 3 independent experiments). (O and P) Cytotoxicity LDH release assays on DT6066 cells were conducted using different ratios of CD3 + CD8 + T cells that had been either pre-treated with the indicated treatments (O) or co-cultured with DCs pre-treated with the indicated treatments (P) ( n = 3 independent experiments). (A–L) One-way ANOVA. (M–P) Two-way ANOVA. See also Figure S3 .

Article Snippet: rabbit mAb against mouse CD3 , CST , Cat# 78588S; RRID:AB_2889902.

Techniques: Activation Assay, In Vitro, Expressing, Derivative Assay, Cell Culture

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: cNP DT6066 @MV DC treatment converts immune-cold pancreatic tumors into hot tumors, reduces hypoxia, and enhances blood vessel function (A) FACS analysis of CD11c + CD86 + DCs in orthotopic DT6066 pancreatic tumors after treatments, with a bar chart showing their percentage relative to total lymphocytes ( n = 3 mice per group). (B) FACS analysis of CD3 + CD4 + CD69 + T cells in orthotopic pancreatic tumors after treatments, with a bar chart showing their percentage relative to total CD3 + CD4 + T cells in each group ( n = 3 mice per group). (C) FACS analysis of CD3 + CD8 + CD69 + T cells in orthotopic pancreatic tumors after treatments, with a bar chart showing their percentage relative to total CD3 + CD8 + T cells in each group ( n = 3 mice per group). (D) Representative images of CD11c and CD86 co-immunostaining in tumor sections from each treatment group. Bar chart shows the quantification of CD11c + CD86 + DC × 10 3 per cm 2 in each group ( n = 3 mice per group). (E and F) Representative images of co-immunostaining for CD3 and CD4 (E) or CD8 (F) in tumor sections from each treatment group ( n = 3 mice per group). (G) FACS analysis of CD3 − NK1.1 + NK cells or CD3 − NK1.1 + CD69 + NK cells in orthotopic pancreatic tumors after treatments. Bar charts show their percentage relative to total CD3 − cells (left) or CD3 − NK1.1 + NK cells (right) in each group ( n = 3 mice per group). (H–J) Cytotoxicity LDH release assays of DT6066 cells after co-culture with various ratios of CD3 + CD8 + T cells isolated from tumors (H), lymph nodes (I), or spleens (J) of orthotopic DT6066 pancreatic tumor-bearing mice after treatments ( n = 3 mice per group). (K) FACS analysis of migratory DCs in orthotopic pancreatic tumors and TDLNs from mice treated as indicated. Bar charts show CD86 + CD103 + DCs as a percentage of CD11c + DCs in each group ( n = 3 mice per group). (L) Representative images of CD11c/CD86/CD103 triple immunostaining on paired tumor and TDLN sections in each group. Bar chart shows the percentage of CD11c + CD86 + CD103 + migratory DC × 10 3 per cm 2 in tumors (left) or TDLNs (right) from each group ( n = 3 mice per group). (M–O) Representative microbubble contrast ultrasound images are shown, and bar charts show quantification across the entire tumors, including tumor cores ( n = 3 mice per group). (P) Representative IHC staining of endomucin in tumor sections from each treatment group. Bar chart shows blood vessel diameter (left) or number of blood vessels (right) per cm 2 in each group ( n = 3 mice per group). (Q) Representative image of GLUT1 and endomucin co-immunostaining in tumor sections from each group. Bar chart shows the relative GLUT1 intensity per group ( n = 3 mice per group). (A–G, K, L, and N–Q) One-way ANOVA. (H–J) Two-way ANOVA. Scale bars in (D), (E), (F), and (L) represent 50 μm. (M) 1 cm. (P and Q) 100 μm. See also Figures S5 and .

Article Snippet: rabbit mAb against mouse CD3 , CST , Cat# 78588S; RRID:AB_2889902.

Techniques: Immunostaining, Co-Culture Assay, Isolation, Triple Immunostaining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet:

Article Snippet: rabbit mAb against mouse CD3 , CST , Cat# 78588S; RRID:AB_2889902.

Techniques: Virus, Recombinant, Negative Staining, Lysis, Control, Phospho-proteomics, Cell Isolation, DNA Purification, DNA Extraction, Bicinchoninic Acid Protein Assay, Protein Purification, Magnetic Beads, Software

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: Migratory DCs associate with tumor immune status, patient prognosis, and immunotherapeutic response in patients with cancer (A and B) Representative images of multiplex immunostaining of migratory DC and T cell markers on tumor sections derived from immune-“hot” and “cold” patients with PDAC/NSCLC are given. (C and D) Correlation analysis between migratory DC population and overall survival across our collected PDAC and NSCLC patient cohorts ( n = 66 patients with PDAC, n = 65 patients with NSCLC). (E and F) ROC curves represent the accuracy of migratory DCs and CD3 + CD8 + T cells in predicting overall survival in patients with PDAC and NSCLC. (G and H) Spatial analysis of multiplex IF images from each group. Magnified images are given. (I and J) Stack bar charts show the density of CD3 + CD8 + T cells at varying distances from migratory DCs within both hot and cold tumors from patients with PDAC/NSCLC. (K) Representative images of multiplex immunostaining of migratory DC and T cell markers on tumor sections from NSCLC immunotherapy non-responder and responder. (L) High population of migratory DCs correlated with better progression-free survival in patients with advanced NSCLC treated with anti-PD-1 ( n = 19 patients). (M) ROC curves illustrate the predictive accuracy of migratory DCs and CD3 + CD8 + T cells for progression-free survival in patients with advanced NSCLC treated with anti-PD-1. (N) Spatial proximity analysis of multiplex IF images from each group. Magnified image is given. (O) Stack bar chart illustrates the density of CD3 + CD8 + T cells at different distances from migratory DCs in each group. (C, D, and L) Log rank (Mantel-Cox). Scale bars in (A), (B), and (K) represent 50 μm. See also Figure S1 and Tables S1 , , and .

Article Snippet: rabbit mAb against human CD3 , CST , Cat# 85061; RRID:AB_2721019.

Techniques: Multiplex Assay, Immunostaining, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: cNP cancer cell @MV DC treatment triggers DC maturation and CD8 + T and NK cell activation in vitro (A and B) FACS analysis measured CD86 expression in immature human/mouse DCs after treatments. Bar charts display the percentage of CD11c + CD86 + DCs per group ( n = 3 independent experiments). (C and D) FACS analysis examined human/murine CD3 + CD8 + T cell proliferation after treatments ( n = 3 independent experiments). (E–H) FACS analysis assessed human/murine CD3 + CD8 + T cell activation after treatments. Bar charts show the percentage of CD3 + CD8 + CD69 + T cells or CD3 + CD8 + IFN-γ + T cells per group ( n = 3 independent experiments). (I–L) FACS analysis assessed human/murine NK cell activation after treatments. Graphs show the percentage of CD3 − CD56 + CD69 + , CD3 − NK1.1 + CD69 + , CD3 − CD56 + IFN-γ + , or CD3 − NK1.1 + IFN-γ + relative to the total human/murine NK population in each group ( n = 3 independent experiments). (M and N) Schematic diagrams illustrate LDH release assays conducted on PDAC patient-derived tumor organoids (T, target), using varying ratios of CD3 + CD8 + T cells (E, effector) pre-treated with the indicated treatments (M), or co-cultured with hDCs pre-treated with the indicated treatments (N). Bar charts indicate the killing efficiency percentage at each E:T ratio among different groups ( n = 3 independent experiments). (O and P) Cytotoxicity LDH release assays on DT6066 cells were conducted using different ratios of CD3 + CD8 + T cells that had been either pre-treated with the indicated treatments (O) or co-cultured with DCs pre-treated with the indicated treatments (P) ( n = 3 independent experiments). (A–L) One-way ANOVA. (M–P) Two-way ANOVA. See also Figure S3 .

Article Snippet: rabbit mAb against human CD3 , CST , Cat# 85061; RRID:AB_2721019.

Techniques: Activation Assay, In Vitro, Expressing, Derivative Assay, Cell Culture

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: cNP DT6066 @MV DC treatment converts immune-cold pancreatic tumors into hot tumors, reduces hypoxia, and enhances blood vessel function (A) FACS analysis of CD11c + CD86 + DCs in orthotopic DT6066 pancreatic tumors after treatments, with a bar chart showing their percentage relative to total lymphocytes ( n = 3 mice per group). (B) FACS analysis of CD3 + CD4 + CD69 + T cells in orthotopic pancreatic tumors after treatments, with a bar chart showing their percentage relative to total CD3 + CD4 + T cells in each group ( n = 3 mice per group). (C) FACS analysis of CD3 + CD8 + CD69 + T cells in orthotopic pancreatic tumors after treatments, with a bar chart showing their percentage relative to total CD3 + CD8 + T cells in each group ( n = 3 mice per group). (D) Representative images of CD11c and CD86 co-immunostaining in tumor sections from each treatment group. Bar chart shows the quantification of CD11c + CD86 + DC × 10 3 per cm 2 in each group ( n = 3 mice per group). (E and F) Representative images of co-immunostaining for CD3 and CD4 (E) or CD8 (F) in tumor sections from each treatment group ( n = 3 mice per group). (G) FACS analysis of CD3 − NK1.1 + NK cells or CD3 − NK1.1 + CD69 + NK cells in orthotopic pancreatic tumors after treatments. Bar charts show their percentage relative to total CD3 − cells (left) or CD3 − NK1.1 + NK cells (right) in each group ( n = 3 mice per group). (H–J) Cytotoxicity LDH release assays of DT6066 cells after co-culture with various ratios of CD3 + CD8 + T cells isolated from tumors (H), lymph nodes (I), or spleens (J) of orthotopic DT6066 pancreatic tumor-bearing mice after treatments ( n = 3 mice per group). (K) FACS analysis of migratory DCs in orthotopic pancreatic tumors and TDLNs from mice treated as indicated. Bar charts show CD86 + CD103 + DCs as a percentage of CD11c + DCs in each group ( n = 3 mice per group). (L) Representative images of CD11c/CD86/CD103 triple immunostaining on paired tumor and TDLN sections in each group. Bar chart shows the percentage of CD11c + CD86 + CD103 + migratory DC × 10 3 per cm 2 in tumors (left) or TDLNs (right) from each group ( n = 3 mice per group). (M–O) Representative microbubble contrast ultrasound images are shown, and bar charts show quantification across the entire tumors, including tumor cores ( n = 3 mice per group). (P) Representative IHC staining of endomucin in tumor sections from each treatment group. Bar chart shows blood vessel diameter (left) or number of blood vessels (right) per cm 2 in each group ( n = 3 mice per group). (Q) Representative image of GLUT1 and endomucin co-immunostaining in tumor sections from each group. Bar chart shows the relative GLUT1 intensity per group ( n = 3 mice per group). (A–G, K, L, and N–Q) One-way ANOVA. (H–J) Two-way ANOVA. Scale bars in (D), (E), (F), and (L) represent 50 μm. (M) 1 cm. (P and Q) 100 μm. See also Figures S5 and .

Article Snippet: rabbit mAb against human CD3 , CST , Cat# 85061; RRID:AB_2721019.

Techniques: Immunostaining, Co-Culture Assay, Isolation, Triple Immunostaining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet:

Article Snippet: rabbit mAb against human CD3 , CST , Cat# 85061; RRID:AB_2721019.

Techniques: Virus, Recombinant, Negative Staining, Lysis, Control, Phospho-proteomics, Cell Isolation, DNA Purification, DNA Extraction, Bicinchoninic Acid Protein Assay, Protein Purification, Magnetic Beads, Software