Journal: bioRxiv

Article Title: Transient immune landscape remodelling shapes CD8 T-cell priming during infection

doi: 10.64898/2026.03.18.712682

Figure Lengend Snippet: (A-B) Graphs show XCR1 (A) and SIRPa (B) signal density within innate-infiltrated and non-infiltrated regions on stained spleen sections harvested 24hrs after LM-OVA infection (n=5). C- Mice were adoptively transferred with OTIxGFP T-cells and infected with LM-OVA. After 24hrs, spleen sections were stained for CD169 (red), 33D1 (magenta), XCR1 (cyan), OTI (green). Representative image. (D-F) Mice were infected with LM-OVA for 24hrs and splenocytes were analysed by flow cytometry. Graphs show CD80 MFI ( D , n=5-7), CD86 MFI ( E , n=5-7), and frequency of CD70+ cDC1 and cDC2 ( F , n=4-5). (G-H) Spleen sections from mice infected with LM-OVA for 24hrs were stained for CD169 (blue), CD70 (red), CD11b (white). G- Representative image. H- Graph shows CD70 signal intensity within innate-infiltrated and non-infiltrated regions in WPs (n=5). I-K Mice were infected with LM-OVA and treated with anti-CXCR3 or anti-CD70 antibodies as indicated 6hrs before infection. Graphs show frequencies (I) , absolute numbers (J) and avidity (K) of N4-tetramer+ CD8 T-cells 8 days post-infection (n=12-17 per group). Ratio paired t-tests (A-B, D-F, H) , Welch and Brown-Forsythe one-way ANOVA with Dunnett’s multiple comparisons test (I-K) .

Article Snippet: For CD70 blockade, mice were intraperitoneally administered 300μg anti-CD70 antibody (BioXCell, BE0022) or isotype control antibody (BioXcell, BE0290) 6hr before infection, either alone or in combination with CXCR3 blockade.

Techniques: Staining, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Transient immune landscape remodelling shapes CD8 T-cell priming during infection

doi: 10.64898/2026.03.18.712682

Figure Lengend Snippet: (A-B) Representative image of spleen sections from mice infected with LM-OVA for 24hrs were stained for CD169 (blue), XCR1 (cyan), NKp46 (red) and SIRPa ( A , magenta), or 33D1 ( B , magenta). C- Mice were adoptively transferred with OTIxGFP cells and infected with LM-OVA. Representative image of spleen sections stained for CD169 (red), 33D1 (magenta), XCR1 (cyan), OTI (green) 24hrs post-infection. (D-E) Splenocytes from mice infected with LM-OVA for 24hrs were analysed by flow cytometry. D- Representative gating strategy for splenic cDC1 and cDC2 subsets. E- Representative histograms showing CD80, CD86, and CD70 expression in cDC1 and cDC2.

Article Snippet: For CD70 blockade, mice were intraperitoneally administered 300μg anti-CD70 antibody (BioXCell, BE0022) or isotype control antibody (BioXcell, BE0290) 6hr before infection, either alone or in combination with CXCR3 blockade.

Techniques: Infection, Staining, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Validation of CD70 as a GB target for CAR-T cell therapy (A) Expression of CD70 and selected markers from an in-house glioma patient single-cell RNA seq dataset. (B) CD70 expression in GB samples from Heidelberg University Hospital, analyzed by bulk RNAseq. Each dot represents a patient. A Mann-Whitney test was performed to assess statistical significance. (C) CD70 expression in 20 matched pairs from (B). A two-tailed paired t test was used to assess significance. (D) IHC staining of three Heidelberg University Hospital GB patients from (B) with high CD70 expression based on RNAseq. Scale bars, 200 μm. Patient-1 and Patient-3: primary GB; Patient-2: recurrent GB. (E) Measurement of CD70 protein levels on GB cells by flow cytometry. (F) Measurement of CD70 gene expression levels in glioma cell lines by RT-qPCR. N = 3 technical replicates per cell line. (G) Correlation between CD70 gene expression and protein levels from (E) and (F). (H) Measurement of CD70 on the surface of generated primary GB models by flow cytometry (blue histograms). An isotype control antibody (red histograms) was used. For (E) and (H), data were gated on single live cells. Data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: Biomarker Discovery, Expressing, Single Cell, RNA Sequencing, RNA sequencing, MANN-WHITNEY, Two Tailed Test, Immunohistochemistry, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Generated, Control

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Generation and phenotyping of anti-CD70 CAR-Tcells (A) CAR construct design. (B) Transduction efficiency of primary human T cells by flow cytometry. A non-transduced (NT) sample from each donor was used to determine gating. Data gated on single live CD3 + cells. (C) CD8a/CD4 expression on transduced T cells, determined by flow cytometry. A one-way ANOVA followed by a Dunnett’s multiple comparisons test was performed to assess significance. (D) Expression of exhaustion receptors on transduced T cells, determined by flow cytometry. (E) Quantification of PD1 + /LAG3 + /TIM3 + T cells from (D). (F) Expression of immune memory-associated markers on transduced T cells by flow cytometry. For (C), (D), (E), and (F), N = 3 biological replicates per group. Isotype controls were used to determine gating and displayed data are gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Data presented as mean (SD). A one-way ANOVA followed by a post-hoc Šídák’s multiple comparisons test was performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: Construct, Transduction, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Assessment of anti-CD70 CAR-T cell killing in naturally CD70 expressing cell lines (A) CD70 expression levels (blue histogram) on primary GB cell line MMKI by flow cytometry. Data gated on single live cells. An isotype control (red histogram) was used. (B) Quantification of activation marker CD137 after O/N co-culture of MMKI cells with CD70-targeting CAR-T cells by flow cytometry. N = 4 biological replicates per group. Data gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Isotype control antibodies were used for gating. A one-way ANOVA followed by a Tukey’s multiple comparisons test was applied for significance. (C) Measurement of CD70 (blue histograms) on the surface of generated genetic knockout models by flow cytometry. An isotype control (red histogram) was used. Data gated on single live EGFP + cells. (D) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. Statistical significance was assessed as in B). For (B) and (D), data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: Expressing, Flow Cytometry, Control, Activation Assay, Marker, Co-Culture Assay, Generated, Knock-Out

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vivo evaluation of the anti-CD70 CAR-T cell killing efficacy (A) In vivo pipeline. (B) Measurement of tumor cell signal in mice orthotopically implanted with P3/CD70_NLuc/EGFP cells and subsequently treated ICT with anti-CD70 CAR-T cells by BLI. (C) Quantification of BLI signals from B). N = 8 animals per group. (D) Overall survival of treated mice from B). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0083. (E) Analysis of CAR-T cell persistence and CD70 expression in recurrent tumors of treated mice from (A) by IF. Representative images from N = 3 animals per group. Scale bars, 100 μm.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: In Vivo, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based CAR constructs lead to regression of syngeneic GB tumors in vivo (A) Schematic representation of the syngeneic model in vivo experiment. (B) Evaluation of mCAR expression on transduced murine T cells on treatment day by flow cytometry. A non-transduced sample was used to determine gating. Data gated on single live mCD3 + cells. (C) Weekly tumor volume evaluation in C57BL/6J mice orthotopically implanted with GL261/CD70 cells and subsequently treated ICT with anti-mCD70 CAR-T cells by MRI. A one-way ANOVA with a Dunnett’s multiple comparisons test was used to assess significance. (D) Indicative T2-weighed MRI sections (coronal plane) from N = 3 mice of each treatment group from (C), before and after treatment with anti-mCD70 CAR-T cells. (E) Comparison of tumor volumes among treatment groups from (C). For day-14, a one-way ANOVA followed by a Tukey’s multiple comparisons test was used. For day-21, a Welch’s ANOVA followed by a Dunnett’s T3 multiple comparisons test was used. For day-28, a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used. Data presented as mean (SD). (F) Overall survival of treated mice from (C). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0166. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: For cloning of the lentiviral CD70 knockout construct, the LentiCRISPRv2GFP was a gift from David Feldser (Addgene #82416; http://n2t.net/addgene:82416 ; RRID:Addgene_82416).

Techniques: Construct, In Vivo, Expressing, Flow Cytometry, Comparison

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Validation of CD70 as a GB target for CAR-T cell therapy (A) Expression of CD70 and selected markers from an in-house glioma patient single-cell RNA seq dataset. (B) CD70 expression in GB samples from Heidelberg University Hospital, analyzed by bulk RNAseq. Each dot represents a patient. A Mann-Whitney test was performed to assess statistical significance. (C) CD70 expression in 20 matched pairs from (B). A two-tailed paired t test was used to assess significance. (D) IHC staining of three Heidelberg University Hospital GB patients from (B) with high CD70 expression based on RNAseq. Scale bars, 200 μm. Patient-1 and Patient-3: primary GB; Patient-2: recurrent GB. (E) Measurement of CD70 protein levels on GB cells by flow cytometry. (F) Measurement of CD70 gene expression levels in glioma cell lines by RT-qPCR. N = 3 technical replicates per cell line. (G) Correlation between CD70 gene expression and protein levels from (E) and (F). (H) Measurement of CD70 on the surface of generated primary GB models by flow cytometry (blue histograms). An isotype control antibody (red histograms) was used. For (E) and (H), data were gated on single live cells. Data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Biomarker Discovery, Expressing, Single Cell, RNA Sequencing, RNA sequencing, MANN-WHITNEY, Two Tailed Test, Immunohistochemistry, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Generated, Control

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Generation and phenotyping of anti-CD70 CAR-Tcells (A) CAR construct design. (B) Transduction efficiency of primary human T cells by flow cytometry. A non-transduced (NT) sample from each donor was used to determine gating. Data gated on single live CD3 + cells. (C) CD8a/CD4 expression on transduced T cells, determined by flow cytometry. A one-way ANOVA followed by a Dunnett’s multiple comparisons test was performed to assess significance. (D) Expression of exhaustion receptors on transduced T cells, determined by flow cytometry. (E) Quantification of PD1 + /LAG3 + /TIM3 + T cells from (D). (F) Expression of immune memory-associated markers on transduced T cells by flow cytometry. For (C), (D), (E), and (F), N = 3 biological replicates per group. Isotype controls were used to determine gating and displayed data are gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Data presented as mean (SD). A one-way ANOVA followed by a post-hoc Šídák’s multiple comparisons test was performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Construct, Transduction, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Assessment of anti-CD70 CAR-T cell killing in naturally CD70 expressing cell lines (A) CD70 expression levels (blue histogram) on primary GB cell line MMKI by flow cytometry. Data gated on single live cells. An isotype control (red histogram) was used. (B) Quantification of activation marker CD137 after O/N co-culture of MMKI cells with CD70-targeting CAR-T cells by flow cytometry. N = 4 biological replicates per group. Data gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Isotype control antibodies were used for gating. A one-way ANOVA followed by a Tukey’s multiple comparisons test was applied for significance. (C) Measurement of CD70 (blue histograms) on the surface of generated genetic knockout models by flow cytometry. An isotype control (red histogram) was used. Data gated on single live EGFP + cells. (D) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. Statistical significance was assessed as in B). For (B) and (D), data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Expressing, Flow Cytometry, Control, Activation Assay, Marker, Co-Culture Assay, Generated, Knock-Out

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vivo evaluation of the anti-CD70 CAR-T cell killing efficacy (A) In vivo pipeline. (B) Measurement of tumor cell signal in mice orthotopically implanted with P3/CD70_NLuc/EGFP cells and subsequently treated ICT with anti-CD70 CAR-T cells by BLI. (C) Quantification of BLI signals from B). N = 8 animals per group. (D) Overall survival of treated mice from B). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0083. (E) Analysis of CAR-T cell persistence and CD70 expression in recurrent tumors of treated mice from (A) by IF. Representative images from N = 3 animals per group. Scale bars, 100 μm.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vivo, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based CAR constructs lead to regression of syngeneic GB tumors in vivo (A) Schematic representation of the syngeneic model in vivo experiment. (B) Evaluation of mCAR expression on transduced murine T cells on treatment day by flow cytometry. A non-transduced sample was used to determine gating. Data gated on single live mCD3 + cells. (C) Weekly tumor volume evaluation in C57BL/6J mice orthotopically implanted with GL261/CD70 cells and subsequently treated ICT with anti-mCD70 CAR-T cells by MRI. A one-way ANOVA with a Dunnett’s multiple comparisons test was used to assess significance. (D) Indicative T2-weighed MRI sections (coronal plane) from N = 3 mice of each treatment group from (C), before and after treatment with anti-mCD70 CAR-T cells. (E) Comparison of tumor volumes among treatment groups from (C). For day-14, a one-way ANOVA followed by a Tukey’s multiple comparisons test was used. For day-21, a Welch’s ANOVA followed by a Dunnett’s T3 multiple comparisons test was used. For day-28, a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used. Data presented as mean (SD). (F) Overall survival of treated mice from (C). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0166. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Construct, In Vivo, Expressing, Flow Cytometry, Comparison

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Validation of CD70 as a GB target for CAR-T cell therapy (A) Expression of CD70 and selected markers from an in-house glioma patient single-cell RNA seq dataset. (B) CD70 expression in GB samples from Heidelberg University Hospital, analyzed by bulk RNAseq. Each dot represents a patient. A Mann-Whitney test was performed to assess statistical significance. (C) CD70 expression in 20 matched pairs from (B). A two-tailed paired t test was used to assess significance. (D) IHC staining of three Heidelberg University Hospital GB patients from (B) with high CD70 expression based on RNAseq. Scale bars, 200 μm. Patient-1 and Patient-3: primary GB; Patient-2: recurrent GB. (E) Measurement of CD70 protein levels on GB cells by flow cytometry. (F) Measurement of CD70 gene expression levels in glioma cell lines by RT-qPCR. N = 3 technical replicates per cell line. (G) Correlation between CD70 gene expression and protein levels from (E) and (F). (H) Measurement of CD70 on the surface of generated primary GB models by flow cytometry (blue histograms). An isotype control antibody (red histograms) was used. For (E) and (H), data were gated on single live cells. Data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Biomarker Discovery, Expressing, Single Cell, RNA Sequencing, RNA sequencing, MANN-WHITNEY, Two Tailed Test, Immunohistochemistry, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Generated, Control

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Generation and phenotyping of anti-CD70 CAR-Tcells (A) CAR construct design. (B) Transduction efficiency of primary human T cells by flow cytometry. A non-transduced (NT) sample from each donor was used to determine gating. Data gated on single live CD3 + cells. (C) CD8a/CD4 expression on transduced T cells, determined by flow cytometry. A one-way ANOVA followed by a Dunnett’s multiple comparisons test was performed to assess significance. (D) Expression of exhaustion receptors on transduced T cells, determined by flow cytometry. (E) Quantification of PD1 + /LAG3 + /TIM3 + T cells from (D). (F) Expression of immune memory-associated markers on transduced T cells by flow cytometry. For (C), (D), (E), and (F), N = 3 biological replicates per group. Isotype controls were used to determine gating and displayed data are gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Data presented as mean (SD). A one-way ANOVA followed by a post-hoc Šídák’s multiple comparisons test was performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Construct, Transduction, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Assessment of anti-CD70 CAR-T cell killing in naturally CD70 expressing cell lines (A) CD70 expression levels (blue histogram) on primary GB cell line MMKI by flow cytometry. Data gated on single live cells. An isotype control (red histogram) was used. (B) Quantification of activation marker CD137 after O/N co-culture of MMKI cells with CD70-targeting CAR-T cells by flow cytometry. N = 4 biological replicates per group. Data gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Isotype control antibodies were used for gating. A one-way ANOVA followed by a Tukey’s multiple comparisons test was applied for significance. (C) Measurement of CD70 (blue histograms) on the surface of generated genetic knockout models by flow cytometry. An isotype control (red histogram) was used. Data gated on single live EGFP + cells. (D) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. Statistical significance was assessed as in B). For (B) and (D), data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Expressing, Flow Cytometry, Control, Activation Assay, Marker, Co-Culture Assay, Generated, Knock-Out

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vivo evaluation of the anti-CD70 CAR-T cell killing efficacy (A) In vivo pipeline. (B) Measurement of tumor cell signal in mice orthotopically implanted with P3/CD70_NLuc/EGFP cells and subsequently treated ICT with anti-CD70 CAR-T cells by BLI. (C) Quantification of BLI signals from B). N = 8 animals per group. (D) Overall survival of treated mice from B). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0083. (E) Analysis of CAR-T cell persistence and CD70 expression in recurrent tumors of treated mice from (A) by IF. Representative images from N = 3 animals per group. Scale bars, 100 μm.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: In Vivo, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based CAR constructs lead to regression of syngeneic GB tumors in vivo (A) Schematic representation of the syngeneic model in vivo experiment. (B) Evaluation of mCAR expression on transduced murine T cells on treatment day by flow cytometry. A non-transduced sample was used to determine gating. Data gated on single live mCD3 + cells. (C) Weekly tumor volume evaluation in C57BL/6J mice orthotopically implanted with GL261/CD70 cells and subsequently treated ICT with anti-mCD70 CAR-T cells by MRI. A one-way ANOVA with a Dunnett’s multiple comparisons test was used to assess significance. (D) Indicative T2-weighed MRI sections (coronal plane) from N = 3 mice of each treatment group from (C), before and after treatment with anti-mCD70 CAR-T cells. (E) Comparison of tumor volumes among treatment groups from (C). For day-14, a one-way ANOVA followed by a Tukey’s multiple comparisons test was used. For day-21, a Welch’s ANOVA followed by a Dunnett’s T3 multiple comparisons test was used. For day-28, a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used. Data presented as mean (SD). (F) Overall survival of treated mice from (C). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0166. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Construct, In Vivo, Expressing, Flow Cytometry, Comparison

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Validation of CD70 as a GB target for CAR-T cell therapy (A) Expression of CD70 and selected markers from an in-house glioma patient single-cell RNA seq dataset. (B) CD70 expression in GB samples from Heidelberg University Hospital, analyzed by bulk RNAseq. Each dot represents a patient. A Mann-Whitney test was performed to assess statistical significance. (C) CD70 expression in 20 matched pairs from (B). A two-tailed paired t test was used to assess significance. (D) IHC staining of three Heidelberg University Hospital GB patients from (B) with high CD70 expression based on RNAseq. Scale bars, 200 μm. Patient-1 and Patient-3: primary GB; Patient-2: recurrent GB. (E) Measurement of CD70 protein levels on GB cells by flow cytometry. (F) Measurement of CD70 gene expression levels in glioma cell lines by RT-qPCR. N = 3 technical replicates per cell line. (G) Correlation between CD70 gene expression and protein levels from (E) and (F). (H) Measurement of CD70 on the surface of generated primary GB models by flow cytometry (blue histograms). An isotype control antibody (red histograms) was used. For (E) and (H), data were gated on single live cells. Data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Biomarker Discovery, Expressing, Single Cell, RNA Sequencing, RNA sequencing, MANN-WHITNEY, Two Tailed Test, Immunohistochemistry, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Generated, Control

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Generation and phenotyping of anti-CD70 CAR-Tcells (A) CAR construct design. (B) Transduction efficiency of primary human T cells by flow cytometry. A non-transduced (NT) sample from each donor was used to determine gating. Data gated on single live CD3 + cells. (C) CD8a/CD4 expression on transduced T cells, determined by flow cytometry. A one-way ANOVA followed by a Dunnett’s multiple comparisons test was performed to assess significance. (D) Expression of exhaustion receptors on transduced T cells, determined by flow cytometry. (E) Quantification of PD1 + /LAG3 + /TIM3 + T cells from (D). (F) Expression of immune memory-associated markers on transduced T cells by flow cytometry. For (C), (D), (E), and (F), N = 3 biological replicates per group. Isotype controls were used to determine gating and displayed data are gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Data presented as mean (SD). A one-way ANOVA followed by a post-hoc Šídák’s multiple comparisons test was performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Construct, Transduction, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Assessment of anti-CD70 CAR-T cell killing in naturally CD70 expressing cell lines (A) CD70 expression levels (blue histogram) on primary GB cell line MMKI by flow cytometry. Data gated on single live cells. An isotype control (red histogram) was used. (B) Quantification of activation marker CD137 after O/N co-culture of MMKI cells with CD70-targeting CAR-T cells by flow cytometry. N = 4 biological replicates per group. Data gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Isotype control antibodies were used for gating. A one-way ANOVA followed by a Tukey’s multiple comparisons test was applied for significance. (C) Measurement of CD70 (blue histograms) on the surface of generated genetic knockout models by flow cytometry. An isotype control (red histogram) was used. Data gated on single live EGFP + cells. (D) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. Statistical significance was assessed as in B). For (B) and (D), data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Expressing, Flow Cytometry, Control, Activation Assay, Marker, Co-Culture Assay, Generated, Knock-Out

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vivo evaluation of the anti-CD70 CAR-T cell killing efficacy (A) In vivo pipeline. (B) Measurement of tumor cell signal in mice orthotopically implanted with P3/CD70_NLuc/EGFP cells and subsequently treated ICT with anti-CD70 CAR-T cells by BLI. (C) Quantification of BLI signals from B). N = 8 animals per group. (D) Overall survival of treated mice from B). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0083. (E) Analysis of CAR-T cell persistence and CD70 expression in recurrent tumors of treated mice from (A) by IF. Representative images from N = 3 animals per group. Scale bars, 100 μm.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vivo, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based CAR constructs lead to regression of syngeneic GB tumors in vivo (A) Schematic representation of the syngeneic model in vivo experiment. (B) Evaluation of mCAR expression on transduced murine T cells on treatment day by flow cytometry. A non-transduced sample was used to determine gating. Data gated on single live mCD3 + cells. (C) Weekly tumor volume evaluation in C57BL/6J mice orthotopically implanted with GL261/CD70 cells and subsequently treated ICT with anti-mCD70 CAR-T cells by MRI. A one-way ANOVA with a Dunnett’s multiple comparisons test was used to assess significance. (D) Indicative T2-weighed MRI sections (coronal plane) from N = 3 mice of each treatment group from (C), before and after treatment with anti-mCD70 CAR-T cells. (E) Comparison of tumor volumes among treatment groups from (C). For day-14, a one-way ANOVA followed by a Tukey’s multiple comparisons test was used. For day-21, a Welch’s ANOVA followed by a Dunnett’s T3 multiple comparisons test was used. For day-28, a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used. Data presented as mean (SD). (F) Overall survival of treated mice from (C). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0166. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: Gene expression quantification was achieved on a QuantStudio-3 real-time PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) and TaqMan probes targeting human CD70 (assay ID: Hs00174297_m1, Applied Biosystems) and human GAPDH (assay ID: Hs99999905_m1, Applied Biosystems), or mouse CD70 (assay ID: Mm00441914_m1, Applied Biosystems) and mouse GAPDH (assay ID: Mm99999915_g1, Applied Biosystems).

Techniques: Construct, In Vivo, Expressing, Flow Cytometry, Comparison

Journal: Blood Cancer Discovery

Article Title: CD70-Targeting CAR NK Cells Overcome BCMA Downregulation and Improve Survival in High-risk Multiple Myeloma Models

doi: 10.1158/2643-3230.BCD-25-0130

Figure Lengend Snippet: CD70 expression in patients with multiple myeloma. Comparison of CD70 mRNA levels based on ( A ) RNA levels from the MMRF CoMMpass dataset of different translocations, Amp1q and Del17p, in patients with NDMM and ( B ) the ISS ( n = 561). C, Paired comparison of CD70 mRNA in NDMM and at first relapse (left). The difference between the expression levels is plotted (right, relapsed–NDMM) with a dotted line at the mean of differences (0.609, P = 0.0028, paired t test, n = 61). D, Patients were stratified into tertiles based on CD70 mRNA expression, and overall survival (OS) was plotted ( n = 792). Color shading represents the 95% confidence interval. E, Bone marrow aspirates from patients with multiple myeloma ( n = 46) from the MD Anderson Cancer Center with different translocations involving chromosome 14 were analyzed for CD70 mRNA using the pseudobulk approach on single cell (sc)-mRNA. The control group included samples from n = 3 healthy control donors. The fourth data point in the control group represents nontumor or polyclonal plasma cells (PC) from patients with multiple myeloma identified by the expression of B-cell receptor VDJ using scRNA-seq (see “Methods”); monoclonal plasma cells were similarly defined based on BCR VDJ sequences, and each remaining data point corresponds to an individual patient with multiple myeloma: no translocation (none; n = 20), t(11;14) ( n = 16), t(14;16) ( n = 2), and t(4;14) ( n = 8). F, The sc-mRNA transcriptome data from all samples projected onto a Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP). Individual CD70 mRNA was mapped at single-cell resolution. G, Translocations involving the immunoglobulin heavy chain locus identified by FISH are mapped onto a UMAP. Box and whisker plots show the median and IQR (25th and 75th percentiles), and the whiskers extend to ±1.5 times the IQR. The P values were determined by the Wilcoxon test [ A , B , D (left panel) and E ]. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant. UMI, unique molecular identifier.

Article Snippet: The CD70 antibody (Cell Signaling Technology, clone e3Q1A, cat. #69209S) was used at a 1:50 dilution, incubated for 15 minutes at room temperature, and subsequently detected using the BOND Polymer Refine Detection kit (Leica Biosystems, cat. #DS9800) with diaminobenzidine as the chromogen and counterstained with hematoxylin.

Techniques: Expressing, Comparison, Single Cell, Control, Clinical Proteomics, Translocation Assay, Whisker Assay

Journal: Blood Cancer Discovery

Article Title: CD70-Targeting CAR NK Cells Overcome BCMA Downregulation and Improve Survival in High-risk Multiple Myeloma Models

doi: 10.1158/2643-3230.BCD-25-0130

Figure Lengend Snippet: Phenotypic characterization of CD70 expression in patients with multiple myeloma. A, Bone marrow aspirates from 30 patients with multiple myeloma were stained with anti-CD70 and anti-BCMA antibodies and assayed by flow cytometry. The percentage of positive cells from the FSC/SSC/Single cells/Live-Dead/CD3 − /CD19 − /CD20 − /CD14 − /CD138+ population is plotted, with each dot representing one patient sample. The gating strategy is shown in Supplementary Fig. S3. B, The percentage of positive expression (circle size) and the robust z -score calculated from the mean fluorescence intensity (color) for CD70, BCMA, CD38, CS1, CD56, and CD45 proteins from the same population were plotted in a dot plot for each of the patient samples in ( A ). C, An optimized stochastic neighbor embedding (opt-SNE) plot was generated for each marker in the CD138 + , CD3 − , CD19/20 − , and CD14 − populations from an example patient with amp 1q and loss of one copy of TP53 . Representative images of IHC CD70 staining of the gingiva ( D ) and bone marrow ( E ) clot of a patient with high-grade myeloma. F, CD70 staining of the humerus of a patient who had not responded to the BCMA antibody–drug conjugate belantamab mafodotin. G, CD70 staining from the bone marrow of a patient without multiple myeloma as a control. Scale bar, 100 μm.

Article Snippet: The CD70 antibody (Cell Signaling Technology, clone e3Q1A, cat. #69209S) was used at a 1:50 dilution, incubated for 15 minutes at room temperature, and subsequently detected using the BOND Polymer Refine Detection kit (Leica Biosystems, cat. #DS9800) with diaminobenzidine as the chromogen and counterstained with hematoxylin.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence, Generated, Marker, Control

Journal: Blood Cancer Discovery

Article Title: CD70-Targeting CAR NK Cells Overcome BCMA Downregulation and Improve Survival in High-risk Multiple Myeloma Models

doi: 10.1158/2643-3230.BCD-25-0130

Figure Lengend Snippet: Armoring NK cells with CAR27/IL-15 results in enhanced antitumor activity against multiple myeloma. A, Schematic of the retroviral vector used to transduce CB-NK cells. B, Flow cytometry histogram assay of CD70 surface protein levels in NCI-H929 (blue) and MM.1S (magenta) cells. C, 51 Cr release assay of NT or CAR27/IL-15 NK cells against CD70 + MM.1S or ( D ) CD70 − NCI-H929 tumor cells at different E:T ratios and measured 4 hours after coincubation ( n = 3). E, MM.1S tumor cells transduced to express mKATE were cultured alone or with NT, IL-15, or CAR27/IL-15 NK cells. The number of MM.1S cells was measured over time with sequential images from the Incucyte machine. Representative images from 0 Hours (baseline) and after 24 Hours after coculture are shown. White bar, 400 μm. F, The percentage of initial MM.1S cells from panel ( E ) is plotted for each group over 30 hours. Bar graph (right) shows the AUC analysis. G, The Incucyte killing assay was repeated for NT and CAR27/IL-15 NK cells against the CD70-positive U266B1 myeloma cell line. Bar graph (right) shows the AUC analysis. Data are represented as mean ± SD. Data for the AUC graphs are represented as mean ± SEM. The P values were determined by two-way ANOVA with Sidak’s multiple comparisons ( C ), one-way ANOVA with Tukey multiple comparison ( F ), and unpaired t test ( G ). ***, P ≤ 0.001. ICD, intracellular domain; LTR, long terminal repeats; TMD, transmembrane domain.

Article Snippet: The CD70 antibody (Cell Signaling Technology, clone e3Q1A, cat. #69209S) was used at a 1:50 dilution, incubated for 15 minutes at room temperature, and subsequently detected using the BOND Polymer Refine Detection kit (Leica Biosystems, cat. #DS9800) with diaminobenzidine as the chromogen and counterstained with hematoxylin.

Techniques: Activity Assay, Retroviral, Plasmid Preparation, Transduction, Flow Cytometry, Release Assay, Cell Culture, Comparison

Journal: Blood Cancer Discovery

Article Title: CD70-Targeting CAR NK Cells Overcome BCMA Downregulation and Improve Survival in High-risk Multiple Myeloma Models

doi: 10.1158/2643-3230.BCD-25-0130

Figure Lengend Snippet: In vivo anti-myeloma activity of CD70-targeting CAR NK cells. A, Mice were first injected with MM.1S tumor cells and subsequently treated (day 0) with no additional cells (tumor alone), NT NK cells, CAR27/IL-15 NK cells, CAR27 NK cells without IL-15 (CAR27), and NK cells transduced to express only IL-15 (IL-15 NK). B, BLI was obtained weekly until day 54. Different radiance scales were used for early (days 0–14) and late (days 21–54) imaging due to signal intensities spanning several orders of magnitude. An ‘X’ denotes that all mice in the group have died. C, BLI signal for individual mice plotted over days post-CAR NK cell treatment. D, Percentage of human CD45 + cells (NK cells) detected in the blood at day 12 after NK cell infusion. Only three of the five mice per group were bled for this analysis. E, Kaplan–Meier survival plot. P value significance is denoted for the following comparisons: blue asterisks: NT NK vs. CAR27/IL-15 NK; green asterisks: CAR27 NK vs. CAR27/IL-15 NK; and magenta asterisks: IL-15 NK vs. CAR27/IL-15 NK. F, Weight of the mice in grams over time. Data are represented as mean ± SD. P values were calculated using the log-rank test ( E ). **, P ≤ 0.01.

Article Snippet: The CD70 antibody (Cell Signaling Technology, clone e3Q1A, cat. #69209S) was used at a 1:50 dilution, incubated for 15 minutes at room temperature, and subsequently detected using the BOND Polymer Refine Detection kit (Leica Biosystems, cat. #DS9800) with diaminobenzidine as the chromogen and counterstained with hematoxylin.

Techniques: In Vivo, Activity Assay, Injection, Imaging

Journal: Blood Cancer Discovery

Article Title: CD70-Targeting CAR NK Cells Overcome BCMA Downregulation and Improve Survival in High-risk Multiple Myeloma Models

doi: 10.1158/2643-3230.BCD-25-0130

Figure Lengend Snippet: Loss of BCMA does not affect the anti-myeloma activity of CAR27/IL-15 NK cells. A, Whole-cell lysates from K562 (negative control), MM.1S wild-type (WT), and MM.1S TNFRSF17 KO cells (KO) were analyzed by Western blotting for BCMA expression. β-actin served as the loading control. A representative blot is shown. B, TNFRSF17 KO efficiency analyzed by flow cytometry. C, CD70 expression of MM.1S WT and MM.1S TNFRSF17 KO tumor cells. D, 51 Cr release assay of NT NK cells or CAR27/IL-15 NK cells against MM.1S TNFRSF17 KO cells. E, Incucyte real-time killing assay of MM.1S TNFRSF17 KO tumor cells by NT NK, IL-15 NK, and CAR27/IL-15 NK cells. Bar graph (right) shows the AUC analysis. Data are represented as mean ± SD. Data for the AUC graph are represented as mean ± SEM. The P values were determined by two-way ANOVA with Sidak’s multiple comparisons ( D ) and one-way ANOVA with Tukey multiple comparisons ( E ). ***, P ≤ 0.001.

Article Snippet: The CD70 antibody (Cell Signaling Technology, clone e3Q1A, cat. #69209S) was used at a 1:50 dilution, incubated for 15 minutes at room temperature, and subsequently detected using the BOND Polymer Refine Detection kit (Leica Biosystems, cat. #DS9800) with diaminobenzidine as the chromogen and counterstained with hematoxylin.

Techniques: Activity Assay, Negative Control, Western Blot, Expressing, Control, Flow Cytometry, Release Assay

Journal: Nature Communications

Article Title: A spatially resolved atlas of gastric cancer characterises a lymphocyte-aggregated region

doi: 10.1038/s41467-026-68612-z

Figure Lengend Snippet: a Distinct immune checkpoint ligand-receptor pairs enriched in different spatial regions. * indicates P < 0.001. b Forest plot delineates the association between the axis of CD27-CD70 and donor groups based on effect sizes and their 95% confidence intervals. c Comparison of expression of CD27-CD70 between LAR blocks of Groups A and B ( P < 0.001, two-sided t-test). A total of 520 LAR spatial blocks across 26 samples were used for comparison. For all the box plots in this figure, the centre line of the box denotes the median value, and the horizontal line denotes the 95% confidence interval. d Boxplots compare the expression of inhibitory axes between NCR blocks of Groups A and B ( P < 0.001, two-sided t-test). A total of 1204 NCR spatial blocks across 33 samples were used for comparison. e Dot plot indicates the cellular communications of cDC-LAMP3 with T cell subsets coordinated by the axis of CD27-CD70 inferred from the scRNA-seq data. Benjamini–Hochberg adjusted P values were generated from a one-sided Z-test. f Immunohistochemistry staining of CD8A, PD1, CD27, LAMP3 and CD70 for the sample D11T1. The physical contact between these two cell types was observed within LARs of 11 samples from the main study cohort and five samples from the independent validation cohort. Left panel, the whole slide view of H&E and mIHC images; middle panel, zoom-in views for an LAR region highlighted by the rectangle of the left panel (scale bar, 100 μm); right panel, zoom-in views for the rectangle areas in the middle panel (scale bar, 10 μm). Yellow triangles indicate PD1 + CD27 + CD8 + cells, while white triangles indicate LAMP3 + CD70 + cells. g A Venn diagram illustrates the LAR spatial blocks expressing both axes of CD28 and CTLA4. h , Box plot compares the average distance between PD1 + CD27 + CD8 + and CD70 + LAMP3 + cells and the average distance between DAPI and CD70 + LAMP3 + cells within each LAR in the main study cohort. A total of 19 LAR regions across 10 samples were used for comparison. The Bonferroni-adjusted P value indicates the statistical significance between these two groups ( P < 0.001, two-sided t-test).

Article Snippet: The protein markers used for staining were CD8 (1:400, clone C8/144B, 70306, Cell Signalling Technology), TCF1 (1:200, clone C63D9, 14456, Cell Signalling Technology), Ki67 (1:200, clone SP6, RM-9106-s1, Thermo Scientific), CD27 (1:2000, clone LPFS2/1611, ab268144, Abcam), CD70 (1:400, clone E3Q1A, 69209, Cell Signalling Technology), PD-1 (1:300, clone 29 F.1A12, 135210, Biolegend) and LAMP3 (1:200, clone 1010E1.01, Novus Biologicals).

Techniques: Comparison, Expressing, Generated, Immunohistochemistry, Staining, Biomarker Discovery