Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a, b) ELISA of IFN-β from supernatants of MEFs (WT or Isg15 −/−) (a) and HeLa cells (WT or ISG15 KO) (b) transiently transfected with increasing amounts of FLAG-tagged MDA5 or RIG-I for 40 h. Whole cell lysates (WCLs) were probed by immunoblotting (IB) with anti-ISG15, anti-FLAG, and anti-Actin (loading control). (c) ELISA of IFN-β from supernatants of WT or Isg15 −/− MEFs that were mock-stimulated or transfected with EMCV-RNA (0.1 or 0.4 μg/mL), HMW-poly (I:C) (0.5 μg/mL), or RABVLe (1 pmol/mL), or infected with SeV (10 HAU/mL) for 24 h. (d) Quantitative RT-PCR (qRT-PCR) analysis of IFNB1 and CCL5 mRNA in WT and Isg15 −/− MEFs stimulated as in (c). (e) IRF3 phosphorylation in the WCLs of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABVLe (1 pmol/mL) for 6 h, assessed by IB with anti-pS396-IRF3 and anti-IRF3. (f) ELISA of IFN-β from supernatants of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABVLe (1 pmol/mL), or infected with SeV (10 HAU/mL) for 16 h. (g) ELISA of IFN-β from the supernatants of PBMCs that were transduced for 40 h with the indicated shRNAs and then infected with mutEMCV (MOI 10) or SeV (200 HAU/mL) for 8 h. (h) qRT-PCR analysis of IFNA2 and IL-6 mRNA in PBMCs that were transduced and infected as in (g). Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, b, c, d, f and mean of n = 2 biological replicates in g and h). * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired Student’s t -test). ND, not detected; NS, not significant.

Article Snippet: One-step qRT-PCR was performed using the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) and predesigned PrimeTime qPCR Probe Assays (IDT) on a 7500 Fast Real-Time PCR System (Applied Biosystems).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a) Endogenous MDA5 ISGylation in NHLFs that were mock-treated, transfected with HMW-poly (I:C) (0.1 μg/mL) for 40 h (left), or infected with DENV or ZIKV (MOI 1 for each) for 48 h (right), determined by immunoprecipitation (IP) with anti-MDA5 (or an IgG isotype control) followed by IB with anti-ISG15 and anti-MDA5. WCLs were probed by IB with anti-ISG15 and anti-Actin (loading control). (b) ISGylation of FLAG-tagged MDA5-2CARD and MDA5ΔCARD in transiently transfected HEK293T cells that also expressed V5-ISG15, HA-Ube1L, and FLAG-UbcH8, assessed by FLAG pulldown (PD) and IB with anti-V5 and anti-FLAG forty hours after transfection. WCLs were probed by IB with anti-HA, anti-FLAG, anti-V5, and anti-Actin. (c) Endogenous MDA5 ISGylation in ISG15 KO HeLa cells stably reconstituted with vector, WT ISG15 or ISG15-AA and co-transfected with HA-Ube1L and FLAG-UbcH8 after IFN-β treatment (1,000 U/mL) for 24 h, determined by IP with anti-MDA5 and IB with anti-ISG15 and anti-MDA5. (d) ISGylation of GST-MDA5-2CARD WT and K23R/K43R in HEK293T cells that were co-transfected with V5-ISG15, HA-Ube1L, and FLAG-UbcH8 for 24 h, determined by GST-PD and IB with anti-V5 and anti-GST. (e) ISGylation of FLAG-tagged MDA5 WT and K23R/K43R in HEK293T cells that were co-transfected with V5-ISG15, HA-Ube1L, and FLAG-UbcH8, determined by FLAG-PD and IB with anti-V5 and anti-FLAG. (f) IFN-β-luciferase reporter activity in HEK293T cells that were transfected for 40 h with vector, or FLAG-tagged MDA5 WT or mutants. Luciferase activity is presented as fold induction relative to the values for vector-transfected cells, set to 1. WCLs were probed by IB with anti-FLAG and anti-Actin. (g) qRT-PCR analysis of IFNB1 and CCL5 mRNA in HEK293T cells that were transiently transfected with either vector, or increasing amounts of FLAG-tagged MDA5 WT or K23R/K43R. (h) STAT1 phosphorylation and ISG (IFIT1 and 2) protein abundance in the WCLs of HEK293T cells that were transiently transfected with vector or FLAG-tagged MDA5 WT or K23R/K43R, determined by IB with anti-pY701-STAT1, anti-STAT1, anti-IFIT1, anti-IFIT2, anti-FLAG (expression control) and anti-Actin (loading control). (i) qRT-PCR analysis of IFNB1 , CCL5 , OAS1 , and RSAD2 mRNA in MDA5 KO SVGAs that were reconstituted with either empty vector or FLAG-tagged MDA5 WT, K23R/K43R or S88E. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in f, g, and i). * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired Student’s t -test). NS, not significant.

Article Snippet: One-step qRT-PCR was performed using the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) and predesigned PrimeTime qPCR Probe Assays (IDT) on a 7500 Fast Real-Time PCR System (Applied Biosystems).

Techniques: Transfection, Infection, Immunoprecipitation, Stable Transfection, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

doi: 10.1101/2020.10.26.356048

Figure Lengend Snippet: (a) qRT-PCR analysis of IFNB1 , IFNL1 , ISG15 , MDA5, and RIG-I transcripts in NHLFs that were transfected with the indicated siRNAs for 40 h and then transfected with mock-RNA or SCoV2-RNA (0.4 μg/mL) for 24 h. (b) Binding of SCoV2 Nsp3 to endogenous MDA5 in A549-hACE2 cells that were infected with SCoV2 (MOI 0.5) for 24 h, determined by IP with anti-MDA5 (or an IgG isotype control) followed by IB with anti-PLpro and anti-MDA5. WCLs were probed by IB with anti-PLpro (Nsp3) and anti-Actin. (c) Endogenous MDA5 ISGylation in A549-hACE2 cells that were mock-infected, or infected with SCoV2 or DENV (MOI 0.1 for each) for 48 h, determined by immunoprecipitation (IP) with anti-MDA5 followed by IB with anti-ISG15 and anti-MDA5. Protein abundance of IFIT1, RSAD2, IFITM3, ISG15 and actin in the WCLs were probed by IB. Efficient virus replication was verified by immunoblotting WCLs with anti-PLpro (Nsp3) or anti-NS3 (DENV). (d) qRT-PCR analysis of IFNB1 , CCL5 , IFIT1 transcript, and EMCV genomic RNA (gRNA) in HeLa cells that were transiently transfected for 24 h with vector, or V5-SCoV2 PLpro WT or mutants and then infected with mutEMCV (MOI 0.5) for 12 h. (e) EMCV titers in the supernatant of RIG-I KO HEK293 cells that were transiently transfected for 24 h with vector or FLAG-MDA5 along with V5-SCoV2 PLpro WT, C111A, or R166S/E167R and then infected with EMCV (MOI 0.001) for 16 h, determined by plaque assay. (f) Protein abundance of the indicated ISGs in the WCLs from the experiment in (e), determined by IB with the indicated antibodies. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, d, e). * p < 0.05, *** p < 0.001 (unpaired Student’s t -test).

Article Snippet: One-step qRT-PCR was performed using the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) and predesigned PrimeTime qPCR Probe Assays (IDT) on a 7500 Fast Real-Time PCR System (Applied Biosystems).

Techniques: Quantitative RT-PCR, Transfection, Binding Assay, Infection, Immunoprecipitation, Western Blot, Plasmid Preparation, Plaque Assay

Journal: Nature Communications

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

doi: 10.1038/s41467-026-69796-0

Figure Lengend Snippet: A Experimental CAR structure. scFv = single chain variable fragment. h hinge. TM transmembrane. IC intracellular domain. Created in BioRender. Klepsch, V. ( https://BioRender.com/1s7z4s9 ). B Experimental setup and timeline. Immunocompetent wild type mice were injected subcutaneously with 1 × 10 6 Panc02-EpCAM cells (mosaic: 71% EpCAM positive, 29% EpCAM negative) and treated 2 days later with 3.6 × 10 6 genetically modified 28ζ CAR-T cells using CRISPR/Cas9 (Non-targeting control/NTC vs Nr2f6 crispr −/− .g04 ). Created in BioRender. Humer, D. ( https://BioRender.com/egrvdww ). Average tumor growth curve ( C ) and survival curve ( D ) and tumor growth in single mice ( E ). Tumor area was compared to untreated tumor-bearing mice (PBS) and measured by caliper. 5 of 9 in the Nr2f6 crispr −/− .g04 CAR-T receiving cohort ( n = 9 mice) and 1 of 11 in the NTC CAR-T receiving cohort ( n = 11 mice) showed complete responses. 1 of 9 in the Nr2f6 crispr −/− .g04 CAR-T receiving cohort relapsed and had to be killed on day 100. PBS ( n = 9 mice), pooled from two independent experiments. F–J FACS analysis of NTC and Nr2f6 crispr −/− CAR + T cells d3 after ACT in Panc02-EpCAM tumor-bearing mice. F Percentage of CAR T cells from total tumor digested cells. G gMFI of TCF-1 protein in CAR T cells in the draining lymph node. H Representative plot for TCF-1 protein expression. I , J CAR T cells from spleens were restimulated ex-vivo with Panc02-EpCAM cells and analyzed for intracellular cytokine (INFγ + GzmB) production by FACS. The experiments were done as two ( C – E ) biological replicates / one ( F – J ) biological replicate. F – J n = 5 mice per genotype. Two-way ANOVA ( C ), Log-Rank test ( D ), two-tailed unpaired Student’s t-test [ F – J ]. Data shown as mean ± SEM.

Article Snippet: For quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen, 74106) in a 20 μL reaction using random hexamer as well as Oligo(dt)-primer (Fisher Scientific, 18418012). qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs, M3004E) and pre-designed Nr2f6 Taqman primers (Fisher Scientific, see below) on a 7500 Real-Time PCR machine (Applied Biosystems).

Techniques: Injection, Genetically Modified, CRISPR, Control, Expressing, Ex Vivo, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-026-69796-0

Figure Lengend Snippet: A Flow cytometric analysis of CAR transduction efficiency in wild type and Nr2f6 −/− CAR-T cells. Representative image shows 6 concatenated experiments per genotype; % CAR positive indicated in numbers. B IFNγ production after 24 h co-culture with tumor cells 5 days after T cell isolation assessed by flow cytometry. Control and Nr2f6 -deficient CAR-T cells were stimulated at an effector to target cell ratio of 5:1 with Panc02-EpCAM (100% positive). n = 5 replicates, derived from two independent experiments. C Experimental setup of chronic stimulation assay. Created in BioRender. Klepsch, V. ( https://BioRender.com/sespri5 ). D Cytotoxicity of control and Nr2f6 -deficient CAR-T cells against GFP + Panc02-EpCAM cells was assessed after serial stimulation, beginning five days after T cell isolation at E:T of 5:1 at 48-h intervals in medium without cytokines. n = 9 replicates from four independent experiments, ns = 0.9536. Granzyme B ( E ) and IFNγ ( F ) release in the supernatant after each round of re-stimulation (every 48 h) with GFP + Panc02-EpCAM tumor cells beginning 5 days after T cell isolation assessed by Luminex xMAP technology. n = 6 replicates, derived from two independent experiments. G PCA blot from normalized RNAseq data obtained from wild type and Nr2f6 −/− CAR-T cells isolated on d8 of serial killing ( n = 3 per genotype, derived from one representative experiment). H Gene ontology (GO) terms of differential expressed genes showing significantly enriched pathways in Nr2f6 −/− CAR-T cells compared to wild type control CAR-T cells isolated on day 8 of chronic stimulation. I Gasdermin E cleavage assay. Wild type and Nr2f6 -modified CAR-T cells were chronically stimulated with Panc02-EpCAM tumor cells beginning on day five after T cell isolation at E:T 5:1. 6 h after the 2 nd re-stimulation, Panc02-EpCAM were analyzed for GSDME cleavage using immunoblot. Quantification was done in ImageJ. n = 6 replicates from two independent experiments. B , I Two-tailed unpaired Student’s t test. D two-way ANOVA for each round. E , F multiple two-tailed unpaired Student’s t test. Data shown as mean ± SEM. ns=not significant.

Article Snippet: For quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen, 74106) in a 20 μL reaction using random hexamer as well as Oligo(dt)-primer (Fisher Scientific, 18418012). qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs, M3004E) and pre-designed Nr2f6 Taqman primers (Fisher Scientific, see below) on a 7500 Real-Time PCR machine (Applied Biosystems).

Techniques: Transduction, Co-Culture Assay, Cell Isolation, Flow Cytometry, Control, Derivative Assay, Luminex, RNA sequencing, Isolation, Cleavage Assay, Modification, Western Blot, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-026-69796-0

Figure Lengend Snippet: A Flow cytometric analysis of CD44 and CD62L expression on CAR-T cells on day 4 of chronic stimulation assay. n = 6 replicates from 2 independent experiments. B Representative image (d4) and ratio of progenitor (Tim3 − Ly108 + ) to terminally (Tim3 + Ly108 − ) exhausted T cells on d4 and d8 of chronic stimulation assay of wild type and Nr2f6 −/− CAR-T cells. n = 6 donors, from 2 independent experiments. Tpex = progenitor exhausted T cells; Ttex = terminally exhausted T cells; Text trans = transitory exhausted T cells. C Flow cytometric analysis of Tcf-1 expression in control and Nr2f6 -deficient CAR-T cells on day 4 of chronic stimulation. Representative image (left) and quantification of geometric mean fluorescence intensity (gMFI) (right). n = 6 replicates from 2 independent experiments. A , C Two-tailed unpaired Student’s t test. B two-sided non-parametric Mann-Whitney test. Data shown as mean ± SEM.

Article Snippet: For quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen, 74106) in a 20 μL reaction using random hexamer as well as Oligo(dt)-primer (Fisher Scientific, 18418012). qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs, M3004E) and pre-designed Nr2f6 Taqman primers (Fisher Scientific, see below) on a 7500 Real-Time PCR machine (Applied Biosystems).

Techniques: Expressing, Control, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

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

doi: 10.1038/s41467-026-69796-0

Figure Lengend Snippet: A Real-time mitochondrial stress test profile, oxygen consumption rate and ( B ) extra cellular acidification rate as measured by Seahorse Cell Mito Stress Test of control or Nr2f6 -deficient CAR-T cells under resting and challenge conditions on day 4 of chronic stimulation. n = 6 donors in duplicates from two independent experiments. FCCP = Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone, AA+Rot = AntimycinA + Rotenone. A spare ns = p = 0.16, non mitoch. ns = p = 0.55. C PCA blot from normalized RNAseq data obtained from wild type and Nr2f6 −/− CAR-T cells isolated on d4 of chronic stimulation ( n = 3 replicates, data derived from one representative experiment). D Gene ontology (GO) terms obtained from overrepresentation analysis of differentially expressed genes RNAseq data showing significantly enriched pathways in Nr2f6 −/− CAR-T cells compared to wild type control CAR-T cells. E Gene set enrichment analysis (GSEA) of Nr2f6 -deficient CAR-T cells compared to control CAR-T cells isolated on 4 days of chronic stimulation using the KEGG database. Normalized enrichment scores (NES) and p values are shown. A positive NES indicates that the gene set was enriched in Nr2f6 -deficient cells. A , B two-tailed unpaired Student’s t-test, data shown as mean ± SEM, ns not significant. E Wald statistic from DESeq2.

Article Snippet: For quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen, 74106) in a 20 μL reaction using random hexamer as well as Oligo(dt)-primer (Fisher Scientific, 18418012). qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs, M3004E) and pre-designed Nr2f6 Taqman primers (Fisher Scientific, see below) on a 7500 Real-Time PCR machine (Applied Biosystems).

Techniques: Control, RNA sequencing, Isolation, Derivative Assay, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-026-69796-0

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

Article Snippet: For quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen, 74106) in a 20 μL reaction using random hexamer as well as Oligo(dt)-primer (Fisher Scientific, 18418012). qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs, M3004E) and pre-designed Nr2f6 Taqman primers (Fisher Scientific, see below) on a 7500 Real-Time PCR machine (Applied Biosystems).

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

Journal: Nature Communications

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

doi: 10.1038/s41467-026-69796-0

Figure Lengend Snippet: A Experimental setup and timeline. Created in BioRender. Humer, D. ( https://BioRender.com/bcyeh6x ). B Average tumor growth and ( C ) survival curve. Tumor growth and survival of NTC and Nr2f6 crispr−/−.g04 CAR-T cell receiving complete responders (CR) re-challenged 150 days with 2.5 × 10 6 Panc02-EpCAM pos (left flank) and 1 × 10 6 Panc02-EpCAM neg (right flank) tumor cells (1 st re-challenge). Naïve, age-matched C57BL/6 mice were used as controls (naïve wild type). n = 4 for Nr2f6 crispr−/−.g04 CR, n = 1 for NTC CR and n = 7 for naïve WT from one memory experiment. CR = complete responders, (B) Panc02-EpCAM hi ns = p = 0.385, Panc02-EpCAM neg ns = p = 0.680. D, E Nr2f6 crispr−/−.g04 CAR-T cell-treated long-term CR received a 2 nd re-challenge 150 days later (300 days after initial tumor induction) with 1 × 10 6 Panc02-EpCAM neg tumor cells. Naïve age-matched C57BL/6 mice were used as controls (naïve wild type) ( D ) Tumor growth and survival of Panc02-EpCAM neg -naïve (1 × 10 6 ) tumor-bearing wild type mice adoptively transferred with 1 × 10 7 splenocytes from 2 nd re-challenged CR euthanized on d305 or Panc02-naïve splenocytes. n = 8 for naïve splenocytes, and n = 7 for Nr2f6 crispr−/−.g04 CR splenocytes. E Flow cytometric analysis of IFNγ production after 24 h co-culture of CD8 T cells from CR with tumor cells. Naïve and CR were stimulated at E:T of 5:1 with Panc02-EpCAM pos tumor cells. n = 4 from two independent experiments. EpCAM pos = EpCAM positive, EpCAM neg = EpCAM negative. B – D Two-way ANOVA with Šídák’s multiple comparisons test, ( C , D ) Log-rank rest ( E ) two-tailed unpaired Student’s t-test. Data are presented as mean ± SEM.

Article Snippet: For quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen, 74106) in a 20 μL reaction using random hexamer as well as Oligo(dt)-primer (Fisher Scientific, 18418012). qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs, M3004E) and pre-designed Nr2f6 Taqman primers (Fisher Scientific, see below) on a 7500 Real-Time PCR machine (Applied Biosystems).

Techniques: CRISPR, Co-Culture Assay, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-026-69796-0

Figure Lengend Snippet: A UMAP plots showing cell annotation for naïve vs. CR mice. CD45 + TILs from tumor-bearing Nr2f6 -modified CAR-T cell-treated CR and Panc02-naïve mice were subjected to scRNA- and scTCRseq. CR = complete responder ( B ) Stacked charts showing proportions of each cell cluster ( C , D ) ORA of GO terms showing enriched pathways in intratumoral proliferating CD8 T cells and cDC1. E TCR scRNA-seq; Gini index of TCR clonality ( p = 0.20) and Shannon entropy of diversity ( p = 0.34) were compared in naïve and CR tumor-bearing mice. A – E n = 4 per group (naïve vs CR), one representative experiment. Graphical Summary: Functional advantages and mechanisms underlying Nr2f6 -modified CAR-T cell therapy in a solid tumor model . F In vitro: Conventional CAR-T cells exhibit reduced cytotoxicity, impaired cytokine release, increased exhaustion, and diminished metabolic fitness. In contrast, Nr2f6 -modified CAR-T cells display enhanced tumor-cell killing, increased IFNγ and TNFα production, and improved metabolic performance under repetitive stimulation. G In vivo: Schematic depiction of therapeutic dynamics following CAR-T cell infusion into solid tumor-bearing mice. Conventional CAR-T cells progressively lose efficacy due to antigen heterogeneity, immunosuppressive cues within the tumor microenvironment (TME), and cumulative dysfunction. In comparison, Nr2f6 -modified CAR-T therapy induces immunogenic tumor cell death (ICD), facilitates antigen uptake and presentation by host antigen-presenting cells (APCs), and immune responses beyond the initial CAR target. H Overall concept: The major limitations of conventional CAR-T cells in solid tumors, antigen heterogeneity, a suppressive TME, and limited persistence, are overcome by Nr2f6 -modified CAR-T cells. These cells retain sustained cytotoxic activity, effectively target heterogeneous tumor antigens, and provoke a durable, polyclonal host immune memory response. For the first time, this approach demonstrates that antigenically diverse solid tumors can be efficiently eradicated, bridging the efficacy gap between hematologic and solid malignancies in CAR-T therapy. All schematics are conceptual representations summarizing the cellular and molecular events that collectively underlie the enhanced therapeutic efficacy of Nr2f6 -modified CAR-T cells. Created in BioRender. Klepsch, V. ( https://BioRender.com/sezlqdw ).

Article Snippet: For quantitative real-time PCR (qRT-PCR), total RNA was isolated using the RNeasy Mini kit (Qiagen) according to manufacturer’s instruction and subjected to cDNA synthesis using Omniscript RT kit (Qiagen, 74106) in a 20 μL reaction using random hexamer as well as Oligo(dt)-primer (Fisher Scientific, 18418012). qRT-PCR was performed in duplicates using the LUNA mastermix (New England Biolabs, M3004E) and pre-designed Nr2f6 Taqman primers (Fisher Scientific, see below) on a 7500 Real-Time PCR machine (Applied Biosystems).

Techniques: Modification, Functional Assay, In Vitro, In Vivo, Comparison, Activity Assay, Drug discovery