car t-cells  (Novartis)

Journal: Nature Communications

Article Title: IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma

doi: 10.1038/s41467-026-76972-9

Figure Lengend Snippet: A Schematic for ( B – F ) where CAR T cells and Eμ-myc cells were cocultured with unMac, imMac, M1-like macrophages, or without macrophages. Created in BioRender. Stone, M. ( https://BioRender.com/xpmm6k3 ) B CAR T cell death at 48 h was assessed by 7-aminoactinomycin D (7-AAD) incorporation via flow cytometry ( n = 4 technical replicates from a single experiment, repeated 2 times with similar results). C DNA replication of CAR T cells at 42 h was measured by bromodeoxyuridine (BrdU) incorporation via flow cytometry ( n = 4 technical replicates from a single experiment). D CAR T cell expansion was measured over 48 h by a live cell analysis system. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the corresponding no-macrophage control condition at each time point. E Total CAR expression levels in CAR T cells at 48 h were assessed via flow cytometry. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the corresponding no-macrophage control condition. F Surface CAR expression levels in CAR T cells at 48 h were detected by staining single-chain variable fragments (scFv) of CAR with protein L and analyzed via flow cytometry ( n = 4 technical replicates from a single experiment). G Schematic for ( H – J ) where CAR T cells were isolated after initial coculture with Eμ-myc cells and with unMac or imMac or without macrophages (No Mac) for 48 h and subsequently cocultured with fresh Eμ-myc cells. H , I Levels of IFN-γ and TNF-α in coculture supernatants of CAR T cells and Eμ-myc cells at 36 h were analyzed by an automated enzyme-linked immunosorbent assay (ELISA) ( n = 4 technical replicates from a single experiment). J Lysis of Eμ-myc cells by CAR T cells at 36 h was assessed using a bioluminescence assay. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the corresponding no-macrophage control condition. All data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Bonferroni correction for multiple comparisons ( D , E , J ). Exact P values are provided in the Source Data file. **, P < 0.01; ***, P < 0.001; ### , P < 0.001; #### , P <0.0001; $, P < 0.05; BM bone marrow, MFI mean fluorescence intensity, exp exposed CAR T cells isolated from prior coculture. Source data are provided as a Source Data file.

Article Snippet: The expansion of mCherry + CAR T cells was determined by an IncuCyte S3 live cell analysis system (Essen Bioscience).

Techniques: Flow Cytometry, BrdU Incorporation Assay, Cell Analysis, Control, Expressing, Staining, Isolation, Automated, Enzyme-linked Immunosorbent Assay, Lysis, ATP Bioluminescent Assay, Fluorescence

Journal: Nature Communications

Article Title: IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma

doi: 10.1038/s41467-026-76972-9

Figure Lengend Snippet: A Schematic for ( B – F ) where CAR T cells and Eμ-myc cells were cocultured with unMac or imMac or without macrophages (No Mac) for 24 h. Coculture supernatants were analyzed by global metabolomics using LC-MS. B , C Quantification of relative abundance of metabolites in the supernatants derived from cocultures containing imMac versus No Mac ( B ) or unMac ( C ) ( n = 3 technical replicates from a single experiment). D – F Arginine, ornithine, and citrulline levels in the supernatants of coculture groups ( n = 3 technical replicates from a single experiment). The data were normalized to the mean value of the no-macrophage control condition. G Flow cytometry analysis of expression of ARG-1 and iNOS in unMac or imMac cocultured with Eμ-myc cells in the presence or absence of CAR T cells at 24 h. Data are from n = 3 independent experiments. H NO levels in the supernatants derived from cocultures of CAR T cells and Eμ-myc cells with unMac or imMac or without macrophages at 48 h were analyzed by Griess assay. Data are from n = 3 independent experiments. Data in ( D – H ) are presented as mean ± SD. Statistical significance was determined by unpaired two-sided Student’s t tests ( G ) or one-way ANOVA with Bonferroni correction for multiple comparisons ( H ). Exact P values are provided in the Source Data file. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns not significant, FC fold change, LC-MS liquid chromatography-mass spectrometry, iNOS inducible nitric oxide synthase, ARG-1 arginase-1, NO nitric oxide. Source data are provided as a Source Data file.

Article Snippet: The expansion of mCherry + CAR T cells was determined by an IncuCyte S3 live cell analysis system (Essen Bioscience).

Techniques: Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Control, Flow Cytometry, Analysis, Expressing, Griess Assay, Liquid Chromatography, Mass Spectrometry

Journal: Nature Communications

Article Title: IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma

doi: 10.1038/s41467-026-76972-9

Figure Lengend Snippet: A CAR T cells and Eμ-myc cells were cocultured with unMac, imMac or no macrophages, +/- L-NIL (50 μM). CAR T cell expansion was measured over 48 h using live cell analysis. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the corresponding no-macrophage control condition at each time point. B – D After 48 h coculture described in ( A ), isolated CAR T cells were cocultured with fresh Eμ-myc cells. B Target cell lysis was assessed by luciferase assay at 24 h ( n = 4 technical replicates from a single experiment, repeated 2 times with similar results). C , D IFN-γ and TNF-α in coculture supernatants at 36 h were analyzed by ELISA ( n = 4 technical replicates from a single experiment). E CAR T cells and Eμ-myc cells were cocultured with WT or iNOS −/− unMac or imMac or without macrophages. CAR T cell expansion was measured over 48 h by a live cell analysis ( n = 4 technical replicates from a single experiment, repeated 2 times with similar results). F After 48 h of coculture described in ( E ), isolated CAR T cells were cocultured with fresh Eμ-myc cells and target cell lysis was assessed at 36 h ( n = 4 technical replicates from a single experiment, repeated 2 times with similar results). G CAR T cells and Eμ-myc cells were cocultured with NCX-4016 or 0.01% DMSO (Veh). CAR T cell expansion was measured over 48 h by a live cell analysis. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the corresponding +Veh control condition at each time point. H CAR T cells were cocultured with Eμ-myc cells +/- peroxynitrite (PNT). CAR T cell expansion was measured over 48 h by a live cell analysis. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the corresponding no-treatment control condition at each time point. I After 48 h of initial coculture described in G-H, isolated CAR T cells were cocultured with fresh Eμ-myc cells, and target cell lysis was assessed at 24 h ( n = 4 technical replicates from a single experiment). J CAR T cells and Eμ-myc cells were cocultured with or without imMac +/- carboxyl-PTIO (c-PTIO). CAR T cell expansion was measured over 48 h by live cell analysis. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the corresponding no-macrophage control condition at each time point. All data are presented as mean ± SD. Statistical significance was determined by unpaired two-sided Student’s t tests ( A , G , H , J ). Exact P values are provided in the Source Data file. *, P < 0.05; **, P < 0.01; ***, P < 0.001; exp exposed CAR T cells isolated from prior coculture. Source data are provided as a Source Data file.

Article Snippet: The expansion of mCherry + CAR T cells was determined by an IncuCyte S3 live cell analysis system (Essen Bioscience).

Techniques: Cell Analysis, Control, Isolation, Lysis, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma

doi: 10.1038/s41467-026-76972-9

Figure Lengend Snippet: A – C CAR T cells and Eμ-myc cells were cocultured with unMac or imMac in the presence of anti-IFN-γ (10 μg/mL) or IsoCon (10 μg/mL). (A) Expression of ARG-1 and iNOS in unMac or imMac was analyzed at 24 h via flow cytometry ( n = 3 technical replicates from a single experiment, repeated 2 times with similar results). B NO levels in coculture supernatants at 44 h were analyzed by Griess assay. Data are from n = 3 independent experiments. Within each independent experiment, technical replicates were averaged, and values were normalized to the +imMac+IsoCon control. ( C ) CAR T cell expansion was measured over 48 h by a live cell analysis system ( n = 4 technical replicates from a single experiment, repeated 2 times with similar results). D – F After initial coculture of CAR T cells and Eμ-myc cells with unMac or imMac in the presence of anti-IFN-γ (10 μg/mL) or IsoCon (10 μg/mL) for 48 h, isolated CAR T cells were cocultured with fresh Eμ-myc cells. D Luciferase-based lysis of Eμ-myc cells by CAR T cells was assessed at 24 h ( n = 4 technical replicates from a single experiment). E , F Levels of IFN-γ and TNF-α in the coculture supernatants at 24 h were analyzed by ELISA ( n = 4 technical replicates from a single experiment). G Schematic for ( H ) where WT or IFN-γ −/− CAR T cells were cocultured with Eμ-myc cells in the presence of unMac or imMac. After coculture for 16 h, scRNA-seq was performed on T cells. H Pathway enrichment analysis of differentially expressed genes in CAR T cells from cocultures of WT CAR T cells and imMac, compared to those from WT CAR T cells and unMac or IFN-γ −/− CAR T cells and imMac cocultures ( n = 2 technical replicates from a single experiment). Data in ( A – F ) are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Bonferroni correction for multiple comparisons ( B ). Exact P values are provided in the Source Data file. ***, P < 0.001; ****, P < 0.0001; IsoCon isotype control, NO nitric oxide, exp exposed CAR T cells isolated from prior coculture. Source data are provided as a Source Data file.

Article Snippet: The expansion of mCherry + CAR T cells was determined by an IncuCyte S3 live cell analysis system (Essen Bioscience).

Techniques: Expressing, Flow Cytometry, Griess Assay, Control, Cell Analysis, Isolation, Luciferase, Lysis, Enzyme-linked Immunosorbent Assay, Analysis

Journal: Nature Communications

Article Title: IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma

doi: 10.1038/s41467-026-76972-9

Figure Lengend Snippet: CAR T cells and Eμ-myc cells were cocultured with unMac, imMac or without macrophages (No Mac), +/- L-NIL (50 μM). After coculture for 48 h, proteomics was performed on T cells. A Experimental design. B Principal component analysis (PCA) plot of proteomics data showing distinct clustering of samples ( n = 3 technical replicates from a single experiment). C Pathway enrichment analysis of differentially expressed proteins (DEPs) in CAR T cells cocultured with imMac versus No Mac, unMac, or imMac+L-NIL (50 μM) ( n = 3 technical replicates from a single experiment). D – F The p53 and MYC pathway DEPs in CAR T cells cocultured with imMac versus No Mac ( D ), unMac ( E ), or imMac+L-NIL treatment ( F ) ( n = 3 technical replicates from a single experiment). G DEPs in the p53 pathway ( n = 3 technical replicates from a single experiment). H DEPs in MYC target proteins ( n = 3 technical replicates from a single experiment). Exp exposed CAR T cells isolated from prior coculture. Source data are provided as a Source Data file.

Article Snippet: The expansion of mCherry + CAR T cells was determined by an IncuCyte S3 live cell analysis system (Essen Bioscience).

Techniques: Analysis, Isolation

Journal: Nature Communications

Article Title: IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma

doi: 10.1038/s41467-026-76972-9

Figure Lengend Snippet: A – L CAR T cells and Eμ-myc cells were cocultured with unMac or imMac or without macrophages (No Mac), +/- L-NIL (50 μM). After coculture for 48 h, global metabolomics was performed on T cells. Quantification of relative abundance of metabolites in CAR T cells derived from cocultures with imMac versus No Mac ( A ), unMac ( B ), or imMac+L-NIL treatment ( C ) ( n = 4 technical replicates from a single experiment). D Schematic depicting altered metabolites associated with glycolysis pathway and TCA cycle. ( E – L ) Levels of F1,6BP, G3P, DHAP, citrate, aconitate, succinate, malate, and itaconate ( n = 4 technical replicates from a single experiment). The data were normalized to the mean value of CAR T cells derived from cocultures without macrophages and L-NIL treatment. M CAR T cells were cocultured with Eμ-myc cells in the presence or absence of 4-OI. CAR T cell expansion was measured over 48 h by a live cell analysis system (1928z CAR T ( + Veh), n = 4; +4-OI (500 µM), n = 3; +4-OI (1 mM), n = 4; +4-OI (2 mM), n = 4; technical replicates from a single experiment, repeated 2 times with similar results). N CAR T cells and Eμ-myc cells were cocultured with imMac or without macrophages (No Mac), +/- L-NIL (50 μM). After coculture for 48 h, seahorse assay was performed on CAR T cells ( n = 6 technical replicates from a single experiment). ECAR was measured in response to glucose (Glc), ATP synthase inhibitor (Oligo), or hexokinase II inhibitor (2-DG). OCR was measured in response to Oligo, mitochondrial oxidative phosphorylation uncoupler (FCCP), or electron transport chain complex I/III inhibitor (Rot/AA). Data in ( E – N ) are presented as mean ± SD. F1,6BP fructose 1,6-bisphosphate, G3P glyceraldehyde 3-phosphate, DHAP dihydroxyacetone phosphate, 4-OI 4-octyl itaconate, ECAR extracellular acidification rate, OCR oxygen consumption rate, Oligo oligomycin A, 2-DG 2-deoxy-D-glucose, FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone, Rot/AA rotenone/antimycin A, exp exposed CAR T cells isolated from prior coculture. Source data are provided as a Source Data file.

Article Snippet: The expansion of mCherry + CAR T cells was determined by an IncuCyte S3 live cell analysis system (Essen Bioscience).

Techniques: Derivative Assay, Cell Analysis, Phospho-proteomics, Isolation

Journal: Nature Communications

Article Title: IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma

doi: 10.1038/s41467-026-76972-9

Figure Lengend Snippet: A Experimental settings for ( B – E ) where Eμ-myc cells were intraperitoneally injected into Rag1 −/− mice. Seven days later, WT 1928z, IFN-γ −/− 1928z, or WT 19dz CAR T cells were transferred into mice. Peritoneal lavage cells were obtained 24–40 h after CAR T cell transfer. B – D Flow cytometry analysis of iNOS + cells among CD11b + F4/80 + macrophages ( B ), ARG-1 + cells among CD11b + F4/80 + macrophages ( C ), and CD11b + F4/80 + macrophages among CD45 + cells ( D ) at 24 h after CAR T cell transfer ( n = 6 mice/group). E CD11b + peritoneal myeloid cells were obtained 40 h following WT 1928z CAR T cell transfer. CD11b + cells were then ex vivo cocultured with fresh antigen-naïve CAR T cells and Eμ-myc cells in the presence or absence of L-NIL (50 μM) (coculture ratio, 1:1:1 = CD11b + cell:CAR T:Eμ-myc cell). Expansion of CAR T cells was measured over 48 h by a live cell analysis system (technical replicates from a single experiment, +Veh, n = 4; +L-NIL, n = 4; +CD11b + cells+Veh, n = 5; + CD11b + cells+L-NIL, n = 5). ( F ) Experimental settings for (G). G Percentage of survival of tumor-bearing mice treated with WT 19dz or WT 1928z CAR T cells receiving L-NIL or PBS (vehicle). Results are from two pooled independent experiments (PBS, n = 12 mice; L-NIL, n = 12 mice; 19dz CAR T + PBS, n = 11 mice; 19dz CAR T + L-NIL, n = 12 mice; 1928z+PBS, n = 28 mice; 1928z+L-NIL, n = 34 mice). H Percentage of iNOS + cells among CD45 + CD3 - CD20 - CD56 - CD11b + CD14 + monocytes in pre-lymphodepletion leukapheresis (DR, 19 patients; NDR, 32 patients). I Human CAR T cells were cocultured with Raji cells in the presence of human monocyte-derived unpolarized macrophages (unMac; n = 4 technical replicates from 1 human donor), IL-4, IL-10, IL-13-activated macrophages (imMac; n = 4 technical replicates from 1 human donor) or macrophages activated with serum from patients in complete remission (CR) at 3 months post-treatment or from non-responders (NR) (CR, 3 patients; NR, 7 patients). CAR T cell expansion was measured over 48 h by a live cell analysis system. Area under the curve (AUC) values were compared. Data in ( B – D ) are presented as mean ± SEM and ( E , I ) are presented as mean ± SD. For the box plot in ( H ), the center line represents the median, the bounds of the box indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, with all individual data points shown. Statistical significance was determined by one-way ANOVA with Bonferroni correction for multiple comparisons ( B – D ), unpaired two-sided Student’s t tests ( I ), two-sided log-rank Mantel-Cox test between the 1928z CAR T + PBS and 1928z CAR T + L-NIL groups ( G ), or two-sided Mann-Whitney U test ( H ). Exact P values are provided in the Source Data file. *, P < 0.05. Source data are provided as a Source Data file.

Article Snippet: The expansion of mCherry + CAR T cells was determined by an IncuCyte S3 live cell analysis system (Essen Bioscience).

Techniques: Injection, Flow Cytometry, Analysis, Ex Vivo, Cell Analysis, Derivative Assay, MANN-WHITNEY

Journal: Blood Advances

Article Title: CD19 CAR T-cell outcomes in relapsed/refractory extramedullary B-ALL: a multisite, retrospective cohort review

doi: 10.1182/bloodadvances.2025018604

Figure Lengend Snippet: Relapse and toxicity outcomes after CD19-CAR. (A) Cumulative incidence of relapse in the iBM (n = 21; green), EMD (n = 36; red), and CNS (n = 251; blue) cohorts, calculated using the Kaplan-Meier method. (B) Post-CAR T-cell relapse phenotype was assessed by flow cytometry and was reported as either CD19 + , CD19 − , lineage switch, or unknown. Relapse occurred in 134 patients (EMD n = 7; CNS n = 21; iBM n = 106). There were no statistical differences in the phenotypes among the cohorts. (C) Incidence of CRS in the different patient cohorts. Each pie chart represents 1 of the 3 cohorts as indicated. Displayed are patients with no CRS (blue), grade 1 to 2 CRS (gold), and grade 3 to 5 CRS (beige). CRS was graded by the reporting institution and included 126 of 308 (40.9%) cases graded according to the Lee criteria, 156 of 308 (50.6%) cases graded according to the University of Pennsylvania criteria, and 26 of 308 (8.4%) cases graded according to the American Society for Transplantation and Cellular Therapy guidelines. (D) The percentage of patients who developed neurotoxicity, as diagnosed by reporting institution, in the cohorts is indicated on the x-axis. (E) Reported reason for post-CAR HSCT in each individual cohort. Other encompasses 4 patients in the BM alone cohort who proceeded to transplant because of next-generation sequencing positivity in the absence of flow-detectable disease (n = 2), persistent BM aplasia (n = 1), and relapse with unknown phenotype characterized by the development of a scalp chloroma by day 28 after infusion (n = 1).

Article Snippet: R.A.G. reports a consulting relationship with Moonlight Bio; and royalties from Bristol Myers Squibb related to CAR T-cell technologies.

Techniques: Flow Cytometry, Transplantation Assay, Next-Generation Sequencing

Journal: bioRxiv

Article Title: In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

doi: 10.64898/2026.04.30.721530

Figure Lengend Snippet: NSG mice bearing SKOV3 tumors were treated with a single dose of 2×10 6 HER2-28z or CD19-28z CAR+ T cells (Control CAR-T cell). A ) Tumors were measured at indicated time points. Data are plotted as mean ±SEM of tumor volume (control CAR-T, n=6 tumors) or as individual tumors (HER2-CAR-T, n=7 tumors). Representative of n>5 experiments using different healthy donors. Timepoints at which CAR-TILs were isolated from tumors for use in downstream analyses are indicated. B ) Violin plots showing mean fluorescence intensity of PD-1 (right panel) and Ki67 (left panel) in effective compared to dysfunctional CAR-TILs. Each dot represents a tumor (n=4-5 healthy donors). C ) Ex vivo real-time cytotoxicity analysis of isolated CAR-TILs at effective (upper panel) and dysfunctional (bottom panel) phases against SKOV3 tumor cells (E:T=5:1). Data are plotted as mean ±SEM of normalized cell index of a representative experiment using one healthy donor. Time when T cells were added is indicated. D ) Analysis of IFNγ production by CAR-TILs after 24h of ex vivo co-culture with SKOV3 tumor cells (E:T=3:1). The PD-1/PD-L1 axis was inhibited by the addition of PD-L1 blocking antibody Durvalumab. Data are plotted as mean ±SEM (n=3 in effective, n=2 in dysfunctional and dysfunctional + durvalumab). Each dot represents a tumor used for isolating CAR-TILs. **p<0.01 by one-way ANOVA with Tukey’s multiple comparisons test. E ) IFNγ secretion by CAR-TILs after overnight stimulation with PMA/Ionomycin. Each dot represents a tumor used for isolating CAR-TILs (n=2 healthy donors). Fold change of effective vs dysfunctional is indicated. B and E ) **p<0.01, ***p<0.0001 by two-tailed unpaired t-test. Fold change relative to effective (Ki67, IFNγ) or dysfunctional (PD-1) is shown. F ) Principal component analysis of HER2-28z CAR-T samples from preinfusion, effective, and dysfunctional phases included in the RNA-seq analysis. Number of differentially expressed genes (p-adj. value<0.05 and logFc<1 or logFc<-1) between groups is indicated (n=7 healthy donors for preinfusion; n=6 for effective; n=5 for dysfunctional). G ) Ingenuity Pathway Analysis (IPA) of upregulated genes in dysfunctional compared to preinfusion samples. Significant selected pathways are ranked by p-value of enrichment. Orange bars denote NK cell-related pathways, and red bars indicate pathways of T cell exhaustion. H ) Heatmap showing the expression level of 17 TIL marker genes in preinfusion, effective and dysfunctional samples.

Article Snippet: S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Gilead.

Techniques: Control, Isolation, Fluorescence, Ex Vivo, Co-Culture Assay, Blocking Assay, Two Tailed Test, RNA Sequencing, Expressing, Marker

Journal: bioRxiv

Article Title: In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

doi: 10.64898/2026.04.30.721530

Figure Lengend Snippet: A ) Overlap of upregulated genes across comparisons between preinfusion, effective and dysfunctional CAR-TILs. A custom CRISPR/Cas9 library was generated by selecting 100 genes from early, late, and progressive dysfunction timepoints (indicated by circles). B ) Gene knockout enrichment following the in vivo screen was analyzed from Amplicon-seq data using the MAGeCK algorithm (n=3 healthy donors). The correlation between MAGeCK score and fold change is shown. Significantly enriched genes (MAGeCK score>1 and p-value<0.05) are indicated in black. Candidate genes used for further validation are highlighted. Safe harbour AAVS1 and ROSA26, and positive controls PD-1 and LAG-3 are also depicted. For simplicity of the plot, only genes with a MAGeCK score>1 are shown. C-F ) NSG bearing pre-established SKOV3 tumors were treated with a single dose of 2×10 6 untransduced T cells (UTD), anti-HER2-28z CAR+ T cells knocked out for candidate genes ZC3H12C or TG, or AAVS1. C ) Histogram of CAR expression in CD8+ T cells used for in vivo assessment of antitumor effect. D ) CRISPR/Cas9 efficiency represented as KO score at the end of primary T cell expansion. E ) Tumor volume of mice treated with KO CAR-T cells was analyzed at indicated timepoints. F ) Change in tumor volume on day 30 after CAR-T therapy versus baseline is plotted for individual tumors. G-J ) In a second experiment, the anti-tumor efficacy of ITGB8- or RGS2-KO CAR-T was evaluated as in (C-F). Histogram of CAR expression in CD8+ CAR-T cells used for this experiment ( G ), KO score ( H ), tumor volume of treated mice ( I ) and change in tumor volume at day 26 after CAR-T therapy ( J ). ( E and I ) Data are plotted as mean ±SEM of tumor volume (n=10-12 tumors per group). *p<0.05 by two-way ANOVA with Tukey’s multiple comparisons test at day 26 after CAR-T therapy. ( F and J ) *p<0.05, **p<0.01 by one-way ANOVA at indicated timepoint. K-L ) Analysis of total T cell concentration in the blood of animals treated with ZC3H12C-KO, TG-KO, ITGB8-KO, control AAVS1-KO CAR-T or UTD T cells (two independent experiments) at day 14 ( K ) and 21 ( L ) after T cell injection. Data are plotted as mean ±SEM. Each dot represents a mouse. Fold change relative to AAVS1-KO group is indicated. **p<0.01, ***p<0.001 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Gilead.

Techniques: CRISPR, Generated, Gene Knockout, In Vivo, Amplification, Biomarker Discovery, Expressing, Concentration Assay, Control, Injection

Journal: bioRxiv

Article Title: In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

doi: 10.64898/2026.04.30.721530

Figure Lengend Snippet: A-B ) Uniform manifold approximation and projection (UMAP) plot showing preinfusion CAR-T cells, and effective and dysfunctional CAR-TILs isolated from SKOV3 tumors. Data from one healthy donor independently analyzed. Similar results were observed in three healthy donors. Data are shown with merged timepoints (A) and separated timepoints (B). C ) Violin plots showing expression of ZC3H12C within clusters identified by single-cell RNA-seq analysis. D ) Genome browser tracks showing ATAC-seq signal at ZC3H12C, TOX and PD-1 regulatory regions within each of the clusters represented in A. E ) Motif analysis was performed to identify regulatory elements around the transcription start site (TSS) of differentially expressed genes in ZC3H12C-positive cells. Selected motifs for transcription factors associated with exhaustion from Top24 enriched motifs are shown. F ) T cell subtype classification integrated with the signature dataset reported by Zheng and colleagues . G ) UMAP plots of preinfusion CAR-T cells and effective and dysfunctional CAR-TILs color-coded by the density of expression of ZC3H12C, TG or ITGB8. H ) Dot plot showing expression of ZC3H12C and selected exhaustion-associated genes in meta-clusters identified in CD8+ T cells from Zheng, et al. 2021 . Exhaustion clusters are highlighted in bold. Color indicates effect size (ES) and size indicates the level of statistical significance.

Article Snippet: S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Gilead.

Techniques: Isolation, Expressing, Single Cell, RNA Sequencing

Journal: bioRxiv

Article Title: In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

doi: 10.64898/2026.04.30.721530

Figure Lengend Snippet: A) Schematic diagram illustrating the in vitro assay used to induce dysfunction through repetitive stimulation of CAR-T cells with antigen. B ) Population doublings of AAVS1-KO (left panel) and ZC3H12C-KO (right panel) CAR-T cells during in vitro restimulation with target cells. Each color represents a different healthy donor. C ) Cytotoxicity of AAVS1-KO and ZC3H12-KO CAR-T cells against fresh HCC1954 tumor cells (E:T=1:1) was measured at indicated timepoints during the restimulation assay by using a real-time assay. Mean ±SEM of normalized cell index of one representative donor for each timepoint is shown. Time when T cells were added is indicated. D ) TNF-α, IL-2, and IFNγ production by AAVS1-KO and ZC3H12C-KO CAR-T cells after 24h co-culture with fresh HCC1954 tumor cells (E:T=1:3) was measured at indicated timepoints of in vitro restimulation with tumor cells. Data are plotted as mean ±SEM. Each dot represents a healthy donor (n=5 for day 0 and day 7; n=4 for day 14). Fold increase of cytokine secretion by ZC3H12C-KO versus AAVS1-KO CAR-T cells is indicated. *p<0.05, **p<0.01 by two-tailed paired T-test. E ) Representative flow cytometry plots of intracellular TNF-α and IFNγ staining in AAVS1-KO and ZC3H12C-KO CAR-T cells (gated on live/CD45+) after 24h co-culture with fresh SKOV3 cells (E:T=1:3) on day 7 after repetitive antigen stimulation in vitro (top). Frequencies of single and double-positive T cells for IFN-γ and TNF-α are represented as absolute numbers (bottom). Data are plotted as mean ±SD. Each dot represents a healthy donor (n=5). *p<0.05, **p<0.01 by two-tailed paired T-test. Fold change of ZC3H12C-KO relative to AAVS1-KO is indicated (bottom).

Article Snippet: S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Gilead.

Techniques: In Vitro, Co-Culture Assay, Two Tailed Test, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

doi: 10.64898/2026.04.30.721530

Figure Lengend Snippet: A ) Schematic representing the experimental approach used to analyze the ex vivo function of ZC3H12C-KO CAR-TILs. B ) Analysis of CD8+ T cell infiltration in tumors treated with AAVS1-KO or ZC3H12C-KO CAR-T cells on day 17 after infusion. Representative flow cytometry plots showing CD45 and HER2 staining after excluding dead cells (left panel). Violin plots showing the frequency of live/CD45+ cells in tumors treated with AAVS1-KO or ZC3H12C-KO CAR-T cells (right panel). Each dot represents a tumor (n=2 healthy donors, 7-8 tumors per group). Fold change relative to AAVS1 is indicated. C ) Frequency of PD-1+ LAG-3+ TIM3+ CD8+ CAR-TILs (gated as live, CD45+). *p<0.05 by two-tailed unpaired t-test. D ) Ex vivo cytotoxicity of AAVS1-KO and ZC3H12C-KO CAR-TILs was analyzed against SKOV3 tumor cells (E:T=5:1) using a real-time assay. Time when T cells were added is indicated. Data are plotted as mean ±SEM of normalized index of two duplicates (n=1). E ) Cytokine production by AAVS1-KO and ZC3H12C-KO CAR-TILs after a 24h ex vivo co-culture with SKOV3 tumor cells (E:T=3:1). Data are plotted as mean ±SEM. Each dot represents a healthy donor (n=4). *p<0.05, **p<0.01 by two-tailed paired t-test. Fold change of ZC3H12C-KO relative to AAVS1-KO is indicated.

Article Snippet: S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Gilead.

Techniques: Ex Vivo, Flow Cytometry, Staining, Two Tailed Test, Co-Culture Assay

Journal: bioRxiv

Article Title: In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

doi: 10.64898/2026.04.30.721530

Figure Lengend Snippet: A-C ) NSG mice bearing subcutaneous L55-CD19 tumors were treated with a single dose of 4×10 6 control unstransduced (UTD) T cells, AAVS1-KO or ZC3H12C-KO CAR+ T cells. A ) Tumors were measured at indicated timepoints. Data are plotted as mean ±SEM of tumor volume (n=8-10 tumors per group). *p<0.05, by two-way ANOVA with Tukey’s multiple comparisons test at day 27. B ) Change in tumor volume on day 31 after CAR-T cell therapy. **p<0.01 by two-tailed unpaired t-test. C ) The concentration of total T cells was determined in the blood of animals treated with ZC3H12C-KO, AAVS1-KO ARI-0001 CAR-T cells, or UTD T cells, at day 14 (left panel) and day 21 (right panel) after treatment. Data are plotted as mean ±SD. Each dot represents a mouse (n=1 healthy donor, 8-10 tumors per group). ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. Fold change relative to ZC3H12C-KO group is indicated. D ) Schematic diagram illustrating the in vitro assay using hematological cancer cells. E ) T cell proliferation after an in vitro 7-day co-culture of CAR-T cells with NALM-6 or Ramos cell lines (E:T=1:2) analyzed by flow cytometry. For each cell line, T cell is represented as absolute numbers (left panel) or fold change compared to AAVS1-KO CAR-T cell group (right panel) (n=1 healthy donor). F-G ) NSG mice were intravenously injected with Nalm6-GFP-luciferase cells. After 7 days, mice were randomized and treated with 2×10 6 CAR+ T cells, including UTD control T cells, AAVS1-KO or ZC3H12C-KO ARI-0001 (n=10 mice per group, 2 healthy donors). F ) The Kaplan-Meier survival curves of treated mice are plotted and median survival days calculated. *p<0.05 by Log-rank (Mantel-Cox) test. G ) T cell persistence in the blood of treated animals was determined on days 7 and 14 after CAR-T cell therapy. Each dot represents a mouse. *p<0.05 by one-way ANOVA with Tukey’s multiple comparisons test. Fold change relative to ZC3H12C-KO group is indicated.

Article Snippet: S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Gilead.

Techniques: Control, Two Tailed Test, Concentration Assay, In Vitro, Co-Culture Assay, Flow Cytometry, Injection, Luciferase