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

Domain: chemistry
Published: 2026-02-27
Editorial outcome: major_revision

Abstract
CAR-T cell therapy is effective in hematologic malignancies but remains challenging in solid tumors owing to antigen heterogeneity and tumor microenvironment-induced exhaustion. Here, gene editing of the nuclear receptor NR2F6 restores CAR-T cell functionality, sustaining a TCF1⁺ progenitor-exhausted phenotype, enhancing metabolic fitness, and preserving cytotoxic potency under chronic antigen exposure. In immunocompetent models, Nr2f6-deficient CAR-T cells suppress solid tumor growth and induce robust, polyclonal host antitumor responses that persist after CAR-T clearance, as demonstrated by tumor re-challenge protection. Although infused CAR-T cells disappear within 2 weeks, durable tumor control coincides with epitope spreading and secondary immune responses, likely via dendritic cell reactivation. Protection against antigen-negative tumors and transferable immunity reveal a dual mode of direct cytotoxicity followed by durable immune reprogramming. This broadened host immunity may offset immune escape driven by antigen heterogeneity or loss, establishing NR2F6 inhibition as a promising CAR-T engineering strategy for durable, antigen-agnostic solid-tumor immunotherapy.

Introduction
Chimeric Antigen Receptor (CAR)-T cells combine the antigen recognition ability of antibodies with the cytotoxic effector function of T cells, enabling specific tumor eradication by immune-mediated cytotoxicity independent of the major histocompatibility complex (MHC) 1, 2. CAR-T cells have transformed the treatment landscape of hematologic malignancies, such as B cell acute lymphoblastic leukemia and diffuse large B cell lymphoma 3, 4, 5. Despite these successes, CAR-T cell efficacy in solid tumors remains limited. Tumor antigen heterogeneity, loss or downregulation of CAR target antigens, and the immunosuppressive tumor microenvironment (TME) collectively restrict durable anti-tumor responses 6, 7, 8. Metabolic dysfunction, hypoxia, and nutrient competition within the TME further impair CAR-T cell expansion and persistence by promoting exhaustion 9, 10, 11. Current approaches such as cytokine “armoring,” costimulatory optimization, or checkpoint blockade have achieved encouraging first benefits, but they often lack broad applicability.

Recent work indicates that durable tumor control requires coordinated activity between engineered CAR-T cells and the endogenous immune system. However, the mechanistic understanding of host tumor rejection pathways secondary to CAR-T therapy, particularly the contribution of tumor-derived epitope spreading (ES) that reactivates the non-CAR-T cell immune compartment, remains limited, posing a major challenge for the development of therapies capable of overcoming the profound antigenic heterogeneity characteristic of solid cancers. The phenomenon of ES refers to the induction and secondary amplification of immune responses directed against tumor antigens beyond the original therapeutic CAR target antigen 12, 13. However, the regulators of this secondary host immune activation remain insufficiently defined. In the context of adoptive cell transfer (ACT), a compelling strategy could be to utilize CAR-T cells for the targeting of one surface-expressed antigen while simultaneously inducing a robust endogenous T cell response against additional tumor antigens. This approach has the potential to effectively address the challenges posed by the high intra-tumor heterogeneity and antigen-loss-mediated immune escape. A growing body of evidence indicates that ES can be elicited and may play a role in the overall therapeutic outcome of cancer immunotherapy 14, 15. Armored CAR-T cells, engineered to express immunomodulatory mediators like CD40L or FLT3L, were demonstrated to elicit ES to improve anti-tumor immune response of endogenous immunity 16, 17. In the pre-clinical setting, the majority of CAR-T studies employed immunodeficient mice, which lack an endogenous T cell response. In immunocompetent murine models, CAR-T therapy appears to possess a limited capacity to elicit ES, particularly in solid tumors 18. Importantly, the mechanisms by which ES can be promoted during CAR-T therapy remain poorly understood.

Here, we focus our investigation on the role of the orphan nuclear receptor NR2F6, a member of the COUP TF family of nuclear receptors. Acting as an intracellular checkpoint, NR2F6 represses NFAT- and AP-1-dependent cytokine transcription thresholds in effector T cells. It functions mechanistically as a ligand-independent transcriptional repressor that antagonizes the DNA binding of these transcription factors, thereby suppressing the expression of key pro-inflammatory cytokines, including IL-2, IFNγ, TNF, IL-17, and IL-21 19, 20, 21. The activity of NR2F6 is tightly controlled by PKC-mediated phosphorylation, which modulates chromatin association and transcriptional repression 21. Through this molecular brake, NR2F6 maintains immune homeostasis and prevents excessive effector differentiation into highly activated T lymphocyte subsets 19, 20, 21. Inhibition of Nr2f6 enhances also NK cell effector function 22 and improves pathogen clearance in a preclinical Listeria monocytogenes model 23. Together with our previous studies identifying NR2F6 as an intracellular immune checkpoint in the effector T cell compartment 24, 25, these findings establish NR2F6 as a promising therapeutic target for enhancing anti-tumor immunity. Moreover, NR2F6 expression in melanoma cells contributes to immune evasion and tumor progression, underscoring its dual role in the tumor microenvironment 26. These findings emphasize the translational relevance of targeting NR2F6 in cancer immunotherapy and provide a rationale for exploring Nr2f6-edited CAR-T cells in solid tumor models.

We therefore hypothesized that deletion of Nr2f6 could reprogram CAR-T cells toward a metabolically fitter, less exhausted state, capable of initiating robust adaptive immune amplification through tumor antigen cross priming and epitope spreading. Here, we examine the consequences of Nr2f6 deletion in CAR-T cells directed against solid, syngeneic tumors in fully immunocompetent mice. We demonstrate that CRISPR/Cas9 engineered, Nr2f6-deficient CAR-T cells confer robust anti-tumor cytotoxicity and achieve superior tumor control compared with controls. Importantly, these cells maintain a sustained TCF1⁺ progenitor state, display enhanced metabolic resilience, and preserve potent effector functions under chronic antigen stimulation. As a direct consequence, Nr2f6 edited CAR-T cells provoke secondary, polyclonal host immune responses through immunogenic tumor cell death and activation of antigen presenting cell-mediated epitope spreading that extends beyond the CAR targeted antigen.

Using CRISPR/Cas9‑engineered CAR‑T cells in immunocompetent mouse models, we demonstrate that Nr2f6 deletion establishes durable antitumor immunity marked by sustained TCF1⁺ progenitor states, heightened cytokine production under chronic stimulation, and dendritic‑cell–dependent epitope spreading (ES). This integrated mechanism positions NR2F6 inhibition as a pivotal link between restored CAR‑T‑cell cytotoxicity within the immunosuppressive tumor microenvironment and the induction of antigen‑agnostic host immune memory. To our knowledge, this work provides the first experimental framework showing that gene editing can transform CAR‑T cells into robust catalysts of systemic, polyclonal antitumor immunity capable of overriding adaptive resistance in solid tumors.

Collectively, these findings position NR2F6 as a tractable metabolic checkpoint and articulate a conceptual blueprint for CAR-T engineering strategies that enhance secondary, polyclonal, antigen-agnostic host immune priming against additional tumor antigens beyond the CAR-targeted antigen, thereby overcoming tumor antigen heterogeneity as a central barrier to effective solid-tumor immunotherapy.

Results
CRISPR/Cas9-mediated Nr2f6 deletion enhances CAR-T efficacy across independent solid tumor models
Based on our previously published results, we hypothesized that depletion of Nr2f6 may improve CAR-T cell therapy in solid tumors. To test our hypothesis, we used an investigational second-generation CAR, directed against murine EpCAM, and a solid syngeneic tumor model involving the pancreatic ductal adenocarcinoma cell line Panc02 engineered to overexpress EpCAM in vivo 27. This 28 CAR consists of a scFv fragment, derived from the EpCAM antibody clone G8.8, fused to an intracellular CD28 as well as CD3ζ domain connected via a CD8 hinge and a CD28 transmembrane region (Fig1A). For translational reasons, we edited the Nr2f6 gene in primary mouse CD8+ T cells using CRISPR/Cas9. Synthetic guide (g)RNAs targeting the Nr2f6 locus were introduced by electroporation into Cas9 transgenic (Cas9tg) CD8+ T cells. Nr2f6 gene editing efficiency was determined by tracking of indels by decomposition (TIDE) analysis: Nr2f6.gRNA03 frequently yielded an efficiency >90%, whereas Nr2f6.gRNA04 was less efficient with 40% (Suppl.Fig4A). Of note, and of potential clinical relevance, we have previously demonstrated that even partial loss of Nr2f6, as seen in heterozygous knockout mice, is sufficient to elicit a robust anti-tumor phenotype 24. This indicates that full genetic inactivation may not be required and that haploinsufficiency alone can drive significant therapeutic effects. To assess the anti-tumor efficacy of non-targeting control (NTC) and Nr2f6crispr-/- CAR-T cells, we inoculated fully immunocompetent wild type mice subcutaneously with 70% antigen-positive 1x106 Panc02-EpCAM cells to introduce surrogate heterogeneity. Two days later, once the tumors had become established, we treated the mice intravenously with 3.6 × 106 CAR-T cells (Fig1B). Treatment of tumor-bearing mice with Nr2f6crispr-/- CAR-T cells resulted in reduced tumor growth and prolonged overall survival compared to control animals treated with NTC CAR-T cells (Fig1C-E). Notably, 55% (5/9) of animals in the knockout-treated group showed complete remission after tumor challenge versus 9% (1/11) in the NTC group. Similar results could be obtained in Nr2f6crispr-/- CAR-T-treated tumor bearing mice using an independent non-sequence overlapping gRNA indicating that indeed the knockout of Nr2f6 is responsible for the superior anti-tumor response (Suppl.Fig4B). Nr2f6 gene-edited polyclonal non-CAR transduced CD8+ T cells alone failed to induce an anti-tumor immune response in this harsh solid tumor model, suggesting that potent tumor rejection is dependent on both, CAR expression and deletion of Nr2f6 (Suppl.Fig4C).

To demonstrate broader applicability, we employed two independent CAR-T mouse models expressing an anti-human EGFR 28ζ CAR, together with syngeneic B16-F10 and MC38 tumor cells transduced with human EGFR (hereafter referred to as B16-F10-hEGFR and MC38-hEGFR). In both additional syngeneic tumor models, Nr2f6-depleted CAR-T-treated animals exhibited superior tumor control and prolonged survival compared with NTC CAR-T recipients (Suppl.Fig2A–G). These findings underline the broader relevance of Nr2f6-modified CAR-T cells, which achieve more effective and durable tumor clearance across distinct solid-tumor models.

Several clinical studies have highlighted the importance of CAR-T cell persistence regarding clinical outcome 28, 29. Therefore, we challenged Ly5.1+ mice with Panc02-EpCAM tumors and treated them with Ly5.2+ CAR-T cells to track transferred CAR-T cells. On day 3 after adoptive CAR-T cell transfer (ACT), we observed an increased fraction of Nr2f6crispr-/- CAR-T cells within the tumor compared to NTC-treated controls (Fig1F). Notably, flow cytometry analysis revealed that Nr2f6-depleted CAR-T cells in the tumor-draining lymph node (tdLN) express elevated levels of TCF1 protein (Fig1G, H), a key transcription factor shown to regulate T cell memory formation and stemness 30, 31. To assess their cytotoxic phenotype, we re-stimulated NTC and Nr2f6crispr-/- CAR-T cells from spleens of tumor-bearing mice ex vivo with target tumor cells. Nr2f6-modified CAR-T cells produced significantly higher amounts of interferon gamma (IFNγ) and granzyme B (GrzB) compared to controls (Fig1I, J), supporting their enhanced effector function. Of note, however, we did not observe long-term persistence of the CAR-T cells as they were undetectable in the spleen of all animals after day 13, regardless of treatment regimen (Suppl.Fig4D). Hence, we hypothesized that ACT with Nr2f6-modified (but not control) CAR-T cells might have facilitated a secondary immune response that appears to enable durable tumor rejection. Of note, and focusing on CAR persistence, some reports highlight the crucial role of CD4+ T cells promoting and sustaining CAR-T cell responses in long term responders 28. We transferred a mixture of CD8+ and CD4+ Nr2f6crispr-/- CAR-T cells to evaluate the impact of CD4+ T cell help but were unable to observe a beneficial effect on tumor growth comparing to CD8+ CAR-T cell only therapy (Suppl.Fig2H-K). Surprisingly, we could observe a comparable CD8+ only phenotype when transferring CD4+ Nr2f6crispr-/- CAR-T cells (Suppl.Fig9). Collectively, these results suggest that CRISPR/Cas9-mediated depletion of Nr2f6 in CD8+ CAR-T cells substantially improves therapeutic efficacy.

Nr2f6 deletion enhances CAR-T cytotoxicity
To explain our observed in vivo phenotype, we went back to phenotypically investigate the difference between wild type (WT) and Nr2f6-deficient CAR-T cells. Nr2f6 mRNA expression is markedly induced in WT T cells during CAR-T production (Suppl.Fig5A). This observation is of functional relevance, as NR2F6 is a negative T cell regulator that is induced upon TCR stimulation. That prompted us to test potential functional consequences of Nr2f6 depletion in CAR-T cells in vitro that may explain the superior therapeutic in vivo phenotype. We did not observe any differences in CAR transduction efficiency (Fig2A) or expansion of Nr2f6-/- or WT CD8+ T cells during CAR-T cell production (Suppl.Fig5B). Upon acute stimulation of CAR-T cells with Panc02-EpCAM at an effector to target (E:T) ratio of 5:1 overnight, Nr2f6-/- CAR-T cells produced higher levels of IFN, reflecting the results obtained from ex vivo stimulation (Fig2B). To test whether this is a 28ζ CAR-dependent phenotype, we also stimulated EpCAM CAR-T cells harboring a 41BB signaling domain with Panc02-EpCAM. Consistently with our previous results, Nr2f6-modified BB CAR-T cells produce more IFN compared to WT controls (Suppl.Fig5C).

We next asked if this augmented production is maintained during chronic stimulation. Loss of effector cytokine production represents a hallmark of exhaustion, which appears to be one of the major hurdles of optimal CAR-T cell efficacy against solid tumors in vivo 32, 33. To address this issue, we established an in vitro chronic tumor antigen stimulation assay forcing 28 CAR-T cells into an exhaustion-like state (Fig2C, Suppl.Fig5D). Assessing different E:T ratios, 5:1 particularly induced the expression of multiple inhibitory receptors in WT CAR-T cells, frequently found in exhausted T cells (Suppl.Fig5D). Of note, chronic stimulation similarly increased Nr2f6 mRNA levels in WT CAR-T cells in this setting (Suppl.Fig5E). Nr2f6-/- CAR-T cells challenged over multiple rounds with Panc02-EpCAM cells showed superior killing capacity (Fig2D) accompanied by increased IFN and GrzB secretion when compared to WT CAR-T cells (Fig2E, F). Additionally, we subjected CAR-T cells to bulk RNA sequencing (RNAseq) on day 8 during chronic stimulation to elucidate transcriptional differences. Gene ontology (GO) overrepresentation analysis of differentially expressed genes (DEGs) revealed enrichment of T cell activation and cytotoxic signaling pathways, supporting the enhanced antitumor potential of Nr2f6-modified CAR-T cells (Fig2G, H).

To exclude off-target killing via death receptors like Fas or NKG2D, we co-cultured wild type CAR-T cells with Panc02 tumor cells expressing or not expressing the CAR-targeted antigen EpCAM. We could confirm that only CAR antigen-positive Panc02 cells were killed in a CAR-dependent manner (Suppl.Fig5F). CAR-T cells were shown to primarily kill their targets in a perforin-granzyme B-dependent manner, which in turn leads to activation of pro-apoptotic caspase 3, but also to cleavage of gasdermin E (GSDME) ultimately resulting in tumor cell death. The active amino-terminal domain of GSDME has membrane pore-forming properties converting non-inflammatory apoptosis into a highly immunogenic form of cell death, termed pyroptosis 34, 35. To further validate the superior killing phenotype of Nr2f6-/- CAR-T cells at the tumor target cell level, we assessed GSDME clipping on day 4 of chronic stimulation using Western blot. Quantification revealed that tumor cells co-cultured with Nr2f6-modified CAR-T cells showed a significantly increased GSDME clipping activity of full- length GSDME to N-GSMDE when compared to wild type CAR-T cells (Fig2I). Together, these data provide convincing evidence that Nr2f6 deletion in CAR-T cells enhances their anti-tumor potency, which is associated with enhanced cytotoxic activity against solid tumors in vitro and preserved functionality during repetitive stimulation.

Nr2f6 ablation preserves TCF1⁺ stem-like progenitors and metabolic fitness
Next, we set out to determine a deeper phenotypic characterization of our CAR-T cells during chronic stimulation in vitro. Loss of Nr2f6 resulted in a notable increase in the number of CD62L-CD44 double-positive cells (Fig3A), with a shift toward a central memory (CM)-like phenotype. We next asked whether this increase in double-positive CAR-T cells with persistent cytokine secretion would also result in a difference in the manifestation of exhaustion. Indeed, when we examined PD-1+ CAR-T cells, we identified a significant difference in the ratio of CD8+ progenitor exhausted T cells (Tpex, Ly108+Tim3-) to terminally exhausted T cells (Ttex, Ly108-Tim3+) (Tpex/Ttex) (Fig3B) as another underlying mechanism for the benefit of Nr2f6-modified CAR-T cells. In addition, FACS analysis revealed that Nr2f6-/- CAR-T cells expressed more TCF1 on day 4 of co-culture (Fig3C) consistent with previously published data showing that Tpex cells remain TCF1 positive 36. Collectively, these data demonstrate that Nr2f6 loss is associated with decreased terminal differentiation and increased expression of TCF1 maintaining a progenitor exhausted state.

To maintain their cytotoxic machinery, especially in the TME, CD8+ T cells require high metabolic activity. Therefore, we next assessed the metabolic profile of Nr2f6-deleted and WT CAR-T cells during chronic stimulation using the Seahorse Cell Mito Stress Test on day 4 of co-culture, when we observed the greatest difference in Tpex/Ttex ratio. Analysis showed that Nr2f6-modified CAR-T cells exhibited a superior resilience towards stress, which is paralleled by higher basal and maximum oxygen consumption rates (OCR) when compared to WT CAR-T cells (Fig4A). In addition, knockout CAR-T cells have a higher extracellular acidification rate (ECAR) due to an increased glycolytic potential (Fig4B). Consistent with this phenotype, GO overrepresentation analysis of bulk RNAseq data from day 4 of chronic stimulation showed enrichment in key metabolic pathways (Fig4C-D). Gene set enrichment analysis (GSEA) between knockout and WT CAR-T cells revealed that gene sets associated with OXPHOS, glycolysis and fatty acid oxidation (FAO) were significantly up-regulated in Nr2f6-modified CAR-T cells (Fig4E). Collectively, all in vitro characterization results indicate that Nr2f6 knockout in CAR-T enhances performance by increasing cytotoxic capacity, maintaining effector cytokine production and preventing terminal exhaustion/differentiation, thereby preserving a pool of Tpex cells in vitro. Mechanistically, loss of the transcription factor NR2F6 leads to metabolic reprogramming, increasing both OXPHOS and glycolysis potential to meet energy demands during repetitive stimulation.

Nr2f6 ablation facilitates immunogenic tumor cell death and activation of host immunity
Since we did not use lymphodepletion and because CAR-T cells disappeared over time, we next concentrated on better understanding the long-term cancer control triggered by ACT of Nr2f6-modified CAR-T cells with a particular focus on the role of the endogenous immune system. More specifically, we determined whether Nr2f6crispr-/- CAR-T cells boost the endogenous immune system by “warming up the TME” leading to ES. Immunogenic cell death (ICD) is known to promote priming and activation of the endogenous immune system, ultimately resulting in polyclonal and durable tumor cell clearance. To test this hypothesis of an ES-mediated secondary host immune activation event by the Nr2f6-modified CAR-T treatment regimen, we injected immunodeficient Rag1-/- recipients, who lack an endogenous adaptive immune compartment, with Panc02-EpCAM followed by treatment with Nr2f6crispr-/- or NTC CAR-T cells two days later (Fig5A). In strict contrast to the results obtained with immunocompetent wild type recipients (shown above in Fig1C-E), treatment with Nr2f6-modified CAR-T cells did not result in a tumor growth inhibition or a survival benefit (Fig5B, C). The loss of therapeutic effects in Rag1-/- again indicates that an endogenous adaptive immune cell response is required for the superior tumor control mediated by Nr2f6-modified CAR-T cells, promoting a secondary response by the endogenous immune system, which is present only in immunocompetent WT mice.

To further validate these findings, we isolated splenocytes from Nr2f6crispr-/- and NTC CAR-T-receiving immunocompetent WT animals 8 days after tumor injection and subjected them to FACS analysis (Fig5D). Analysis revealed that immunocompetent mice treated with Nr2f6crispr-/- CAR-T cells showed an enrichment of conventional dendritic cell type1 (cDC1) cells in the spleen (Fig5E), as well as increased IL-12p40 in the serum, the master cytokine to prime T cell effector function (Fig5H). cDC1 and their cytokines have been shown to be essential for anti-tumor immunity, primarily by priming and supporting the differentiation of cytotoxic T lymphocytes (CTLs) 37. To further support our hypothesis that the secondary host immune response is triggered by the superior cytotoxic activity of Nr2f6-modified CAR-T cells due to ES, we performed ELISpot experiments. In the absence of the CAR-targeted tumor antigen EpCAM, polyclonal anti-Panc02 T cell responses were evaluated. Using previously established Panc02 neoantigens 38, an ELISpot assay was performed using isolated endogenous CD3+ T cells from spleens from tumor-bearing animals on d19 after treatment with either NTC or Nr2f6crispr-/- CAR-T cells (Fig.5D). The host CD3+ T cells were stimulated ex vivo with DCs loaded with either peptide pools of the described Panc02 neoantigens (Suppl.Table1), primary tumor samples or Panc02 cell line lysates. As a result, we were able to validate DC-mediated ES via T cell priming. Treatment with Nr2f6crispr-/- CAR-T cells, but not, or at least to a much lesser extent, with NTC CAR-T cells, resulted in a significantly enhanced endogenous polyclonal T cell response as evidenced by increased GrzB production (Fig5G, H). Therefore, this durable tumor control observed after the apparent disappearance of CAR-T cells can be explained by ES and the subsequent induction of a secondary, tumor-antigen–specific host T cell response.

To confirm this host T cell response in more detail, we focused on CD8+ T cells, because they were shown to be the most relevant cell type for anti-cancer adaptive immunity. Therefore, we again treated immunocompetent Panc02-EpCAM tumor-bearing mice with either NTC or Nr2f6crispr-/- CAR-T cells and subjected spleens, tdLNs and tumors to FACS analysis 3 days after ACT. We discovered an increased fraction of endogenous CD8+ T cells in the spleen of Nr2f6-modified CAR-T-treated animals (Suppl.Fig3A). Furthermore, these CD8+ T cells showed an attenuated exhaustion phenotype (more Ly108+Tim3- cells) compared to CD8+ T cells from NTC receiving mice (more Ly108-Tim3+ cells) (Suppl.Fig3B-E). Importantly, Nr2f6-depleted CAR-T-treated mice exhibited an increased Tpex/Ttex ratio among CD8+ T cells in the spleen compared to control-treated animals (Suppl.Fig3D). Analysis further revealed that Nr2f6crispr-/- CAR-T-treated animals showed an increased number of NK cells as well as increased IFN-producing endogenous CD8- T cells in the tdLN compared to control-treated mice (Suppl.Fig3F, G). Since recruitment of immune cells is dependent on cytokines and chemokines, we performed a cytokine array assay using lysates from whole spleens. The assay revealed that several inflammatory cytokines (IL13, MIP-1, MIP-2, IL-12) were elevated in Nr2f6crispr-/- CAR-T-treated mice compared to controls (Suppl.Fig3H). Overall, we observe an apparently facilitated cooperative anti-tumor effect of Nr2f6-modified CAR-T therapy and endogenous innate and adaptive immune cell compartments, initiating a cascade of endogenous immune priming.

Epitope spreading facilitates antigen-agnostic durable immunity
Crucially, to ascertain whether Nr2f6crispr-/- CAR-T cell therapy promotes ES and subsequently the activation of the endogenous immune system in vivo, we simultaneously challenged the Nr2f6-modified CAR-T-treated complete responder (CR) cohort with EpCAM-positive and EpCAM-negative Panc02 tumors injected in the right and left flank, respectively (Fig6A). In line with our hypothesis, each Nr2f6-modified CAR-T complete-responder mouse eradicated both EpCAM-positive and EpCAM-negative Panc02 tumors (Fig6B) and showed superior survival compared with tumor-bearing control mice (Fig6C). It is important to note that this secondary immune response apparently was EpCAM independent, which provides strong evidence in support of ES as the underlying process in driving effective polyclonal host anti-tumor immune responses. Consistently, we hypothesized that cDC1-mediated ES may have occurred in the Nr2f6crispr-/- CAR-T treated CR cohort as a mechanistic basis for the efficient rejection of EpCAM-negative tumors. We again exposed the CR cohort to re-challenge with EpCAM-negative Panc02 tumors (Fig6A) and evaluated the endogenous immune response ex vivo using CD8+ T cells from CR (Fig6E) as well as with 10X single-cell RNA and TCR sequencing (scRNA-seq, scTCR-seq). To assess whether anti-Panc02 tumor immune protection could be transferred to WT mice bearing Panc02-EpCAM negative tumors, we performed an ACT using Nr2f6-modified CAR-T cell-treated CR splenocytes. The results demonstrated that these splenocytes were capable of effectively controlling tumor growth and exhibited a survival benefit when directly compared to animals treated with splenocytes from Panc02 antigen-naïve tumor-bearing mice (Fig6D). An ex vivo recall assay demonstrated that endogenous polyclonal CD8+ T cells from Nr2f6-modified CR exhibited heightened IFN production compared to those from Panc02 tumor antigen-naïve tumor-bearing mice when re-stimulated with tumor cells ex vivo (Fig6E). The combined data set provides evidence of a long-lasting CAR-targeted EpCAM antigen-independent immune response accompanied by a transferrable superior tumor rejection phenotype in antigen-naïve tumor-bearing mice when using solely splenocytes isolated from Nr2f6crispr-/- CAR-T-treated CR mice. It is therefore evident that the augmented effector functions of Nr2f6-modified CAR-T cells play a central role in the initial control of tumors. After this initial phase, the role of ES becomes pivotal in sustaining durable endogenous immunity and preventing tumor relapse.

Integrated remodeling of the tumor–immune landscape by Nr2f6-edited CAR-T therapy
To mechanistically understand architectural changes of TILs in the Nr2f6-modified CAR-T cell treated CR cohort, we analyzed scRNA- and paired α/β scTCR-seq data obtained from CD45+ TILs on d305 after initial tumor injection and CAR-T cell application (Fig6A). We identified several clusters representing prominent immune cell subtypes (Fig7A) based on their characteristic expression profiles (Suppl.Table2). Strikingly, we observed a marked difference in cell composition between Nr2f6-modified CAR-T cell treated CR and Panc02 tumor antigen-naïve mice. Nr2f6-modified CAR-T cell-treated CR had reduced numbers of exhausted CD8+ and regulatory T cells and showed, among other changes, an increase in proliferating CD8+ T cells and cDC1s (Fig7B). It is noteworthy that, in the Nr2f6-modified CAR-T cell-treated CR cohort, no CAR+ T cells were detected among TILs, thereby firmly excluding their residual contribution to tumor clearance. Consistent with our working hypothesis, GO analysis of DEGs revealed pathway changes in both proliferating CD8+ T cells (Fig7C) and cDC1s (Fig7D). CD8+ proliferating T cells exhibited an enrichment of pathways linked to positive regulation of T cell differentiation, IL-2 and type II interferon production as well as activation of  T cells (Fig7C). cDC1s showed overrepresentation of terms associated with T cell cytokine production, activation, and differentiation as well as response to type II interferon (Fig7D). To further validate whether ES had occurred, we determined TCR clonality and diversity by scTCRseq. Compared to Panc02 tumor antigen-naïve mice, Nr2f6-modified CR showed a decrease in clonality with a concomitant increase in diversity, indicating that T cells were primed against various epitopes without a few being prominent (Fig7E). This pattern was not observed during the initial CAR-T treatment phase; however, enrichment of CD3⁺ T cells following Nr2f6-edited CAR-T therapy (Suppl.Fig8A-C) supports progressive enhancement of endogenous immunity over time, consistent with recent reports39, and underscores that broadening and adaptation of the endogenous TCR repertoire are predominantly manifested during the memory phase after CAR-T therapy rather than during immediate effector phase.

Safety and translational implications
Although EpCAM is expressed on normal tissue, no evident immune-related adverse effects were observed in mice treated with Nr2f6crispr-/- α EpCAM CAR-T cells. Treated animals showed stable weight and serum cytokine levels comparable to controls (Suppl. Fig1A–C). These data indicate an absence of detectable acute toxicity and suggest a favourable short-term safety profile. Importantly, non-lymphodepleting treatment retained efficacy, implying that Nr2f6 deletion may enhance CAR-T cell activity without increasing immune toxicity. Together, these findings provide a preliminary basis for further evaluation of NR2F6 as a potentially safe intracellular target for augmenting CAR-T therapy in solid tumor models (see schematic cartoon in Fig7F-H).

Methods
Animals
Male and female C57BL/6N (purchased from Charles River; RRID:IMSR_CRL:027) , B6(C)-Gt(ROSA)26Sorem1.1(CAG-cas9*,-EGFP)Rsky/J (purchased from Jackson; RRID:IMSR_JAX:028555), Pep Boy (B6.SJL-Ptprca Pepcb/BoyJ, B6 CD45.1, Ly5.1) (purchased from Jackson; RRID:IMSR_JAX:002014), Rag1-/- (B6.129S7-Rag1tm1Mom/J; purchased from Jackson; RRID:IMSR_JAX:002216), and Nr2f6-/- (described previously in 55) mice were housed under specific pathogen-free (SPF) conditions, light/dark cycle (12/12), 21-23°C ambient room temperature with 45% humidity, and the mice had ad libitum access to water and food. Animals were controlled frequently by the staff of the animal facility and us. All animal experimentation was performed according to European guidelines and approved by the Austrian federal ministry of education, science and research (2023-0.203.973). When necessary, mice were euthanized using carbon dioxide (CO ) and cervical dislocation.

Cell lines
The 3-methylcholanthrene-induced mouse pancreatic ductal adenocarcinoma cell line Panc02 (RRID: CVCL_D627) was stably transduced to express murine EpCAM and was a kind gift of Prof. Kobold (LMU, Munchen) and has been previously described 56, 57. Panc02-EpCAM negative tumor cells were enriched using flow sorting. Panc02-EpCAM cells expressing the fluorescent protein GFP were generated by retroviral transduction using the pMP71-GFP construct 58. GFP+ Panc02-EpCAM cells were enriched using flow sorting. B16-F10-hEGFR and MC38-hEGFR were generated in-house by lentiviral transduction using the PWPXLD-EGFR-WT construct (kind gift of Chay Kuo, Addgene, #133749). Mouse tumor cell lines were maintained in DMEM + 10% fetal calf serum (FCS) + 2mM L-Glutamine (L-Glut) + 100U/mL penicillin + 100mg/ml streptomycin, further referred to as DMEM+++, The PlatinumE (PlatE) cells were obtained from Cell Biolabs, Inc. (RV-101; RRID: CVCL_B488) and were tested negative for mycoplasma (Invivogen, rep-mys-20). They were cultured in DMEM+++, supplemented with 1 µg/mL puromycin (Merck, P8833) and 10 µg/mL blasticidin (Sigma, 15205), to ensure transgene expression. This medium is referred to as PlatE medium. All cell lines were cultured in T175 tissue culture flasks (Sarstedt, 833912002), passaged every two days, and routinely tested for mycoplasma, with negative results.

Cell line passaging and harvest
Adherent cell lines were passaged as follows: cell culture vessel, typically a T175 tissue culture flask (Sarstedt, 833912002), was washed once with 6mL warm 1x phosphate buffer saline (PBS) and then dissociated using 3mL TrypLE Express (Thermofisher, 12604021)for a few minutes at 37°Cuntil cells started to float. Reaction was stopped by adding twice the amount of cell culture medium. Cell suspension was transferred to a Falcon tube and centrifuged at 300 × g/rcf for 5 minutes at room temperature (RT). Cells were then used for in vitro assays, in vivo experiments or resuspended in 1mL cell culture medium and split accordingly: tumor cells typically 1:10 and PlatE 1:5 depending on application.

Primary mouse T cell isolation
Primary mouse T cells were isolated by negative selection using primarily the CD8a+ T cell isolation kit (Miltenyi, 130-104-075) or once the untouched CD4 isolation kit (Miltenyi, 130-104-454). In brief, mice were euthanized and spleen as well as lymph nodes were excised. After pressing organs through a sieve to achieve a single cell suspension, cells were resuspended in 3mL red blood cell (RBC) lysis buffer (0.15M NH Cl, 10mM KHCO , 0.1mM ethylenediaminetetraacetic [ETDTA] dehydrate in ddH O) per spleen and incubated for 5min at RT. Reaction was stopped by adding twice the amount of buffer C (PBS supplemented with 0.5% bovine serum albumin [BSA] and 2mM EDTA). Splenocytes were counted and incubated with biotinylated antibodies and magnetic beads according to manufacturer’s instructions. CD8+ or CD4+ T cells were enriched by applying the cell suspension on LS columns (Miltenyi, 130-042-401) placed onto a strong magnet. Columns were flushed twice with 3mL of buffer C and flow through was collected. Enriched T cells were then counted with LUNA automated cell counter (Logos Biosystems) and used for downstream applications. Enriched T cells were checked for purity using FACS analysis.

Plasmids and constructs
For retroviral production, the pMP71 and MSCV backbones (provided by C.Baum, Hannover or purchased from Vectorbuilder) were used for transfections. The second-generation chimeric antigen receptor (CAR) targeting murine EpCAM on the pMP71 backbone was previously described 56, 57. The CAR targeting human EGFR on a MSCV backbone was purchased from Vectorbuilder. Briefly, the CARs consist of a single-chain variable fragment (scFv) (EpCAM: clone G8.8, EGFR: Cetubixmab) fused to a CD8 hinge and a CD28 transmembrane region. Intracellularly, they consist of a CD28 co- (UniProt entry P31041; amino acids 151–218) and a CD3zeta stimulatory domain (UniProt entry P24161; amino acids 52–164). CAR detection was either possible by a myc-tag between the scFv and the hinge region or by GFP expression, which was separated from the CAR by a P2A self-cleavage sequence. The pMP71-GFP plasmid 58 was used as a control and to transduce tumor cell lines. The PWPXLD-EGFR-WT was a gift from Chay Kuo (Addgene plasmid # 133749,RRID:Addgene_133749) and was used to transduce the melanoma tumor cell line B16-F10 and the colorectal cancer cell line MC-38. All plasmids were visualized on https://benchling.com.

Transfection of PlatE cells and virus production
For optimal viral production, PlatE cells were split three times a week and cultured not more than 12 weeks after thawing as recommended by the supplier. The day prior to transfection, 8 × 105 PlatE cells were seeded into tissue culture-treated 6-well plates (Szabo Scandic, 35 3046) in PlatE medium and incubated overnight at 37°C and 5% CO to reach a confluency of around 70%. On the day of transfection, PlatET medium was discarded and 3mL DMEM supplemented with 10% FCS and 2mM L-Glut without antibiotics was added. Transfection was carried out using the calcium precipitation method: per well, 15µL calcium chloride (2.5M) was mixed with 18µg of transfer plasmid (pMP71-CAR or pMP71-CAR-GFP) and ddH 0 to a final volume of 150µL. This was slowly added into 150µL transfection buffer (1.6g NaCl (Merck, 1064041000), 74mg KCl (VWR, 1049361000), 50mg Na HPO (Sigma Aldrich, 255793), 1g HEPES (Sigma Aldrich, H0887), in 100mL H O, pH 7.1) in a dropwise manner while vortexing constantly. The mix was incubated for 30min at RT. 300µL of the transfection mix was then added onto PlatE cells and incubated for 6 hours at 37°C 5% CO . After incubation, medium was discarded and 3mL DMEM+++ were added and plates were put at 37°C 5% CO for 42 hours.

T cell activation and culturing for CAR transduction
12-well plates (Fisher Scientific, 150628) were incubated with 5µg/mL CD3 antibody (BioXCell, CP082) solution overnight at 4°C. After negative selection on day 0, CD8 or CD4 T cells were resuspended at 3 × 106/mL in CAR RPMI (RPMI 1640 + 10% FCS + 100U/mL penicillin + 100mg/ml streptomycin (M&B Sticker, P06-07100) + 2mM L-Glutamine (M&B Sticker, P04-80050) + 1mM sodium pyruvate (Sigma Aldrich, S8636) + 1mM HEPES (Sigma Aldrich, H0887) + 50µM 2-mercaptoethanol (Sigma Aldrich, M3148)). Coated plates were washed twice with 1x PBS before seeding 3 × 106 T cells into pre-coated wells. Medium was supplemented with 1µg/mL CD28 antibody (BioXCell, CP042) and 10ng/mL IL-2 (Biolegend, 589106). Plates were incubated overnight at 37°C and 5% CO .

Retroviral transduction of T cells and CAR detection
In general, CAR-T cells used for in vivo experiments were transduced with a CAR together with a GFP reporter, whereas CAR-T cells for in vitro experiments were transduced with a CAR only. For viral transduction, non-tissue culture-treated 24-well plates (Corning, 351147) were coated with 12.5µg/mL Retronection solution (Takara, T 100B) overnight at 4°C. The next day, plates were blocked with ddH O supplemented with 2% BSA fraction V (Roth, T844.2) for 30min at 37°C. In the meanwhile, supernatant containing CAR viral particles was harvested from PlatE cells and filtered through a 0.45µm cellulose-acetate syringe filter (Integra Biosciences, 158015). 2mL fresh DMEM+++ was added to PlatE cells to harvest supernatant again after 24 hours. After blocking, plates were washed with 1x PBS supplemented with 25mM HEPES and finally 2mL of viral supernatant was added to each well of the Retronectin-coated plate and spun at 3000 × g for 2 hours at 4°C. One hour before the end of the spin, T cells were prepared. Approximately 22 hours after plate-bound activation, T cells were harvested and counted using a LUNA automated cell counter. T cells were adjusted to 1 × 106/mL in CAR RPMI supplemented with 10ng/mL hIL-2 and mouse CD3/CD28 activation beads (Thermo Fisher, 11453D) at a ratio of one bead per T cell (25µl bead solution/mL). After the spin, viral supernatant was discarded, and 1mL T cell suspension was added per well. Plates were spun at 800 × g for 30min at 32°C and put at 37°C 5% CO overnight. The next day, viral supernatant was harvested again from PlatE cells and filtered through a 0.45µm cellulose-acetate syringe filter (Integra Biosciences, 158015). 0.5mL of the viral particle containing supernatant was mixed with 0.5mL CAR RPMI and 1mL of the mix was added directly to T cells. Plates were then spun a second time at 800 × g for 30min at 32°C and incubated for another 5 hours. After this last incubation, T cells were harvested, activation beads were removed, and T cells were counted. Cells were then adjusted to 1.5 × 106/mL in CAR RPMI supplemented with 10ng/mL interleukin 15 (IL-15, Biolegend, 566302) as well as interleukin 7 (IL-7, Biolegend, 577804) and seeded into 24- or 12-well plates. Cultures were maintained at 1.5 × 106/mL in IL-15 and IL-7 containing medium. Transduction efficiency of αEpCAM CAR was assessed either by using a c-myc FITC antibody (Miltenyi; clone: REA1287, 130-116-485) or GFP by flow cytometry. For detection of αEGFR CARs, T cells were stained with recombinant biotinylated EGFR (Creative Biolabs, CARD-LX025) and Streptavidin coupled to bv421 (Biolegend, clone 3A20.2 405226). For in vitro experiments CAR-T were used on the 5th day after isolation, whereas for in vivo experiments CAR-Ts from the 7th day were used.

Genome editing of CAR-T cells using CRISPR/Cas9
For acute depletion of Nr2f6 in CD8+ and CD4+ (CAR) T cells, the CRISPR/Cas9 system was applied. In this case, (CAR) T cells from Cas9 transgenic mice were electroporated with synthetic Nr2f6 targeting guide (g)RNAs (Horizon Discovery, see below and Suppl.Table4) 48 hours after T cell activation using the Amaxa mouse T cell nucleofactor kit (Lonza, VVPA-1006) on the nucleofector 2b device (Lonza Biosciences). In this study, different kinds of guides with different targeting sites in the Nr2f6 gene were used. sgRNA.3 (sg03) was used directly, whereas crispr (cr)RNA.4 (crRNA.4) and the non-targeting control (NTC) crRNA were annealed with a corresponding tracr RNA to yield a fully competent gRNA. Therefore, 200µM crRNA and 200µM tracrRNA solution in a ratio of 1:1 were heated to 95°C in a thermocycler and left to cool at RT to allow sufficient complexing. Five hours after the second spin transduction, transduced T cells were harvested and counted. 1 × 107 T cells were pelleted and transfected with 3.1µM Nr2f6 or NTC gRNA solution. T cells were electroporated using the X-001 program on a Nucleofector 2b device (Lonza) and immediately recovered in transfection medium supplemented with 10% FCS, 2mM L-Glut, 100IU/mL Pen/Strep and medium component A and B in a 12-well plate according to manufacturer’s protocol. After a 1-hour rest, 1mL CAR RPMI containing 20ng/mL IL-7 (Biolegend, 577804) and IL-15 (Biolegend, 566302) was added per 1mL T cells. T cells were passaged the next day. Gene editing efficiency was analyzed using the Tracking of Indels by Decomposition (TIDE) algorithm 59. Nr2f6-crRNA.4 (CM-045088-04-0010; 5’ CTCAAGAAGTGCTTCCGGGT 3’); Nr2f6-gRNA.3 (SG-045088-03-0010; 5’ CCGCAATCTCAGCTACACCT 3’), NTC-gRNA (U-007501-01-20), tracrRNA (U-002005-50). See also Suppl.Table4.

Tumor inoculation and ACT with gene-edited CAR-T cells
Panc02-EpCAM, B16-F10-hEGFR or MC38-hEGFR were cultured as described above and were checked for EpCAM or EGFR expression by flow cytometry prior to injection. The day before, tumor cells were split 1:2 to ensure ~70% confluency on day of injection for sufficient engraftment in vivo. Mice used for in vivo experiments were shaved a few days before the injection of tumor cells to mitigate stress. For Panc02-EpCAM experiments, 1 × 106 syngeneic tumor cells were injected subcutaneously (s.c) into the right flank of 8- to 12-week-old immunocompetent female mice. Both homogenous, e.g. 100% EpCAM positive or 100% EpCAM negative, and heterogeneous, e.g. 70% EpCAM positive + 30% EpCAM negative, tumor loads were used, respectively. For B16-F10-hEGFR and MC-38-hEGFR experiments, 1 × 106 (B16-F10-hEGFR) or 5 × 105 (MC-38-hEGFR) tumor cells were injected subcutaneously (s.c) into the right flank of 8- to 12-week-old immunocompetent female wild-type mice (Charles River, 027C57BL/6). Two days (Panc02-EpCAM) or 5 days (B16-F10-hEGFR, MC38-hEGFR) later when tumors reached an approximate size of 50 mm3, tumor bearing mice were randomly assigned into different treatment groups based on their tumor size and injected intravenously (i.v.) with 3.6 × 106 (Panc02-EpCAM and MC-38-hEGFR) or 1 × 106 (B16-F10-hEGFR) NTC or Nr2f6 CRISPR/Cas9 gene-edited CD8+ CAR-T cells in 50µL PBS (used for almost all experiments), or 4 × 106 Nr2f6 CRISPR/Cas9 gene-edited CD8+ + CD4+ CAR-T cells (Suppl.Fig2H-K). As a control, NTC as well as Nr2f6-depleted polyclonal CD8+ T cells were injected i.v. into Panc02-EpCAM tumor bearing mice. Tumor growth and mouse weight were assessed three times per week using a digital caliper and a scale, respectively, and tumor size was calculated with the following equation: 𝑉 = 1 ⁄ 𝑥 (𝑙𝑒𝑛𝑔𝑡ℎ 𝑥 𝑤𝑖𝑑𝑡ℎ2). Tumor measurement was performed in a double-blinded fashion. Mice were euthanized using CO and cervical dislocation when tumors reached 1500mm3, a 20 percent weight loss was observed or when animals were multimorbid. For survival analysis, data were collected in Kaplan-Meier plots. For experiments using immunodeficient Rag1-/- mice, animals were inoculated with 5 × 105 Panc02-EpCAM cells and treated with 1 × 106 gene-edited CD8+ CAR-T cells on day two after tumor injection.

Detection of CAR-T cells and analysis of innate immune cells in tumor bearing mice using multicolor flow cytometry
Ly5.1 recipient mice were challenged with Panc02-EpCAM tumors and treated with NTC or Nr2f6 gene-edited CAR-T cells two or three days later as stated above. For CAR-T cell detection over time, blood was collected from tumor-bearing mice using a lancet and an EDTA (Sigma Aldrich, 03690) containing tube on day 6 and 13 after tumor injection. Red blood cell (RBC) lysis was performed using 40µL blood and in-house Erylysis buffer (described above). Cells were washed thoroughly with buffer C before FcR-block (BD Biosciences; Clone 2.4G2, 553142) was added to prevent unspecific binding. Cells were then stained for viability using fixable viability stain (FVS)780 (BD Biosciences; 565388; 1:2000) and the following surface antibodies: CD45.1 Pacific blue (Biolegend; clone: A20, 110721), CD45.2 PE (Biolegend; clone: 104, 109807), CD4 V500 (BD Biosciences; clone: RM4-5, 560782), CD8 APC (Biolegend; clone: 53-6.7, 100712), CD3 PE-Cy7 (eBiosciences; clone: 17A2, 25-0032-82). Samples were run on a BD FACS Canto II on medium flow rate. CAR-T cells were identified by CD45.1- CD45.2+ GFP+ staining in the FlowJo software (v10.9.0).

For analysis of the innate immune cell compartment, we used a multicolor FACS panels. Therefore, mice were euthanized as indicated in the figure legends. Spleens were harvested and meshed through a sieve to yield a single cell suspension. After RBC lysis, splenocytes were counted, 2 × 106 were transferred to a 96-well round bottom plate (Szabo Scandic, 35 3077) and first stained for viability using FVS440UV according to manufacturer’s instructions (1:1000; BD Biosciences; 566332). Next, FcR-block (BD Biosciences; Clone 2.4G2, 553142) was added followed by the antibody mix (Suppl.Table3) in a total volume of 50µL. Of note, brilliant stain buffer (1:10; BD Biosciences, 566349) was added to the antibody mix to prevent clogging of brilliant dyes. After 20min of incubation, cells were washed twice and acquired on a Cytek Aurora machine (5 laser configuration). Unmixing was performed using a mix of cell and bead samples, respectively, and checked using single stains. Populations were verified using fluorescence minus one (FMO) controls. Data was analyzed in FlowJo (v10.9.0).

ELISpot
Immunocompetent Ly5.1+ recipient mice were injected with 1 × 106 Panc02-EpCAM tumor cells and treated with 3.6 × 106 CD45.2+ CAR+ T cells two days later. On day 21 after tumor injection, mice were euthanized, spleens were harvested and pressed through a sieve to yield a single cell suspension. After RBC lysis, splenocytes were counted and CD45.2+ CAR-T cells were depleted using a biotinylated CD45.2 antibody (Miltenyi; clone: REA1223, 130-124-089). Subsequently CD3+ T cells were negatively isolated using the pan T cell isolation kit (Miltenyi, 130-095-130) according to manufacturer’s recommendations and subjected to ELISpot assay.

For antigen presentation, mouse bone-marrow-derived dendritic cells (BMDCs) were generated as described elsewhere 60 and either left unstimulated or stimulated with an activating antibody against CD3 (Mabtech, Nacka Strand, Sweden, 3605-1-50) as positive control as well as a set of peptides and lysates. For peptide stimulations, dendritic cells were loaded with a pool of control peptides (peptide control) consisting of mouse-unspecific CEF/CEFTA as well as SARS-CoV-2 spike peptide pools (Miltenyi Biotec, Bergisch Gladbach, Germany). Additionally, tumor specific peptides were combined into two different peptide pools (Peptide pool 1 and Peptide pool 3). Lyophilized peptides were reconstituted in DMSO at a stock concentration of 10 mg/ml and used at a final concentration of 2 µg per 100 µL. Similarly, dendritic cells were loaded with lysates from the human monocytic cell line THP-1 as a control (Lysate control), as well as with lysates from the tumor cell line both before (Cell lysate) and after (Tumor lysate) injection and growth within the mice at a 1:10 dilution. Peptide and Lysate loading was performed in culture medium (RPMI with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin; all reagents obtained from Sigma Aldrich, St. Louis, MO, USA) for 2h at 37°C and 5% CO . To detect activated and specific T cells, we used a Mouse Granzyme B ELISpot Kit (EL1865, BioTechne, Minneapolis, MN, USA). The precoated plates were initially washed three times with sterile-filtered 1x PBS and blocked for 30 minutes using CAR RPMI. During this time, the previously loaded or unstimulated BMDCs (2 × 104 cells per well) were washed three times with 1x PBS to remove excess peptides or lysate and combined for antigen presentation with the rested CD3+ T cells (2 × 105 cells per well). The cell suspensions were then transferred onto the respective ELISpot plates and incubated over night at 37°C and 5% CO . Further detection and development were performed according to the manufacturer’s instructions. In brief, cells were discarded and plates washed five times with sterile-filtered PBS or the designated wash buffer. After the last wash, detection antibody was added and plates incubated for 2h at room temperature. Detection solution was discarded, and plates washed again five times as described above. Next, Streptavidin-AP (Granzyme B kit) was added, and plates incubated in the dark for 1h at room temperature. Following another set of washes, the Granzyme B plates were incubated with BCIP/NBT substrate (BioTechne, SEL002) for 1h and then washed thoroughly with tap water. All plates were left to dry before further analysis. Imaging, counting and quality control of ELISpot plates were performed using the Immunospot Analyzer S6 Ultra M2 (CTL, Cleveland, OH, USA).

Tumor re-challenge of relapse-free complete responder mice
Prior to re-challenge, tumor cells were enriched for EpCAM positive and EpCAM negative cells using flow sorting and maintained in DMEM+++. Six months after first tumor challenge, mice, which showed complete response (CR) after treatment with NTC and Nr2f6 acute gene-edited CAR-T cells, were injected with 2.5 × 106 EpCAM positive and 1 × 106 EpCAM negative Panc02 tumor cells into the right and left flank, respectively. Panc02-naïve female C57BL/6 mice were used as controls and injected with an identical tumor load. Monitoring of tumor-bearing mice was performed as described above.

Immune memory transfer from complete responders to CAR-antigen naïve mice, ex vivo recall assay and cell preparation for scRNAseq
Relapse-free complete responder (CR) mice, which survived the 1st re-challenge, were injected again with 1 × 106 Panc02-EpCAM negative tumors. Panc02 antigen-naïve C57BL/6 mice were used as controls and injected with the same tumor load. Five days later, mice were euthanized and spleens and tumors were harvested. Single cell suspensions of spleens were prepared as described above. 1 × 107 splenocytes from CR or antigen-naïve mice were transferred into Panc02-EpCAM negative tumor-bearing mice 2 days after tumor initiation. Tumor growth and survival were assessed, and mice were euthanized if abort criterion were met. Remaining splenocytes were subjected to CD8 T cell isolation using the untouched CD8a+ T cell isolation kit (Miltenyi, 130-104-075). Enriched CD8 T cells from CR and antigen-naïve mice were then co-cultured with Panc02-EpCAM in an E:T ratio of 5:1 for 24 hours at 37°C and 5% CO . The next day, Golgi Plug and Stop (1:1000 [555029] and 1:1500 [554724]; BD Bioscience) were added and incubated for 5 hours. After incubation T cells were harvested and subjected to intracellular FACS analysis.

For scRNA-seq, tumors were digested in DMEM+++ supplemented with Liberase TL (Merck, 5401020001, 10mg/mL) and DNAse I (Roche, 11284932001, 20mg/mL) for 30min at 37°C 5% CO . 0.5M EDTA (Sigma Aldrich, 03690) was added for 5min before meshing tumors through a 20µm cell strainer (Corning, 156499). Tumor single cell suspensions were stained using CD45 PE (Thermo Fisher, clone: I3/2.3, 12045183) for 20min at 4°C. CD45+ leukocytes were flow sorted at the FACS Core Facility of the Medical University of Innsbruck. Library preparation and scRNA-seq was performed by the MulitOmics Core Facility of the Medical University of Innsbruck.

Single-cell sequencing
Single-cell suspensions were processed at the Medical University of Innsbruck MultiOmics Sequencing Core Facility. Adequate Cell viability and concentration was verified with trypan blue (Biorad, 1450021) staining and TC10 automated cell counting (Biorad) complemented with hemocytometer visual inspection. Final cell suspensions were processed with a Chromium single cell controller (10xGenomics, 1000127) and Chromium Next GEM Single Cell 5' Kit v2 (10xGenomics, 1000269) chemistry, targeting 4000-6000 cells per sample. The resulting total RNA and V(D)J libraries were multiplexed and sequenced with Illumina Hi-Seq technology, generating in total approximately 700 M read pairs.

Retroviral and lentiviral transduction of tumor cell lines
Retrovirus containing supernatant was produced as described above. For Panc02-EpCAM-GFP, PlatE cells were transfected with 18µg of transfer plasmid (pMP71-GFP) to produce viral particles. The day prior transduction, 2 × 105 Panc02-EpCAM were seeded into 6-well plates (Szabo Scandic, 35 3046) and incubated overnight at 37°C and 5% CO . The next day, medium was replaced by 2mL DMEM+++ supplemented with 8µg/mL polybrene (Sigma-Aldrich, TR-1003-G). Then 2mL of filtered viral supernatant was added directly and the plates were spun at 800 × g for 2 hours at 32°C. Transduction efficiency was assessed by flow cytometry. GFP+ cells were enriched by flow sorting. Lentiviral supernatants for B16-F10-hEGFR and MC38-hEGFR production were generated as follows: the day prior to transfection, HEK 293T cells were seeded in 10cm dishes (Thermo Scientific, 353003) at 5 × 106 per dish and incubated overnight at 37°C 5% CO . On the next day, medium was replaced with DMEM + 10% FCS + 2mM L-Glutamine and cells were transfected with 5.75µg pHELP1 (PLP1), 5.75µg pHELP2 (PLP2), 3.45µg pHELP3 (VSVG) and 18µg PWPXLD-EGFR-WT (kind gift of Chay Kuo, Addgene, 133749) per dish using Lipofectamine 2000 reagent (Thermo Fisher Scientific, 11668019) according to manufacturer instructions. Supernatant was harvested 48 and 72 hours after transfection and concentrated using the Lenti-X-Concentrator (Takara, 631232) according to manufacturer instructions. Concentrated virus was pooled and frozen at -80°C. The day prior transduction, 2 × 105 B16-F10 or MC-38 were seeded into 6-well plates (Szabo Scandic, 35 3046) and incubated overnight at 37°C and 5% CO . The next day, medium was replaced by 2mL DMEM+++ supplemented with 8µg/mL polybrene (Sigma-Aldrich, TR-1003-G). Then 20µL of concentrated lentiviral supernatant was added directly and the plates were spun at 800 × g for 2 hours at 32°C. Transduction efficiency was assessed by flow cytometry using αEGFR-AF647 (Biolegend, clone AY13, 352917). EGFR+ cells were enriched by flow sorting.

Chronic stimulation assay and live cell imaging
To ensure sufficient target expression, Panc02-EpCAM-GFP+ were subjected to flow sorting the week prior to the assay. Therefore, tumor cells were stained with αEpCAM-APC (Miltenyi; clone: REA977, 130-123-810) and the EpCAM-high GFP-positive population was sorted directly in DMEM+++ on a FACS Aria (BD Biosciences) machine at the FACS Core facility of the Medical University Innsbruck. Flow sorted tumor cells were cultured at 1 × 106 cells per T175 tissue culture flask (Sarstedt, 833912002). On the first day of the assay, 6 × 104 Panc02-EpCAMhi-GFP+ cells were seeded into 96-well flat bottom plates (Szabo Scandic, 353072) in CAR RPMI and left to adhere for a few hours. After CAR-T production and assessment of transduction efficiency, bulk transduced T cells were counted and adjusted to 3 × 106 CAR+/mL. 3 × 105 CAR-T cells were then added at an effector:target ratio (E:T) of 5:1 to tumor cells in CAR RPMI without cytokines. Live cell imaging was performed in an Incucyte live cell analysis system (Sartorius) using the 10x objective. Fluorescence pictures were taken every two hours for up to 10 days. Every 48 hours, co-cultures were taken from the Incucyte machine and CAR-T cells were re-stimulated with fresh target cells. For this, Panc02-EpCAMhi-GFP+ cells were harvested from T175 tissue culture flasks (Sarstedt, 833912002) and resuspended at 6 × 105/mL in CAR RPMI. 100µL of medium was removed from the co-cultures and stored at -20°C for cytokines assessment. Then, 100µL tumor cell suspension was added, and plates were put back into the Incucyte. It was made sure that at least one hour lies between re-stimulation and the next scan to allow sedimentation of tumor cells. Cytotoxicity was determined by decreasing GFP fluorescence and plotted relative to the first scan after each re-stimulation (green integrated intensity per mm2 relative to first scan after re-stimulation). Analysis was performed using the built-in analysis software (Sartorius). CAR-T cells from single mice in duplicates were considered as biological replicates.

Surface and intracellular antibody staining of mouse CAR-T cells
Staining was performed with up to 1 × 106 (CAR) T cells in a 96-well round bottom plate (Szabo Scandic, 353077). For viability assessment, T cells were washed twice with Hanks buffered salt solution (HBSS) containing Magnesium and Calcium (Pan Biotech, P04-32505). Then, 100µL HBSS supplemented with FVS780 (BD Biosciences, 565388) at a final dilution of 1:2000 was added and incubated for 10 minutes at RT in the dark. After the incubation, cells were washed with buffer C (described above) and subjected to surface antibody staining. Surface antigens were stained for 20 minutes at 4°C in a 40µL reaction in buffer C using the following antibodies: c-myc FITC (Miltenyi, clone: REA1287, 130-116-485), Ly108 Pacific blue (Biolegend, clone: 330-AJ, 134608), PD-1 bv510 (Biolegend, clone: 29F.1A12. 135E241), Tim3 PerCp-Cy5.5 (Biolegend, clone: B8.2C12, 134011), Lag3 PE-Cy7 (Biolegend, clone: C9B7W, 125225), CD39 PE (Biolegend, clone: Duha59, 143804), CD45 APC (Fisher Scientific, clone: I3/2.3, 17-0451-82), CD44 PE-Cy7 (Biolegend; clone: IM7, 103030), CD62L APC (BD Biosciences; clone: MEL-14, 553152). For intracellular (IC) cytokine staining, CAR-T cells were transferred to 96-well round bottom plates (Szabo Scandic, 353077) and stained with surface antibodies as stated above. Thereafter cells were fixed using the fixation buffer from Biolegend (420801) for 20 minutes at RT. After fixation, cells were permeabilized by washing twice with the kit’s Perm/Wash buffer (1x, Biolegend, 421002). Intracellular proteins were stained for at least 30 minutes to one hour at 4°C in Perm/Wash buffer using the following antibodies: IFN PE-Cy7 (Biolegend; clone: XMG1.2, 505826), TNF PerCpCy5.5 (Biolegend; clone: MP6-XT22, 506322), IL-2 APC (BD Biosciences; clone: JES6-5H4, 554429), GZMB PE (Biolegend; clone: QA18A28, 396406). For transcription factor staining, CAR-T cells were chronically stimulated with Panc02-EpCAMhi-GFP+. On day four of co-culture, wells were harvested and transferred to a 96-well round bottom plate (Szabo Scandic, 353077) and stained for surface antigens as described above before fixation using the Foxp3 fixation kit (eBiosciences, 00-5523-00) for 30 minutes at 4°C. Cells were permeabilized twice with 1x Perm/Wash and stained with IC antibodies for one hour at 4°C: TCF1 Pacific blue (Cell Signaling; clone: C63D9, 9066S) and TOX APC (Miltenyi; clone: REA473, 130-118-335). After antibody staining cells were wash twice and resuspended in 200µL buffer C before acquisition on a FACS Canto II (BD Biosciences). For absolute cell counts, precision count beads (Biolegend, 424902) were applied. Therefore, cells were stained directly in culture medium for 20 minutes at 4°C. Plates were spun and cells were resuspended in 100µL buffer C. 100µL precision count beads were added per sample. Total cell counts were calculated according to the manufacturer’s instructions. Data was analyzed using FlowJo software (BD Biosciences, version 10.9.0).

Cytokine production assay
On day 5 of CAR-T production, 6 × 104 Panc02-EpCAMhi-GFP+ cells were seeded into 96-well flat bottom well plates (Szabo Scandic, 353072) and incubated for a few hours to allow attachment. Then, 3 × 105 CAR+ T cells were added (E:T ratio of 5:1) and incubated overnight in CAR RPMI without cytokines. The next day, Golgi Plug and Stop (1:1000 [555029] and 1:1500 [554724]; BD Bioscience) was added for 6 hours before cells were subjected to antibody staining and IC FACS analysis.

Gasdermin E clipping assay
1.5 × 106 CAR-T cells were co-cultured with Panc02-EpCAMhi-GFP+ in a 24-well plate (Szabo Scandic, 35 3047) at an E:T of 5:1. CAR-T cells were re-stimulated every second day with 3 × 105 fresh tumor cells. On day 4, wells were harvested by trypsinization 6 hours after re-stimulation. Briefly, cell culture medium was collected in a 15mL Falcon tube, wells were wash with 1x PBS and 400µL TrypLE Express (Fisher Scientific, 12604013) was added per well. When adherent cells started to detach, 800µL CAR RPMI was added to stop the reaction and medium was transferred to 15mL tube. After a wash step with ice-cold 1x PBS, cells were resuspended in 30µL RIPA buffer (Sigma-Aldrich, R0278) supplemented with 5mM EDTA (Sigma Aldrich, 03690) and 1x Halt protease and phosphatase inhibitor cocktail (ThermoFisher, 78444/10137963) and incubated for 30 minutes on ice while vortexing frequently. Then, lysates were spun for 15 minutes at 15000 × g at 4°C. Supernatant was transferred to a new 1.5mL Eppendorf tube, supplemented with 5µL Pierce Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific, 11809340/39000) and frozen at -80°C. Samples were thawed, heated to 95°C and subjected to immunoblotting using the following antibodies: DFNA5/GSDME (Abcam, clone: EPR19859, ab215191, 1:1000), beta-actin (Santa Cruz; clone: sc-47778, 1:1000). Bands were quantified using ImageJ (v1.51).

Cytokine detection in supernatant
Cytokine release was measured in supernatants using the Luminex xMAP technology. In brief, frozen supernatants were thawed and diluted in CAR RPMI. Assays for IFN (Biorad, 171-G5017M) and GZMB (ThermoFisher, EPX010-26074-901/17886521) detection were performed according to manufacturer’s instruction on a Bio-Plex 200 machine (Biorad). Absolute concentrations were determined using a standard curve.

Mouse cytokine array
WT mice were treated on d4 after tumor injection (1 × 106 Panc02-EpCam) with 3.5 × 106 NTC or Nr2f6 acute gene-edited CAR-T cells. On day 3 after CAR ACT mouse spleens were harvested and half a spleen was homogenized in 500µl PBS with complete-proteinase inhibitor cocktail, (Fisher Scientific, 78444/10137963) and 1% TritonX100 (Sigma Aldrich, T8787 – 100) on ice. After one freezing step at -70°C lysate was centrifuged at 10.000 rcf for 5 min to remove cellular debris and protein concentration was measured by Bradford assay (BioRad). Protein equivalents (300µg) were used to probe cytokines/chemokines using the Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D Systems, ARY006) according to the manufacturer´s instructions. Quantification of the spot intensity on the blot membranes was done after background subtraction with QuickSpots software (IdTeal Eyes Systems, Inc.).

RNA isolation and qRT-PCR
To assess Nr2f6 mRNA induction during CAR-T production, 1-2 × 105 T cells were harvested, washed once with 1x PBS and resuspended in 350µL RLT buffer (Qiagen, 74106) and stored at -80°C. For co-culture samples, cells were harvested, and live (CAR) T cells were enriched by density gradient centrifugation. Briefly, cells were resuspended in an 80% Percoll solution (Sigma-Aldrich, GE17-0891-01) in HBSS (Thermo Fisher Scientific, 14185052). A 40% Percoll layer was loaded on top, and cells were spun at 250 × g for 30min without a break. After the spin, the interface layer was harvested and subjected to CAR positive selection using myc-Biotin (Biolegend; 1µg/mL, 908805) and Biotin microbeads (Miltenyi; 130-090-485, 20µL for 107 cells) according to manufacturer’s instructions. 1-2 × 105 CAR+ T cells were then resuspended in 350µL RLT buffer and stored at -80°C.

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 20µL 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). Target gene expression was normalized to Rlp13a (see below) using the ct method. Ct values over 32 were considered as not expressed. Primers used for qPCR-RT: Mm01340321_m1, Mm01612986_gH.

Seahorse Cell Mito Stress assay
For analysis of oxidative phosphorylation (OXPHOS), 2 × 105 CAR-T cells were seeded into poly-lysine pre-coated Seahorse culture plates in XF RPMI without phenol red (Agilent, 103681) supplemented with 20mM glucose, 2mM glutamine and 1mM pyruvate (all Agilent, 103681-100). Assay was performed using the Cell Mito Stress Test Kit (Agilent, 03010-100) on a Seahorse XFp device (Agilent). Briefly, Seahorse plates were put in a CO -free incubator for one hour to allow equilibration. Mitochondrial respiration was analyzed first by assessing the basal respiration, then injecting 1.5µM of the ATP synthase inhibitor oligomycin (Enzo Life Sciences, ALX-380-037-M005), 1µM of the uncoupler carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP, Enzo Life Sciences, BML-CM120-0010) and finally 0.5µM of rotenone/antimycin A (Enzo Life Sciences, ALX-380-075-M010). Results were analyzed using the Seahorse Wave Software (Agilent).

Flow sorting of tumor and CAR-T cells
Panc02-EpCAM, B16-F10-hEGFR, MC-38-hEGFR tumor cells were harvested as described above. Cells were transferred to FACS tubes and up to 1 × 107 cells were stained in 100µL buffer C containing EpCAM-APC (Miltenyi, clone: REA977, 130-123-810) or αEGFR-AF647 (Biolegend, clone AY13, 352917) antibodies for 20 minutes at 4°C. After incubation, tumor cells were washed twice with buffer C and finally resuspended in ice-cold DMEM+++ supplemented with 2mM EDTA at 1 × 107/mL. CAR-T cells from co-culture were harvested and transferred to a FACS tube for antibody staining. Up to 107 cells were resuspended in 100µL buffer C containing c-myc FITC (Miltenyi, clone: REA1287, 130-116-485), CD45 APC-Cy7 (Biolegend, clone: 30-F11, 103116) and EpCAM APC (Miltenyi, clone: REA977, 130-123-810) and incubated for 20 minutes at 4°C. Then, cells were washed twice with buffer C (described above) and resuspended at 1 × 107/mL ice-cold CAR RPMI supplemented with 2mM EDTA. Cells were flow sorted using a BD FACS Aria (BD Biosciences) at the FACS Core Facility at the Medical University Innsbruck. For viability staining, 10µL 4′,6-diamidino-2-phenylindole (DAPI, 422801, 10.9M) was added to 1mL of cell suspension straight before acquisition. Viable EpCAM or EGFR positive tumor cells were directly sorted into DMEM+++ and seeded into T175 flasks (Szabo Scandic, 353112) immediately after sort. 1 × 105 viable CD45+ CAR+ T cells were directly sorted into 200µL RLT buffer (Qiagen, 74106) supplemented with 350mM 2-mercaptoethanol (bME, Sigma Adlrich, M3148) and 2.5µL RNAsin (Promega, N2515) in a RNAse-free 0.5mL Eppendorf tube, immediately frozen at dry ice and subsequently subjected to RNA isolation and bulk RNA sequencing (RNAseq).

Bulk RNAseq
For bulk RNAseq, total RNA was isolated using the RNeasy Micro kit (Qiagen, 74106) according to manufacturer’s instructions. RNA was eluted in the kits elution buffer and stored at -80°C before quality control (QC). Purified total-RNA was submitted for transciptome analysis at the Medical University Innsbruck MultiOmics Sequencing Core for the purpose of gene-expression profiling, using the QuantSeq 3′ mRNA-Seq Library Prep method (Lexogen GmbH, Vienna Biocenter). Quality-validated, barcoded libraries were multiplexed and sequenced using Illumina NovaSeq technology (GeneWiz/Azenta, Leipzig, Germany).

Analysis of CAR-T cell bulk RNAseq
The nf-core/rnaseq (v3.14.0) 61 pipeline was used to align the raw reads to the mouse genome (GRCm39) with STAR and to assess the read counts on the gene models from GENCODE version M31 with Salmon. The pipeline was run using default parameters, but to accommodate the QuantSeq 3’-mRNA library data, the option ‘--extra_star_align_args’ was modified as recommended in the documentation and ‘--noLengthCorrection’ was used for gene quantification with Salmon 62. DESeq (v1.42.1) was employed to identify differentially expressed genes between Nr2f6-/- CAR and WT CAR, applying a fold change threshold of 2 and a false discovery rate (FDR) of 0.1 after Independent Hypothesis Weighting (IHW) 63. Enriched GO terms were calculated using the R package topGO (v2.54.0) 64 and the significant differentially expressed genes (|log2foldchange| > 1, FDR < 0.1) as input. Enrichment scores were defined as -10 × log10(p.value_elim) and plotted as barplots. Pathway Gene set enrichment analysis (GSEA) was performed with the clusterProfiler 65 R package (v4.10.1) and the KEGG pathway database using genes ranked by the Wald statistic (stat) from DESeq2.

scTCRseq and scRNAseq analysis of CD45+ tumor-infiltrating leukocytes from antigen-naïve recipients and Nr2f6crispr-/- CAR-treated complete responder
Pre-processing of 10x VDJ and GEX sequencing files was performed with cellranger (v7.1.0) in the “cellranger multi” mode using the cellranger-vdj GRCm38 and gex-mm10-2020 reference files (10x Genomics). Raw count matrices were imported into AnnData and processed with scverse tools 66. Ambient RNA was removed using scAR, and doublets were identified and removed using SOLO, both implemented in scvi-tools 67, 68. Denoised count data underwent quality control in scanpy 69, involving two stages: 1. Pre-filtering: Cells were initially selected based on having more than 500 transcripts and over 200 genes. 2 . Outlier removal: Cells showing excessive deviation (more than 5 times the median absolute deviation or MAD) in the number of transcripts, number of genes, or percentage of counts in the top 20% of genes were excluded. Additionally, cells with a high proportion of mitochondrial transcripts (deviating by more than 3xMAD or exceeding 8%) were also removed. Batch correction was performed as described above but “batch” (date of sample collection) was used as “batch_key” together with “donor” (individual animal) as “categorical_covariate_key”. After unsupervised clustering with the Leiden algorithm, cell types were annotated based on known marker genes (see Suppl. Table 2 with marker genes). Pseudo-bulk profiles for each cell type were generated in Python with decoupler (git commit hash 9da10c9) summing up the counts and using the following filtering options: “min_cells=10” and “min_counts=1000”. Differential expression analysis between responder (memory) and antigen-naïve samples was performed for each cell type using PyDESeq2 (v0.4.1) 70. To examine which biological processes were enriched in the significant differentially expressed genes (|log2foldchange| > 1, p-adjusted < 0.1), gene ontology overrepresentation analysis was conducted using 'decoupler'. Dotplots were created in which the x-axis represents the 'combined score', calculated as the negative logarithm of the p-value multiplied by the odds ratio, providing a measure of the enrichment strength.

The TCRs were analyzed using the scirpy package (v0.13.1) 71. Cells that did not have a full pair of receptor sequences were filtered out. Clonotypes were defined based on VJ and VDJ CDR3-sequence identity. As a metric for the TCR diversity and TCR clonality the normalized Shannon entropy and the Gini index respectively was calculated.

Statistical analysis
Statistical analysis was performed using GraphPad Prism software (v9.5.1). Data are represented as mean ± standard error of the mean (SEM) unless stated otherwise. Comparisons between two groups were calculated using two-tailed unpaired Student’s t-test or non-parametric Mann-Whitney test as indicated in the figure legends. Comparisons between two or more groups with different variables were analyzed performing two-way ANOVA. In case of multiple comparison, the Šídák method was used. Bulk RNAseq and scRNAseq data were analyzed as stated above. Survival data was collected in a Kaplan-Meier curve and tested using log-rank tests. P-values <0.05 were considered significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Humer et al report that ablation of the TF NR2F6 in CD28-co-stimulated CAR-T cells empowers them with superior metabolic fitness, effector activity, retention of stemness, and reduced T cell exhaustion. Accordingly, Nr2f6-modified CAR-T cells are superior at controlling solid tumor sin vivo.\nThe same authors (e.g., PMID: 26387951, 26992368, 28779026, 31937317, 33589606) have previously reported the role of NR2F6 as a checkpoint for cancer immune surveillance. It is therefore not surprising that ablation of this TF enhances the anti-tumor activity of CAR T cells. In addition, other authors have previously reported multiple gene editing approaches to enhance the efficacy of cancer cell therapies (e.g., PMID: 34673274, 35750052, 36356142, 38387457). There is therefore limited conceptual or technical novelty in this study. Furthermore, CRISPR is a powerful research tool but there are more specific and novel gene editing techniques.\nThe authors also use a single tumor model (no general applicability demonstrated in other tumor) and an antigen (Epcam) that is expressed in healthy epithelial cells. The challenges of targeting specific antigens on the tumor cell surface and heterogeneity of expression, which are the major limitations in the field, are not addressed at all in this study.\nOn the other hand, the demonstration of a boost in polyclonal pre-existing, endogenous T cell responses, while not unexpected or blatantly novel (previous studies have shown that for other CAR T cells), is interesting nevertheless.\nOTHER MAJOR COMMENTS\n-The phenotypes (e.g., GZMs, TCF1) shown in vitro in Figures 2-4 need to be shown in vivo in TILs. The problem is that CAR T cells do not persist in vivo at day 13, edited or not, which is a major concern.\nThis reviewer could not figure out the origin of the scFv used to re-direct CAR T cells against Epcam, but it is likely that it could be recognized as ‘foreign” in immunocompetent mice.\n-Toxicity and on target, off-tumor effects are important concerns that are not addressed.", "reviewer_2": "Review NCOMMS-24-76863-T\nThe authors are investigating the role of transcription factor NR2F6, recently identified as T cell checkpoint, in antigen spreading (AS) in the context of CAR T cell therapy. They employ NR2F6 KO to address their thesis.\nThe more common term for AS is epitope spreading, defined as diversification of epitope specificity, and it typically requires cross-presentation of exracellular antigens in MHC-I context (https://doi.org/10.1038/nri724; https://doi.org/10.1158/1078-0432.CCR-20-0305).\nThe manuscript is well written, and experimental results are compelling. However, some results are in my opinion overstated/overinterpreted. You focus on AS as the main MOA (mechanism of action) of the NR2F6 KO in T cells rejecting tumors in vivo, but the experimental proof of AS being primarily responsible for the observed anti-tumor effect needs to be stronger for such statements. It would be nice to see direct data on broadening of Panc.02-relevant T cell repertoire in mice after vs before CAR therapy with NR2F6 KO cells to strengthen your main hypothesis.\nCan you rule out other mechanisms, such as heightened anti-tumor reactivity, increased viability, proliferation, metabolic fitness, increased secretion of pro-inflammatory cytokines (IFNg) or lower exhaustion of KO effector T cells vs AS, or other?\nWhich of these mechanisms were previously reported for NR2F6 KO T cells, and which are novel?\nIs the AS benefit mediated only by the modified (NR2F6 KO and CART+) CD8T cells, or is it also perpetrated by non-modified autologous T cells in the mice? This may shed light on the MOA.\nMajor comments:\nAbstract and Introduction\nYou speak of treatment of solid tumors in general, but only employ one model of pancreatic cancer: e Panc02 tumors.\nPlease qualify your statements accordingly to your scope of work.\nPlease define your hypothesis more precisely: (e.g. KO of NR2F6 promotes AS in the context of CAR T cell therapy? Or promotes other/additional effects..), and include a corresponding sentence in the abstract and intro.\nReference to relevant literature: NR2F6 appears to play a role both in cancer cells (e.g. AML, glioma, lung cancer, melanoma…) and in immune (T ) cells. To provide a balanced intro, please briefly allude also to the role of this factor in tumor cells, vs the immune compartment.\nA recent report described both tumor intrinsic and tumor-extrinsic benefits of NR2F6 down-modulation in the context of immunotherapy of melanoma tumors DOI: 10.1126/sciadv.adf6621. How does your current work differentiate itself and add to the existing knowledge on this subject? Please add in intro or discussion vs this or similar recent references.\nResults:\nIn your experimental design, did you modify only CD8 T cells, or all T cells with NR2F6 KO and CAR- ? please clarify.\nPage 4:” We did not observe any differences in CAR transduction efficiency (Fig2A) or expansion of Nr2f6-/- or wild type CD8+ T cells during CAR-T cell production (Suppl.Fig2B).”\nDid you look at CD4 T cells? Do they contribute to the beneficial effects you observed in vitro or in vivo? Please report.\nPage 7:” Therefore, the observed immune defense against the tumor after the apparent disappearance of CAR-T cells can be well explained by AS and the subsequent induction of a strong secondary tumor antigen-specific T cell response.\nOverall, we demonstrate a cooperative anti-tumor effect of Nr2f6-modified CAR-T therapy and endogenous immune cells.”\nYou purport AS, but present little direct data of AS in vivo or ex vivo… Were cDC1 you identified involved in prolongation of anti-tumor responses after T cells were gone? Were other cells recruited? What was their phenotype?\n“Therefore, the observed immune defense against the tumor after the apparent disappearance of CAR-T cells can be well explained by AS and the subsequent induction of a strong secondary tumor antigen-specific T cell response. Overall, we demonstrate a cooperative anti-tumor effect of Nr2f6-modified CAR-T therapy and endogenous immune cells.”\n– please consider revising this statement to match the strength of your data. If I understood correctly, you only showed the ability of cDC to generate enhanced T cell priming of NR2F6 KO CD8 T cells in vitro, but not proven that this happened during your in vivo experiment and was indeed responsible for accelerated tumor clearance.\n“The data support the concept that NR2F6-modified CAR-T therapy apparently facilitates the occurrence of AS, which results in a robust secondary polyclonal immune response induced by the crosstalk of endogenous innate and adaptive immune cell compartments.”\n- Again, please prove that a robust secondary polyclonal immune response occurred in vivo, or rephrase.\nFigure 8 is very general and doesn’t point out the specificMOA through which NR2F6 KO benefits CAR T cell therapy in your proposed model. Please revise.\nDiscussion:\nAre your findings regarding NF2F6 function in cancer immunotherapy confined to the specific mouse-strand you used, or can they be generalized, why? How broadly can they be generalized to mouse experimental systems? To human health?\nPlease speculate or provide data/refs to support your points.\nWhat happens with CD4 T cells in your models? Is NR2F6 important for CD4 T cells?\nDoes AS in your model systems occur only via the modified KO T cells, CAR+ T cells, both modifications only, or also is carried out by “naïve”, non-modified host’s T cells?\nDoes AS work via cytokine secretion, boosting the activity of cDCs, or decreasing activation threshold in T cells (modified or not), or other mechanisms? Please propose specific and detailed mechanisms as you see fit and describe in the discussion.\nMinor comments\nPlease revise for clarity/brevity:” …Of note, and relevant to a potential clinical setting, we have previously shown haploinsufficiency for the NR2F6 gene to be sufficient for the anti-tumor phenotype indicating that deletion of one allele will be sufficient (20).”", "reviewer_3": "The manuscript presents highly interesting research on the advantages of Nr2f6 knockout in enhancing the therapeutic efficacy of CAR-T cell responses. To further strengthen this work, we would like to offer some additional considerations regarding the chosen methodology:\n1. Firstly, regarding Nr2f6, its function in T lymphocytes could be more clearly described in the introduction, even if briefly, to provide better context.\n2. Another aspect highlighted in some reports is the crucial role of CD4⁺ T cells in promoting and sustaining CAR-T cell responses in long-term responders. Carl June’s retrospective studies over a decade post treatment have demonstrated the persistence of predominantly CD4⁺ CAR-T cells. In this regard, we believe it would be valuable to evaluate the proposed therapeutic modification in this population, rather than focusing solely on CD8⁺ T cells. While the proposed mechanism of action—through antigen spreading—may be primarily driven by granzyme and perforin release from CD8⁺ T cells, the CD4⁺ subpopulation could also play a significant role in the overall response. Since recent published data show that long term persistence of CAR-T cells in the CD19+ tumor setting is found on CD4+ cells in patients and the authors show that the CD8+ CAR-T cell used do not last, it will be very important and informative to test solely transfering CD4+ CAR-T cells and also mixing CD4+ and CD8+ CAR-T cells to evaluate the impact on CAR-T cell persistence, antigen spreading and memory response formation.\n3. Additionally, although the proposed therapy is intended for solid tumors, hematological malignancies remain the only currently approved indication for CAR-T cell therapy and still require further optimization. In this context, we believe it would be important to evaluate the proposed approach either in this setting or in an alternative tumor model. Although the claim of the authors is based on the antigen spread phenomenon to explain the improved anti tumor effect, testing the effect of Nr2f6 abrogation in a more classical setting such as a CD19+ leukemia or lymphoma could add potential clinical - validated implications to the manuscript.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Subject: Appeal Submission for Manuscript: NCOMMS-24-76863-T. Addendum (Policy Statement for Reviewers): As Nature Communications permits only one appeal per manuscript, and the decision following this process is final, we recognize that this represents our sole opportunity to request reconsideration for manuscript NCOMMS-24-76863-T. We sincerely thank you for your time, thoughtful evaluation, and the constructive feedback provided during the review process. We have addressed each point with care and believe that the revisions have strengthened the manuscript considerably. We fully understand that the final editorial decision rests with the journal, and therefore we hope that our comprehensive responses and careful revisions will be well received.\nDear Reviewers\nWe would like to thank you for your constructive and insightful comments. Your feedback has greatly improved the clarity and scientific rigor of our manuscript, which we have retitled \"NR2F6 deletion revives CAR-T cell function and induces antigen-agnostic immunity in solid tumors.\" We have carefully revised the manuscript in accordance with each reviewer’s evaluation and provided comprehensive, point-by-point responses to address all major concerns. We are confident that these revisions strengthen our principal conclusions, which are now supported by additional experimental data and improved organization. As we were kindly invited to do earlier this year, we formally request that our revised submission be considered an appeal. We respectfully resubmit our manuscript for reconsideration in Nature Communications.\nJustification for Appeal: We are submitting this appeal because we believe that the original decision did not fully reflect the strengthened evidence and conceptual clarity of our revised study. We have incorporated major new data, including two additional tumor models and expanded mechanistic validation confirming the role of NR2F6 as an intracellular checkpoint in CAR-T cells. In parallel, we have made substantial textual and structural clarifications to enhance readability and interpretation. These revisions significantly extend the scope, depth, and translational relevance of our findings beyond those of the originally reviewed version. Therefore, we believe the initial decision underestimated the study's robustness and impact, and the new data and clarifications merit reconsideration on scientific grounds.\nOur study provides the first demonstration that targeted deletion of NR2F6 in CAR-T cells not only restores cell-intrinsic cytotoxicity within the immunosuppressive tumour microenvironment, but also initiates a secondary, epitope-spreading-driven immune cascade in immunocompetent hosts. By linking metabolic reprogramming, sustained TCF1⁺ progenitor states, and dendritic cell-mediated cross-priming, our data reveal a dual mechanistic axis through which NR2F6 inhibition converts transient CAR-T activity into durable, antigen-agnostic tumor immunity. To our knowledge, this work is the first to connect intracellular checkpoint editing with systemic immune re-education against heterogeneous solid tumours. We believe that these expanded results and improvements will strongly interest Nature Communications’ readership and align well with the journal’s focus on mechanistic advances with translational relevance.\nOur detailed responses below outline the specific improvements and corresponding new data.\nThank you very much for your time and consideration.\nYours sincerely, Univ.-Prof. Gottfried Baier (on behalf of all authors)\nP.S.: Major textual changes are marked in blue within the revised manuscript.\nRevised Manuscript:\nNR2F6 deletion revives CAR-T cell function and induces antigen-agnostic immunity in solid tumors\nCorresponding authors: Victoria Klepsch and Gottfried Baier\nReviewer #1:\nComment 1) Humer et al report that ablation of the TF NR2F6 in CD28-co-stimulated CAR-T cells empowers them with superior metabolic fitness, effector activity, retention of stemness, and reduced T cell exhaustion. Accordingly, Nr2f6-modified CAR-T cells are superior at controlling solid tumor sin vivo. The same authors (e.g., PMID: 26387951, 26992368, 28779026, 31937317, 33589606) have previously reported the role of NR2F6 as a checkpoint for cancer immune surveillance. It is therefore not surprising that ablation of this TF enhances the anti-tumor activity of CAR-T cells. In addition, other authors have previously reported multiple gene editing approaches to enhance the efficacy of cancer cell therapies (e.g., PMID: 34673274, 35750052, 36356142, 38387457). There is therefore limited conceptual or technical novelty in this study.\nResponse: We appreciate the reviewer highlighting our foundational work on NR2F6. While previous studies have demonstrated the role of NR2F6 as an immune checkpoint, our current study reveals a distinct mechanism. Nr2f6-deficient CAR-T cells trigger host-mediated epitope spreading in immunocompetent tumor-bearing recipients, thereby inducing antigen-agnostic secondary immune responses.\nOur study extends prior work by demonstrating how Nr2f6-modified CAR-T hyperactivity initiates robust immunogenic tumor cell death. This, in turn, catalyzes critical secondary host immune responses. The resulting in situ \"self-vaccination\" effect establishes polyclonal immune memory against tumor antigens, a mechanism with the potential to overcome major barriers that have historically limited CAR-T efficacy in solid tumors, such as antigenic heterogeneity and antigen downregulation. This process has the potential to deliver durable tumor control, particularly against highly heterogeneous tumors. Furthermore, this process represents a novel therapeutic strategy that eliminates the need for additional interventions, such as lymphodepletion, thereby underscoring its significant translational relevance.\nComment 2) Furthermore, CRISPR is a powerful research tool but there are more specific and novel gene editing techniques.\nResponse: We acknowledge the emergence of new gene-editing technologies and are exploring their potential for future studies. For this project, we used CRISPR/Cas9, a well-established technology that remains the benchmark for immunotherapy research. A recent review (Hudecek et al. Nat Rev Clin Oncol, 2025, DOI: 10.1038/s41571-025-01072-4), reaffirm the continuing value and feasibility of CRISPR/Cas9 in this field. While we recognize the advances offered by base editors, prime editors, and miniaturized Cas variants, for our proof-of-concept objectives, CRISPR/Cas9 provided the optimal balance of efficacy, robustness, and translational relevance. We are enthusiastic about novel developments and are actively exploring their potential for future studies.\nComment 3) The authors also use a single tumor model (no general applicability demonstrated in other tumor) and an antigen (Epcam) that is expressed in healthy epithelial cells.\nResponse: We agree with the reviewer that demonstrating efficacy in additional tumor models is essential to strengthening the generalizability of our findings. Accordingly, we expanded our analysis to include two independent in vivo models using B16-F10 melanoma and MC38 colon cancer cell lines that were engineered to express human EGFR (NEW Supplementary Figures 2A–G). In both syngeneic settings, Nr2f6-knockout CAR-T therapy achieved superior tumor control and improved survival. This confirms that its applicability extends beyond the initial Panc02-EpCAM model. These data demonstrate that our approach is not restricted to a single tumor type or antigen.\nManuscript addition (lines 217-223): \"To demonstrate broader applicability, we employed two independent CAR-T mouse models expressing an anti-human EGFR 28ζ CAR, together with syngeneic B16-F10 and MC38 tumor cells transduced with human EGFR (hereafter referred to as B16-F10-hEGFR and MC38-hEGFR). In both additional models, Nr2f6-depleted CAR-T-treated animals exhibited superior tumor control and prolonged survival compared with NTC CAR-T recipients (Suppl.Fig2A–G). These findings underline the broader relevance of Nr2f6-modified CAR-T cells, which achieve more effective and durable tumor clearance across distinct solid-tumor models.”\nRegarding EpCAM expression in healthy epithelial cells: Previous preclinical and clinical studies have reported limited off-tumor toxicity for EpCAM-directed CAR-T therapies, with ongoing clinical trials confirming tolerable safety and encouraging efficacy profiles (PMID: 32923168; DOI: 10.1126/sciadv.adg9721; NCT02915445).\nComment 4) The challenges of targeting specific antigens on the tumor cell surface and heterogeneity of expression, which are the major limitations in the field, are not addressed at all in this study.\nResponse: To model clinically relevant tumor antigen heterogeneity, we used Panc02 tumor cells that were approximately 70% EpCAM-positive. This created a stringent setting to assess both direct and indirect anti-EpCAM CAR-T effects. Using this approach, we were able to demonstrate epitope spreading and host immune involvement in controlling EpCAM antigen-negative tumor variants. We have revised the Methods and Results sections to clarify our rationale for selecting heterogeneous target cells, and confirm that this information now appears explicitly in the manuscript.\nManuscript addition (lines 202-205): “To assess anti-tumor efficacy of non-targeting control (NTC) and Nr2f6crispr-/- CAR-T, we inoculated immunocompetent mice with Panc02-EpCAM tumor cells comprising approximately 70% antigen-positive cells to introduce tumor heterogeneity and model clinically relevant challenges.”\nComment 5) On the other hand, the demonstration of a boost in polyclonal pre-existing, endogenous T cell responses, while not unexpected or blatantly novel (previous studies have shown that for other CAR-T cells), is interesting nevertheless.\nResponse: We thank the reviewer for highlighting the significance of the secondary, polyclonal, host anti-tumor immune memory response that extends immunity well beyond the initial CAR target. Previous studies have shown that combining vaccination with CAR-T therapy can enhance endogenous T cell responses (Ma et al., Cell, 2023, DOI: 10.1016/j.cell.2023.06.002). Additionally, recent publications from 2025 underscore a rapidly growing consensus that driving secondary, host-mediated, antigen-agnostic immune control is a critical frontier for advancing CAR-T therapy against heterogeneous solid tumors, thereby directly validating our mechanistic strategy. For instance, armored CAR-T cells engineered to express cytokines such as IL-7, CCL19, IL-12, or IL-18 have demonstrated enhanced recruitment and activation of dendritic cells and endogenous T cells, promoting epitope spreading (Mohamed et al., JITC, 2025, DOI: 10.1136/jitc-2024-00999; Li et al., J Immunother Cancer, 2025, DOI: 10.1136/jitc-2024-010581). Similarly, chemokine-secreting CAR-T cells, such as XCL1, have been shown to recruit cDC1s and boost endogenous CD8+ T cell responses (Li et al., J Immunother Cancer, 2025, DOI: 10.1136/jitc-2024-010581).\nIn a distinct and unprecedented advance over these multi-component engineering approaches, our study is the first to show that targeted NR2F6 gene editing in CAR-T cells alone; without additional cytokine expression, vaccination, or combinatorial manipulations, is sufficient to robustly and reproducibly drive broad, polyclonal host immunity and effective immune control of antigenically diverse solid tumors in vivo. This streamlined, single-gene editing strategy not only establishes a new mechanism, distinct from current approaches, but also highlights an innovative and clinically actionable path toward next-generation CAR-T therapies with enhanced efficacy for solid tumor treatment.\nOTHER MAJOR COMMENTS\nComment 6) -The phenotypes (e.g., GZMs, TCF1) shown in vitro in Figures 2-4 need to be shown in vivo in TILs. The problem is that CAR-T cells do not persist in vivo at day 13, edited or not, which is a major concern.\nResponse: We acknowledge the reviewer’s concern regarding CAR-T cell persistence in vivo. While it is true that CAR-T cells in our model do not persist beyond day 13, this limited persistence is not necessarily detrimental. Rather, the transient presence of highly functional Nr2f6-knockout CAR-T cells effectively prime and empower the endogenous immune system, leading to a durable, polyclonal anti-tumor response. Such a mechanism of CAR-T-induced activation of endogenous host immunity has been increasingly recognized as a key route to achieve long-term tumor control despite limited CAR-T persistence (von G López-Cantillo et al. , Front. Immun., 2022; DOI: 10.3389/fimmu.2022.878209; Guerra et al., Int. J. Mol. Sci., 2021, DOI: 10.3390/ijms23010405).\nTo substantiate our findings, we now include in vivo phenotyping data (NEW Fig. 1F–J, day 3 post-therapy) demonstrating enhanced CAR-T infiltration into tumors, elevated TCF-1 expression in tumor-draining lymph nodes, and increased IFNγ and granzyme B production upon ex vivo restimulation. These observations confirm that Nr2f6-depleted CAR-T cells exhibit early, functionally potent effector and memory-linked phenotypes that initiate a host-driven immune cascade resulting in sustained tumor control. The results demonstrate that Nr2f6-depleted CAR-T cells exert early, potent effector-linked functions in vivo, which are linked to the initiation of a host-driven immune cascade that establishes sustained tumor control, even in the absence of long-term CAR-T cell persistence.\nManuscript addition (lines 227–236): “On day 3 after adoptive CAR-T cell transfer (ACT), we observed an increased fraction of Nr2f6crispr-/- CAR-T cells within the tumor compared to NTC-treated controls (Fig1F). Notably, flow cytometry analysis revealed that Nr2f6-depleted CAR-T cells in the tumor-draining lymph node (tdLN) express elevated levels of TCF1 protein (Fig1G, H), a key transcription factor shown to regulate T cell memory formation and stemness (Escobar et al., 2020, Utzschneider et al., 2016). To assess their cytotoxic phenotype, we re-stimulated NTC and Nr2f6crispr-/- CAR-T cells from spleens of tumor-bearing mice ex vivo with target tumor cells. Nr2f6-modified CAR-T cells produced significantly higher amounts of interferon gamma (IFNγ) and granzyme B (GrzB) compared to controls (Fig1I, J), supporting their enhanced effector function.””\nComment 7) This reviewer could not figure out the origin of the scFv used to re-direct CAR-T cells against Epcam, but it is likely that it could be recognized as ‘foreign” in immunocompetent mice.\nResponse: We thank the reviewer for highlighting this important point. The scFv used to redirect CAR-T cells against EpCAM originates from the rat anti-EpCAM monoclonal antibody clone G8.8 (IgG2a). As expected from its non-murine origin, this scFv is immunogenic in immunocompetent mice and likely contributes to the limited persistence of CAR-T cells observed in our model. Importantly, rather than impairing therapeutic outcome, this transient CAR-T presence was sufficient to induce potent tumor rejection, underscoring the capacity of Nr2f6-deficient CAR-T cells to trigger host-mediated immune amplification. The observed tumor control despite immunogenic CAR-T depletion firmly supports our proposed mechanism, in which NR2F6 editing promotes cross-priming and the establishment of durable, antigen-agnostic endogenous immunity.\nManuscript addition (lines 191-193): “This 28ζ CAR comprises an scFv fragment derived from the rat anti-EpCAM antibody clone G8.8, fused to intracellular CD28 and CD3ζ signaling domains connected via a CD8 hinge and CD28 transmembrane region (Fig. 1A).”\nComment 8) -Toxicity and on target, off-tumor effects are important concerns that are not addressed\nResponse: We appreciate the reviewer’s concern regarding potential toxicity and on-target, off-tumor effects. Despite the known lung accumulation of EpCAM-targeted CAR-T cells, we observed no overt signs of toxicity, weight loss, or distress throughout the study (NEW Supplementary Fig. 1A–C). We attribute this favorable safety profile to two key factors: (i) the comparatively low CAR-T cell dose administered (3.6 × 10⁶ cells), which is below standard preclinical dosing regimens, and (ii) the omission of lymphodepletion, thereby minimizing systemic cytokine release and collateral tissue effects.\nConsistent with our findings, previous preclinical and clinical studies have reported limited off-tumor toxicity for EpCAM-directed CAR-T therapies, with ongoing clinical trials confirming tolerable safety and encouraging efficacy profiles (PMID: 32923168; DOI: 10.1126/sciadv.adg9721; NCT02915445).\nCollectively, these data support the translational feasibility of EpCAM-based CAR-T strategies when applied under controlled dosing conditions, such as ours. Importantly, the absence of any pathological or clinical toxicity in our Nr2f6-edited CAR-T treated groups further underscores that the enhanced antitumor efficacy observed is not accompanied by increased on-target, off-tumor activity, highlighting a distinctly favorable therapeutic index.\nManuscript addition (lines 536–538): “Importantly, we observed no toxicity or on-target/off-tumor effects, underscoring that the use of Nr2f6-modified CAR-T cells without lymphodepletion represents a promising strategy with minimal adverse effects while preserving potent endogenous antitumor immunity.”\nReviewer #2:\nThe authors are investigating the role of transcription factor NR2F6, recently identified as T cell checkpoint, in antigen spreading (AS) in the context of CAR-T cell therapy. They employ NR2F6 KO to address their thesis.\nComment 1) The more common term for AS is epitope spreading, defined as diversification of epitope specificity, and it typically requires cross-presentation of extracellular antigens in MHC-I context (https://doi.org/10.1038/nri724; https://doi.org/10.1158/1078-0432.CCR-20-0305).\nResponse: Thank you for this correction. We have replaced 'antigen spreading' with 'epitope spreading' throughout the manuscript to reflect established terminology.\nComment 2) The manuscript is well written, and experimental results are compelling. However, some results are in my opinion overstated/overinterpreted. You focus on AS as the main MOA (mechanism of action) of the NR2F6 KO in T cells rejecting tumors in vivo, but the experimental proof of AS being primarily responsible for the observed anti-tumor effect needs to be stronger for such statements.\nResponse: We sincerely thank the reviewer for the positive evaluation of our study and for raising this important point regarding the mechanistic interpretation of epitope spreading (ES). While alternative mechanisms may also contribute, multiple lines of complementary in vivo and ex vivo evidence now support ES as a key driver of tumor rejection mediated by Nr2f6-deficient T cells. Specifically, we observe the following:\n(i) an increased proportion of host tumor-associated antigen-specific effector CD3⁺ T cells by ELISPOT (see previous Fig.5H–I);\n(ii) elevated frequencies of cross-presenting cDC1 cells and corresponding innate activation signatures in TILs and spleens (see previous Fig.5D–E);\n(iii) robust rejection of EpCAM-negative Panc02 tumors upon rechallenge, indicative of epitope spreading (see previous Fig.6A–C); and\n(iv) adoptive transfer of primed splenocytes conferring tumor control independently of residual CAR-T cells (see previous Fig.6A–E). In addition, single-cell TCR sequencing revealed increased TCR diversity and reduced clonality in tumors from complete responders (see previous Fig.7E). Finally, our new early in vivo phenotyping data (day 3 post-therapy; NEW Fig.1F–J, NEW Supplementary Fig.3A-G) demonstrate enhanced endogenous activation, which is consistent with the initiation of host immunity.\nTaken together, these complementary datasets provide robust evidence in support of ES as a key mechanism of tumor rejection in this model, without ruling out the possibility of additional contributions from other Nr2f6-associated functional effects. We have revised the text to ensure that the mechanistic interpretation remains balanced and consistent with the data presented.\nPlease also see our detailed response to your Comment 11 below.\nComment 3) It would be nice to see direct data on broadening of Panc.02-relevant T cell repertoire in mice after vs before CAR therapy with NR2F6 KO cells to strengthen your main hypothesis.\nResponse: We thank the reviewer for this valuable suggestion. While a comprehensive pre- and post-therapy TCR repertoire analysis was beyond the present study’s technical reach, we did analyse TCR diversity in TILs from our memory/rechallenge experiments, which revealed an increase in TCR diversity among Nr2f6-knockout CAR-T treated animals (see Fig.7E). Recent CAR T cell studies (Cheloni et al., Nat Comm, 2025) demonstrate that endogenous TCR repertoire changes are generally not observed early after CAR T cell therapy; substantial alterations within the host T cell pool (including repertoire broadening, increased diversity, and enrichment of tumor-relevant clonotypes) typically emerge only at later time points, particularly in long-term memory or rechallenge settings. Consistent with this, our memory/rechallenge experiments revealed a marked increase in TCR diversity among Nr2f6-knockout CAR-T treated animals (see Fig. 7E). Focused analyses of high-frequency tumor-reactive clones in blood samples did not show pronounced compositional shifts 12 days after therapy, supporting published work indicating that early endogenous TCR repertoire remains largely unchanged. Notably, enrichment of CD3⁺ T cells following Nr2f6-edited CAR-T therapy (Reviewer Fig. 1) supports the enhancement of endogenous immunity over time.\nTogether, these results, in line with recent reports, underscore that endogenous TCR repertoire broadening and adaptation are mainly manifest during the memory phase following CAR T therapy, and not in the immediate effector phase.\nReviewer Figure 1 Analysis of T cell abundance and TCR repertoire in blood pre- and post-CAR-T therapy. (A) CD3⁺ T cell frequencies among lymphocytes before therapy (white), after non-targeting control CAR-T (gray), and after Nr2f6-knockout CAR-T (green), with individual mouse data and statistical comparison. (B–C) Dominant TCR Vβ family usage among CD8⁺ (B) and CD4⁺ (C) T cells pre- and post-therapy (12 days after CAR-T transfer) for both groups. Color legend denotes TCR Vβ segments; gray indicates undefined clones. Data show no major changes in dominant TCR clones post-therapy, but increased CD3⁺ T cell frequencies in the Nr2f6-edited group, supporting enhanced endogenous T cell responses.\nComment 4) Can you rule out other mechanisms, such as heightened anti-tumor reactivity, increased viability, proliferation, metabolic fitness, increased secretion of pro-inflammatory cytokines (IFNg) or lower exhaustion of KO effector T cells vs AS, or other?\nResponse: We thank the reviewer for highlighting the importance of defining the mechanisms underlying the enhanced antitumor activity of Nr2f6-deficient T cells. Previous work, including from our group, has established NR2F6 as an intracellular immune checkpoint that limits T cell effector function, cytokine production (IFNγ, IL-2, TNFα), viability, and metabolic activity, while promoting exhaustion (Klepsch et al., Nat Commun, 2018; Jakic et al., Cell Death Dis, 2021; H. Miftah et al., Front Immunol, 2023). These well-characterised cell-intrinsic effects explain the heightened anti-tumor reactivity of Nr2f6-deficient conventional T cells.\nIn the current work, we extend these mechanisms to CAR-T cells and uncover additional, previously underexplored features, including improved metabolic fitness, reduced exhaustion, and robust early effector cytokine production within the solid tumor microenvironment. Most importantly, our in vivo data now demonstrate that these hyper-reactive Nr2f6-deficient CAR-T cells can efficiently prime endogenous immunity, triggering durable, polyclonal host responses through epitope spreading (NEW Fig.1F-J, previous Fig.5D-H, Fig.6A-E, Fig.7E, NEW Supplementary Fig.3A-H). Thus, while superior intrinsic viability and cytokine output clearly contribute to improved direct cytotoxicity, our findings reveal a distinct and functionally separable mechanism: Nr2f6-modified, metabolically fit CAR-T cells serve as in situ immune catalysts that amplify secondary host anti-tumor responses through effective cross-priming. This represents a conceptual advance beyond earlier studies and supports Nr2f6 inhibition as a dual-acting strategy, enhancing both CAR-T cell potency and the endogenous immune response to achieve more durable tumor control.\nComment 5) Is the AS benefit mediated only by the modified (NR2F6 KO and CART+) CD8T cells, or is it also perpetrated by non-modified autologous T cells in the mice? This may shed light on the MOA.\nResponse: Our data suggest that the epitope spreading benefit requires modified Nr2f6-knockout CAR-T cells, since CAR transgene un-transduced T cells did not have any effect (see previous Supplementary Figure 1 / NEW Supplementary Figure 4C). Although endogenous T cells contribute to sustained downstream responses, the CAR-modified population is essential for initiating this cascade.\nMajor comments/Abstract and Introduction.\nComment 6) You speak of treatment of solid tumors in general, but only employ one model of pancreatic cancer: e Panc02 tumors. Please qualify your statements accordingly to your scope of work.\nResponse: We thank the reviewer for this important comment. As addressed in our response to Reviewer 1, Comment 3, we have extended our experimental scope beyond the Panc02 pancreatic cancer model to additionally include two independent solid tumor models—B16-F10-hEGFR melanoma and MC38-hEGFR colon carcinoma (see NEW Supplementary Fig. 2A–G). These models were deliberately chosen because they represent immunologically distinct tumor microenvironments: B16-F10 melanomas are poorly immunogenic with low T cell infiltration, whereas MC38 colon tumors are comparatively immune-responsive.\nThe demonstration of consistent Nr2f6-deleted CAR-T efficacy across these diverse systems confirms the broader applicability of our findings and strengthens the translational relevance of the mechanism described. We have also qualified manuscript statements accordingly to reflect this expanded scope.\nManuscript addition (lines 217-223): “To demonstrate broader applicability, we employed two independent CAR-T mouse models expressing an anti-human EGFR 28ζ CAR, together with syngeneic B16-F10 and MC38 tumor cells transduced with human EGFR (hereafter referred to as B16-F10-hEGFR and MC38-hEGFR). In both additional models, Nr2f6-depleted CAR-T-treated animals exhibited superior tumor control and prolonged survival compared with NTC CAR-T recipients (Suppl.Fig2A–G). These findings underline the broader relevance of Nr2f6-modified CAR-T cells, which achieve more effective and durable tumor clearance across distinct solid-tumor models.”\nComment 7) Please define your hypothesis more precisely: (e.g. KO of NR2F6 promotes AS in the context of CAR-T cell therapy? Or promotes other/additional effects..), and include a corresponding sentence in the abstract and intro.\nResponse: We thank the reviewer for this valuable suggestion to refine and explicitly define our working hypothesis. As requested, we have clarified the conceptual framework in both the Abstract and Introduction of the revised manuscript. Our hypothesis is that Nr2f6 deletion enhances the intrinsic activation state and effector function of CAR-T cells, which in turn promotes a secondary, endogenous anti-tumor immune response through antigen-agnostic epitope spreading. This is consistent with recent findings highlighting that targeting NR2F6 or its signalling pathway can potentiate CAR-T efficacy by inducing immunogenic tumor cell death and promoting systemic epitope spreading. This dual mechanism integrates the direct, cytotoxic effects of Nr2f6-edited CAR-T cells with their ability to prime endogenous host immunity.\nManuscript additions: Abstract (lines 60-61): “We postulate that the intrinsic benefits of Nr2f6-modified CAR-T cells enhance anti-tumor immunity by promoting secondary endogenous responses through epitope spreading.”\nIntroduction (lines 161–163): “We therefore hypothesized that deletion of Nr2f6 could reprogram CAR-T cells toward a metabolically fitter, less exhausted state, capable of initiating robust adaptive immune amplification through tumor antigen cross priming and epitope spreading.”\nComment 8) Reference to relevant literature: NR2F6 appears to play a role both in cancer cells (e.g. AML, glioma, lung cancer, melanoma…) and in immune (T ) cells. To provide a balanced intro, please briefly allude also to the role of this factor in tumor cells, vs the immune compartment. A recent report described both tumor intrinsic and tumor-extrinsic benefits of NR2F6 down-modulation in the context of immunotherapy of melanoma tumors DOI: 10.1126/sciadv.adf6621. How does your current work differentiate itself and add to the existing knowledge on this subject? Please add in intro or discussion vs this or similar recent references.\nResponse: We thank the reviewer for this valuable comment and have expanded both the Introduction and Discussion to provide a balanced perspective on NR2F6’s dual functions in cancer biology. Recent work has established that NR2F6 exerts both tumor-intrinsic and immune-extrinsic effects. In melanoma, tumor cell-intrinsic NR2F6 represses interferon-γ–regulated genes and immune-stimulatory pathways, enabling immune evasion, whereas Nr2f6 loss sensitizes tumors to PD-1 blockade (H. Kim et al., Sci. Adv., 2023; DOI: 10.1126/sciadv.adf6621). These findings complement earlier reports identifying NR2F6 as a T cell-intrinsic immune checkpoint controlling NFAT/AP-1–dependent cytokine transcription and effector function (Klepsch et al. Nat. Commun., 2018, DOI: 10.1038/s41467-018-04004-2). Building on these discoveries, our present study focuses exclusively on the immune cell-intrinsic axis of NR2F6 regulation in engineered CAR-T cells. In contrast to tumor-intrinsic Nr2f6 inhibition, our work demonstrates that targeted deletion of Nr2f6 in CAR-T cells alone is sufficient to enhance early effector function and—critically—to initiate epitope spreading-driven endogenous immunity in solid tumors. This approach provides a complementary and translationally distinct framework for immunotherapeutic NR2F6 targeting.\nManuscript addition (Introduction, lines 156-158): “Moreover, NR2F6 expression in melanoma cells contributes to immune evasion and tumor progression, underscoring its dual role in the tumor microenvironment (Kim et al., 2023).”\nResults:\nComment 9) In your experimental design, did you modify only CD8 T cells, or all T cells with NR2F6 KO and CAR- ? please clarify.\nResponse: We used CD8+ T cells exclusively for Nr2f6 knockout and CAR transduction in the experiments reported. During revision, we included limited CD4+ CAR-T data (see NEW Supplementary Figure 2H–K) in response to questions from you (comment 10 + 16) and Reviewer 3 (comment 2).\nComment 10) Page 4:” We did not observe any differences in CAR transduction efficiency (Fig2A) or expansion of Nr2f6-/- or wild type CD8+ T cells during CAR-T cell production (Suppl.Fig2B).” Did you look at CD4 T cells? Do they contribute to the beneficial effects you observed in vitro or in vivo? Please report.\nResponse: We thank the reviewer for this insightful question regarding the role of CD4+ T cells. While the original manuscript did not address CD4+ T cells, prompted by reviewer feedback, we analysed CD4+ CAR-T cells. Our data show that CD4+ CAR-T cells do not enhance the beneficial tumor phenotype mediated by CD8+ CAR-T cells (see NEW Supplementary Figure 2H–K). This may reflect the transient presence of CAR-T cells in our model, where the effects of long-term CD4+ support cannot be observed.\nManuscript addition (lines 240-245): “Of note, and focusing on CAR persistence, some reports highlight the crucial role of CD4+ T cells promoting and sustaining CAR-T cell responses in long term responders (Melenhorst et al., 2022). We transferred a mixture of CD8+ and CD4+ Nr2f6crispr-/- CAR-T cells to evaluate the impact of CD4 help, but could not observe a beneficial effect on tumor growth comparing to CD8 only therapy (Suppl.Fig2H-K).'\nComment 11) Page 7:” Therefore, the observed immune defense against the tumor after the apparent disappearance of CAR-T cells can be well explained by AS and the subsequent induction of a strong secondary tumor antigen-specific T cell response. Overall, we demonstrate a cooperative anti-tumor effect of Nr2f6-modified CAR-T therapy and endogenous immune cells.” You purport AS, but present little direct data of AS in vivo or ex vivo… Were cDC1 you identified involved in prolongation of anti-tumor responses after T cells were gone? Were other cells recruited? What was their phenotype?\nResponse: We thank the reviewer for this crucial question and the opportunity to clarify our interpretation of epitope spreading (ES) as one principal mechanism in our model. Recognizing the inherent complexity of demonstrating ES directly in vivo, we employed multiple complementary in vivo and ex vivo approaches aligned with established definitions of ES in cancer immunotherapy—the expansion and diversification of endogenous T cell responses to secondary tumor antigens beyond the initial CAR target, coordinated primarily by antigen-presenting cells such as cDC1s, and associated with durable anti-tumor immunity.\nOur principal findings include:\n• Increased tumor-associated antigen-specific CD3⁺ T cells by ELISPOT after CAR-T therapy, using re-stimulation with Panc02 peptide-pulsed dendritic cells, tumor lysate, or cell line antigens (previous Fig5.G, H).\n• Elevated frequencies of cDC1 cross-presenting dendritic cells in TILs and spleens, providing essential cross-priming for CD8⁺ T cell activation (previous Fig.5E, F).\n• Rejection of EpCAM-negative Panc02 tumors upon rechallenge in complete responder mice, functionally demonstrating control of antigen-loss variants, a hallmark of productive ES (previous Fig.6 A-C).\n• Adoptive transfer of splenocytes from complete responders conferring tumor control in naïve mice, independent of CAR-T cells, with high IFNγ recall responses ex vivo, indicating induction of durable, host-mediated memory (previous Fig.6D, - E).\n• Single-cell TCR sequencing showing increased diversity and reduced clonality in TILs from responders, consistent with repertoire broadening triggered by CAR-T-induced immunogenic tumor cell death (previous Fig.7E).\n• Early post-therapy phenotyping (day 3) revealing enhanced activation of endogenous CD8⁺ Tpex cells (NEW Supplementary Fig.3A-E), increased CD8-IFNg+ producing cells (NEW Supplementary Fig.3F), and elevated NK cell numbers in tumor-draining lymph nodes (NEW Supplementary Fig.3G), supporting elimination of antigen-negative targets.\n• Regarding other immune cell recruitment, single-cell RNA sequencing analysis revealed increased populations of proliferating CD8+ T cells, Th2 cells, macrophages, and cDC1s in treated animals (previous Fig.7B, D).\nWhile alternative mechanisms may also contribute, multiple lines of complementary in vivo and ex vivo evidence now align with a mechanistic model in which ES acts as a central determinant of durable tumor control. Our data show that Nr2f6-deleted CAR-T cells synergize with endogenous antigen-presenting and effector cell networks to broaden the antitumor immune repertoire, sustain tumor rejection beyond CAR-T cell persistence, and limit antigenic escape. We have revised the manuscript to clarify this cooperative interplay between Nr2f6-edited CAR-T cells, epitope spreading, and both adaptive and innate immune recruitment as the integrated mechanistic forces that drive durable and diversified antitumor immunity in our model.\nComment 12) “Therefore, the observed immune defense against the tumor after the apparent disappearance of CAR-T cells can be well explained by AS and the subsequent induction of a strong secondary tumor antigen-specific T cell response. Overall, we demonstrate a cooperative anti-tumor effect of Nr2f6-modified CAR-T therapy and endogenous immune cells.” – please consider revising this statement to match the strength of your data. If I understood correctly, you only showed the ability of cDC to generate enhanced T cell priming of NR2F6 KO CD8 T cells in vitro, but not proven that this happened during your in vivo experiment and was indeed responsible for accelerated tumor clearance.\nResponse: We thank the reviewer for this important feedback and fully agree that direct in vivo proof of secondary antigen-specific T cell induction and the exclusive role of cDC1s remains correlative in our current study. In line with your suggestion, we have revised the statement in the text to accurately reflect the strength and scope of our data.\nOur evidence supporting ES as a principal mechanism relies on a robust combination of complementary in vivo and ex vivo readouts:\n• Elevated cDC1 levels in tumors and spleens, together with increased IL-12p40, supporting increased cross-presentation capacity and a pro-inflammatory microenvironment (previous Fig.5E-F).\n• Adoptive transfer experiments, where splenocytes from complete responders provided antigen-independent tumor control—strongly indicative of durable endogenous immunity (previous Fig.6D).\n• Early and sustained phenotypic recruitment of endogenous adaptive and innate immune populations (CD8+, Th2, cDC1s, NK, macrophages) by flow cytometry and single-cell sequencing (previous Fig. 5E, previous Fig.7A-D, NEW Supplementary Figure 3 A-G).\nWe acknowledge that, due to the phenotypic diversity of antigen-presenting cells within the tumor microenvironment, it is plausible that other APC subsets, such as monocyte-derived DCs, macrophages, or B cells, also contribute to amplifying endogenous T cell responses in this setting. Although we did not find a definitive phenotypic change in these cell types in our current datasets, this possibility is explicitly discussed as a potential avenue for follow-up mechanistic studies:\nManuscript addition (lines 554-557): “While our data highlight the critical role of cDC1s in this process, we do not exclude potential contributions from other antigen-presenting cell populations, such as monocyte-derived dendritic cells, macrophages, or B cells, which warrant further investigation in future studies.\nComment 13) “The data support the concept that NR2F6-modified CAR-T therapy apparently facilitates the occurrence of AS, which results in a robust secondary polyclonal immune response induced by the crosstalk of endogenous innate and adaptive immune cell compartments.”\n- Again, please prove that a robust secondary polyclonal immune response occurred in vivo, or rephrase.\nResponse: We thank the reviewer for this important comment and have carefully revised our manuscript statements to reflect the scope and strength of our in vivo evidence for a robust, secondary polyclonal immune response. In our model, mice treated with Nr2f6-deficient CAR-T cells not only completely rejected antigen-positive Panc02 tumors, but also exhibited tumor rejection upon rechallenge with antigen-negative variants (previous Fig.6A–C), at a time point when CAR-T cells were no longer detectable. This functional rejection of antigen-loss variants, which lack the CAR target, provides strong evidence for durable, CAR-independent endogenous tumor immunity—a phenomenon widely accepted as a hallmark of productive epitope spreading and polyclonal T cell response activation.\nComplementing these rechallenge data, TCR repertoire sequencing in complete responder mice revealed decreased clonality and increased TCR diversity (previous Fig.7E), strongly supporting expansion and diversification of the host T cell repertoire consistent with polyclonal immunity. While these findings collectively provide compelling evidence for a broad, secondary endogenous T cell response, we fully acknowledge that the precise cellular and antigenic determinants require further delineation and have moderated our interpretations accordingly throughout the text.\nManuscript addition (Conclusion on p19, lines 570-576):This study identifies NR2F6 as a T-cell-intrinsic metabolic checkpoint that limits CAR-T cell function in solid tumors. Deletion of Nr2f6 enhances exhaustion resilience and sustains antitumor activity by coupling cytotoxic reinvigoration with dendritic cell-dependent cross-priming and epitope spreading (ES). These preclinical findings provide a rationale for targeting NR2F6 to overcome immunosuppression and antigenic heterogeneity in solid tumors, with potential relevance for improving CAR-T and other cell-based immunotherapies in human cancer.”\nComment 14) Figure 8 is very general and doesn’t point out the specific MOA through which NR2F6 KO benefits CAR-T cell therapy in your proposed model. Please revise.\nResponse: We have comprehensively revised Figure 8 to highlight the mechanistic pathways through which Nr2f6 knockout enhances CAR-T therapy, including improved cytotoxicity, epitope spreading and endogenous memory formation.\nGraphical Summary in Figure 8: Functional advantages and mechanisms underlying Nr2f6-modified CAR-T cell therapy in a solid tumor model: A. 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 production, and improved metabolic performance under repetitive stimulation. B. 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, facilitates antigen uptake and presentation by host antigen-presenting cells (APCs), and immune responses beyond the initial CAR target. C. 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.\nDiscussion:\nComment 15) Are your findings regarding NF2F6 function in cancer immunotherapy confined to the specific mouse-strand you used, or can they be generalized, why? How broadly can they be generalized to mouse experimental systems? To human health? Please speculate or provide data/refs to support your points.\nResponse: Thank you for raising this important question regarding the generalizability of our NR2F6 findings in cancer immunotherapy. We have expanded the Discussion to clarify that NR2F6 exhibits a high degree of homology and functional conservation between mouse and human, supporting the broader relevance of our data. Previous work from our group and others (Klepsch et al., Nat. Commun., 2018; DOI: 10.1038/s41467-018-04004-2; Hermann-Kleiter et al., Cell Rep., 2015, DOI: 10.1016/j.celrep.2015.08.035) has demonstrated that NR2F6 ablation leads to augmented effector function and hyper-reactivity in both mouse and human T cells, underscoring its conserved immunoregulatory role. Notably, Nr2f6-deficient mice consistently display enhanced tumor control and effector responses across distinct genetic backgrounds, indicating that these effects are not limited by mouse strain.\nClinical relevance is supported by recent human melanoma studies that correlate NR2F6 expression with impaired anti-tumor immunity and show that its targeting can restore effector function and responsiveness to immunotherapy (Kim et al., Sci Adv, 2023; DOI: 10.1126/sciadv.adf6621). Collectively, these data argue that the anti-tumor effects of Nr2f6 deletion identified in our study reflect a broadly conserved mechanism not restricted to a single mouse strain and provide compelling support for translational application in human cancer immunotherapy. These points have been clarified with new references in the revised Discussion.\nComment 16) What happens with CD4 T cells in your models? Is NR2F6 important for CD4 T cells?\nResponse: We thank the reviewer for raising this important point. NR2F6 plays a well-established immunoregulatory role in CD4⁺ T cells, particularly in modulating helper T cell differentiation and cytokine expression. Previous studies have shown that NR2F6 functions as a transcriptional repressor of NFAT:AP-1-dependent genes and limits the production of pro-inflammatory cytokines such as IL-2, IL-17, and IFNγ, thereby restraining Th17 differentiation and effector CD4 responses (Hermann-Kleiter et al., Cell Rep, 2015; DOI: 10.1016/j.celrep.2015.08.035; Klepsch et al., Nat Commun, 2018. DOI: 10.1038/s41467-018-04004-2; Klepsch et al., Cell Commun Signal, 2021, DOI: 10.1186/s12964-019-0454-z). Mechanistically, NR2F6 directly interferes with NFAT:AP-1 DNA binding and acts as a PKC-regulated transcriptional checkpoint, maintaining appropriate activation thresholds in CD4⁺ T cells.\nHowever, our new experiments show that, within our CAR-T context, CD4⁺ CAR-T cells do not significantly contribute to the enhanced tumor-rejection phenotype mediated by Nr2f6-deficient CD8⁺ CAR-T cells (see NEW Supplementary Fig.2H–K). These data indicate that, while NR2F6 remains functionally important in CD4 T cell biology, its contribution to the observed therapeutic effects in this specific CAR-T setting appears secondary to that of CD8⁺ effector cells. We have clarified this distinction in the manuscript to reflect the cell-type specificity of NR2F6 function.\nManuscript addition (discussion, lines 516-518): NR2F6 exerts immunoregulatory control in both CD4⁺ and CD8⁺ T cells, but our data indicate that the enhanced antitumor activity is primarily mediated by CD8⁺ CAR-T cells (Suppl.Fig2H–K).”\nComment 17) Does AS in your model systems occur only via the modified KO T cells, CAR+ T cells, both modifications only, or also is carried out by “naïve”, non-modified host’s T cells?\nResponse: Thank you for highlighting this mechanistic point. Our experiments demonstrate that the augmented epitope spreading and polyclonal, endogenous immune response in our models requires the presence of Nr2f6-knockout CAR-T cells. This effect is not observed with Nr2f6-depleted un-transduced, non-CAR-T cells (see NEW Supplementary Fig.4C / OLD Suppl.Fig.1). These findings indicate that only the combination of CAR expression and Nr2f6 inhibition in T cells enables sufficient tumor cell killing and antigen release to potentiate secondary, host-mediated immune priming and robust epitope spreading. While we cannot fully exclude limited activation of endogenous host T cells, our data show that the observed durability of the anti-tumor response is not generated by naïve or non-modified T cells alone. Instead, the combinatorial effect of Nr2f6-knockout together with CAR modification is required to initiate the cross-priming and expansion of secondary tumor-antigen–specific T cell clones seen in our model.\nManuscript addition (Discussion, lines 518–522): “ The observed ES effect likewise depends mainly on Nr2f6-modified CAR-T cells, as untransduced bystander T cells failed to confer this benefit (Suppl.Fig4C). Still, we cannot fully exclude minor supportive contributions from endogenous, non-modified T cells that may be secondarily activated following CAR-T-mediated tumor recognition.””\nComment 18) Does AS work via cytokine secretion, boosting the activity of cDCs, or decreasing activation threshold in T cells (modified or not), or other mechanisms? Please propose specific and detailed mechanisms as you see fit and describe in the discussion.\nResponse: We appreciate this mechanistic question. Based on our results and supported by current literature, we propose that epitope spreading in our model is driven by Nr2f6-deficient CAR-T cells through three main mechanisms: (1) enhanced cytokine secretion (e.g., IFNγ, that boosts dendritic cell activity and cross-priming); (2) a reduced T cell exhaustion threshold, enabling stronger and faster CAR-T function; and (3) subsequent recruitment and activation of endogenous innate and adaptive immune cells. These effects jointly facilitate immunogenic tumor cell death, dendritic cell activation, and broadening of the host T cell repertoire—a process known to underlie durable, polyclonal antitumor responses in CAR-T therapy.\nManuscript addition (lines 557-562): “ Mechanistically, epitope spreading is likely orchestrated through multiple complementary pathways: enhanced IFNγ secretion by Nr2f6-deficient CAR-T cells amplifies cDC1 activation; loss of NR2F6 lowers the activation threshold of CAR-T cells, inducing a hyper-functional state that strengthens and prolongs direct cytotoxic responses; and immunogenic cell death (ICD) triggered by these effector cells broadens the tumor antigen repertoire, thereby enhancing cross-priming and diversifying host T cell responses.”\nComment 19) Minor comments Please revise for clarity/brevity:” …Of note, and relevant to a potential clinical setting, we have previously shown haploinsufficiency for the NR2F6 gene to be sufficient for the anti-tumor phenotype indicating that deletion of one allele will be sufficient (20).”\nResponse: Thank you for highlighting this point. We have revised the text for clarity and clinical relevance. Previous studies show that even partial reduction of NR2F6 function—such as in Nr2f6⁺/⁻ (heterozygous) mice—elicits the same antitumor phenotype as full knockout, confirming haploinsufficiency of the gene (Klepsch et al., Nat Commun, 2018, DOI: 10.1038/s41467-018-04004-2). This indicates that complete loss of NR2F6 activity is not required for therapeutic benefit, an encouraging finding for translational strategies where partial inhibition or monoallelic editing may be more achievable.\nManuscript addition (Introduction, lines 198–201): “Of note, and of potential clinical relevance, we have previously demonstrated that even partial loss of NR2F6, as seen in heterozygous knockout mice, is sufficient to elicit a robust anti-tumor phenotype (Klepsch et al., 2018b).”\nReviewer #3:\nThe manuscript presents highly interesting research on the advantages of Nr2f6 knockout in enhancing the therapeutic efficacy of CAR-T cell responses. To further strengthen this work, we would like to offer some additional considerations regarding the chosen methodology:\nResponse: We thank the reviewers for their positive feedback and appreciate their suggestions to further strengthen the manuscript.\nComment 1) Firstly, regarding Nr2f6, its function in T lymphocytes could be more clearly described in the introduction, even if briefly, to provide better context.\nResponse: We thank the reviewer for this helpful suggestion. We have added a concise description of NR2F6’s immunoregulatory function in T lymphocytes to the Introduction, for improved context.\nManuscript addition (Introduction, lines 140-160): “Here, we focus our investigation on the role of the orphan nuclear receptor NR2F6, a member of the COUP-TF of transcription factors, acting as an intracellular immune checkpoint, repressing NFAT- and AP-1–dependent cytokine transcription thresholds in effector T cells. It functions mechanistically as a ligand-independent transcriptional repressor that antagonizes the DNA binding of these transcription factors, thereby suppressing the expression of key pro-inflammatory cytokines, including IL-2, IFNγ, TNFα, IL-17, and IL-21 (Hermann-Kleiter et al., 2008, Hermann-Kleiter et al., 2012, Hermann-Kleiter et al., 2015). The activity of NR2F6 is tightly controlled by PKC-mediated phosphorylation, which modulates chromatin association and transcriptional repression (Hermann-Kleiter et al., 2015). Through this molecular brake, NR2F6 maintains immune homeostasis and prevents excessive effector differentiation into highly activated T lymphocyte subsets (Hermann-Kleiter et al., 2008, Hermann-Kleiter et al., 2012, Hermann-Kleiter et al., 2015). Inhibition of NR2F6 enhances also NK cell effector function (Woelk et al., 2025) and improves pathogen clearance in a preclinical Listeria monocytogenes model (Jakic et al., 2021). Together with our previous studies identifying NR2F6 as an intracellular immune checkpoint in the effector T cell compartment (Klepsch et al., 2018b, Klepsch et al., 2018a), these findings establish NR2F6 as a promising therapeutic target for enhancing anti-tumor immunity. Moreover, NR2F6 expression in melanoma cells contributes to immune evasion and tumor progression, underscoring its dual role in the tumor microenvironment (Kim et al., 2023). These findings highlight the translational relevance of targeting NR2F6 in cancer immunotherapy and provide a rationale for exploring Nr2f6-deficient CAR-T cells in solid tumors.\nComment 2) Another aspect highlighted in some reports is the crucial role of CD4⁺ T cells in promoting and sustaining CAR-T cell responses in long-term responders. Carl June’s retrospective studies over a decade post treatment have demonstrated the persistence of predominantly CD4⁺ CAR-T cells. In this regard, we believe it would be valuable to evaluate the proposed therapeutic modification in this population, rather than focusing solely on CD8⁺ T cells. While the proposed mechanism of action—through antigen spreading—may be primarily driven by granzyme and perforin release from CD8⁺ T cells, the CD4⁺ subpopulation could also play a significant role in the overall response. Since recent published data show that long term persistence of CAR-T cells in the CD19+ tumor setting is found on CD4+ cells in patients and the authors show that the CD8+ CAR-T cell used do not last, it will be very important and informative to test solely transferring CD4+ CAR-T cells and also mixing CD4+ and CD8+ CAR-T cells to evaluate the impact on CAR-T cell persistence, antigen spreading and memory response formation.\nResponse: We thank the reviewers for emphasizing the importance of CD4⁺ CAR-T cells in sustaining long-term CAR-T persistence and function. Clinical and preclinical studies have shown that cytotoxic CD4⁺ CAR-T cells can exert direct antitumor activity via IFNγ–dependent mechanisms and play critical helper roles by shaping dendritic cells and CD8⁺ T cell activation. In response, we generated and tested Nr2f6-deficient CD4⁺ CAR-T cells, both alone and in combination with CD8⁺ CAR-T cells. Mixed or combined CD4⁺/CD8⁺ CAR-T infusions did not further improve tumor control compared to CD8⁺ CAR-T cells alone (see NEW Supplementary Fig.2H–K). We have clarified these results and their contextual relevance in the revised manuscript.\nManuscript addition (discussion, lines 516–518): “NR2F6 exerts immunoregulatory control in both CD4⁺ and CD8⁺ T cells, but our data indicate that the enhanced antitumor activity is primarily mediated by CD8⁺ CAR-T cells (Suppl.Fig2H–K).”\nNotably, CD4⁺Nr2f6-KO CAR-T cells alone delayed tumor progression relative to unmodified controls (Reviewer Figure 2), supporting a contributory, but secondary, role for this subset that warrants further mechanistic exploration in the future.\nReviewer Figure 2: Tumor growth kinetics following adoptive transfer of CD4⁺ or mixed CD4⁺/CD8⁺ Nr2f6crispr-/- CAR-T cells. Tumor-bearing mice received PBS (gray line), CD4⁺ Nr2f6crispr-/- CAR-T cells (solid green line), or mixed CD4⁺/CD8⁺ Nr2f6crispr-/- CAR-T cells (dashed green line) on day 5 after tumor inoculation. Tumor volume (mean ± SEM, n ≥ 5/group) was measured over time. CD4⁺ CAR-T cell treatment conferred superior tumor control compared to both the mixed-group and PBS, consistent with recent evidence that cytotoxic and helper CD4⁺ CAR-T cells can drive potent antitumor responses, sometimes independently or in concert with CD8⁺ CAR-T cells.\nComment 3) Additionally, although the proposed therapy is intended for solid tumors, hematological malignancies remain the only currently approved indication for CAR-T cell therapy and still require further optimization. In this context, we believe it would be important to evaluate the proposed approach either in this setting or in an alternative tumor model. Although the claim of the authors is based on the antigen spread phenomenon to explain the improved antitumor effect, testing the effect of Nr2f6 abrogation in a more classical setting such as a CD19+ leukemia or lymphoma could add potential clinical - validated implications to the manuscript.\nResponse: We thank the reviewer for this valuable comment and agree that exploring NR2F6 abrogation in hematologic models, such as CD19⁺ leukemia or lymphoma, would provide additional translational perspective. In the present revision, we have expanded our study to include additional solid tumor models—B16-F10-hEGFR melanoma and MC38-hEGFR colon carcinoma (see NEW Supplementary Figure. 2A–G)—confirming that Nr2f6 knockout enhances CAR-T cell efficacy across distinct solid tumor types.\nWhile evaluation in hematologic malignancies represents important future work, our current focus provides strong proof-of-concept in solid tumors, where therapeutic effectiveness of CAR-T cells remains most challenging. This focus aligns with recent findings showing that Nr2f6 inhibition improves CAR-T cell activation, proliferation, and memory across multiple solid tumor settings. We have clarified these points in the revised manuscript to emphasize both the immediate relevance to solid tumors and the broader translational potential of NR2F6 modulation.\nManuscript addition (Discussion, lines 514-516): “These results were comparable to those obtained with two independent CAR-T mouse models (B16-F10-hEGFR, MC38-hEGFR) (Suppl.Fig2A-G), which demonstrate the broad applicability of the obtained results.”\nReviewer #4:\nComment: I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We appreciate your participation in the Nature Communications peer review training and co-review initiative, and thank you for contributing to the constructive evaluation of our manuscript. We commend the journal’s efforts to recognize and train Early Career Researchers through this program, and fully support such initiatives that foster high-quality, transparent peer review.\nNR2F6 deletion revives CAR-T cell function and induces antigen-agnostic immune memory in solid tumors\nCorresponding authors: Victoria Klepsch and Gottfried Baier\nReviewer #2:\nI am satisfied with this revision by authors.\nResponse: Thank you for your positive feedback.\nReviewer #3:\nThe authors covered most of the requested improvements and the manuscript now is more complete and covering the study limitations.\nResponse: We thank the reviewer for his/her positive feedback and appreciate that our manuscript is more complete and cover the study limitations.\nReviewer #4:\nComment: I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We appreciate your participation in the Nature Communications peer review training and co-review initiative and thank you for contributing to the constructive evaluation of our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_2": "I am satisfied with this revision by authors.", "reviewer_3": "The authors covered most of the requested improvements and the manuscript now is more complete and covering the study limitations.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter