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

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 T cell 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, as shown by protection in tumor re‑challenge experiments. Although infused CAR‑T cells disappear within 2 weeks, durable tumor control coincides with epitope spreading and secondary immune responses, likely via dendritic and other antigen‑presenting 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 loss and tumor heterogeneity, establishing NR2F6 inhibition as a promising CAR‑T engineering strategy for durable, antigen‑agnostic solid‑tumor immunotherapy.

ORGANISM(S): Mus musculus

PROVIDER: GSE284026 | GEO | 2026/01/23

REPOSITORIES: GEO

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Project description:Orphan-Nuclear-Receptors (ONRs) are a group of ligand dependent transcriptional factors belonging to the family of Steroid-Nuclear-Receptors, which includes estrogen, progesterone and retinoic acid receptors. The ONR denomination refers to the fact that the endogenous ligands of these 15 nuclear receptors have not yet been identified. The vast majority of ONRs are expressed not only in normal tissues but also in different types of solid tumors, including various types of breast cancer. In the present study, we define the expression levels of the ONR mRNAs in a large panel of 51 cell-lines, which recapitulate the heterogeneity of breast cancer. Ten ONRs are characterized by a relatively high expression level in the majority of the breast cancer cell-lines considered and this is consistent with the high levels of the transcripts observed in mammary tumor tissues. To get insights into the functional role played by the 10 ONRs in the growth/progression of breast cancer, we silenced each receptor with a siRNA approach. Effective silencing of the NR2F2 receptor in 5 different and representative breast cancer cell-lines with two separate siRNAs results in an increased growth of each cell-line consistent with a tumor-suppressive action of the receptor. The effect is independent of the cell phenotype, as it is observed in Estrogen-Receptor and HER2 positive as well as Triple-Negative cell-lines. By converse, silencing of the NR2F6 receptor reduces the growth of the 5 selected cell-lines characterized by high expression levels of the ONR, suggesting an oncogenic action. Once again, the anti-proliferative effects of NR2F6 silencing are evident in all the cell-lines considered and are not associated with any of the 3 breast cancer phenotypes. Stable silencing of NR2F6 following infection of the Estrogen-Receptor positive MCF-7 and the Triple-Negative MDA-MB231 cell-lines with 2 specific and different shRNAs support the oncogenic properties of the ORF. Indeed, these shRNAs reduce the growth and clonogenic ability of the 2 cell-lines. In a first set of studies, we performed whole-genome RNA-sequencing experiments in the shRNA infected MCF-7 cells to get insights into the molecular mechanisms underlying the oncogenic action of NR2F6. Stable silencing of the NR2F6 mRNA and protein results in an up-regulation of the gene-networks involved in the pathways controlling cell-adhesion and cell-cell interactions. As expected, silencing causes down-regulation of the gene-networks involved in cell-cycle and cell-proliferation. In particular, we identify 50 and 9 genes whose expression is up-regulated and down-regulated by the knock-down of the NR2F6 transcription factor. These genes are likely to be direct or indirect targets of NR2F6 transcriptional activity. In a second set of experiments, we identify the endogenous organic molecules capable of interacting with the NR2F6 receptor using a mass-spectrometry based approach. To this purpose, we used NR2F6 immune-precipitates obtained from MCF-7 cells, which we transfected with a full-length NR2F6 cDNA expression plasmid. These studies permitted us to identify palmitoylethanolamide as the only endogenous organic molecule binding NR2F6 with high affinity and reproducibility.

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

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