The integrated stress response promotes immune evasion through lipocalin 2

Cancer cells activate the integrated stress response (ISR) to adapt to stress and resist therapy. ISR signals converge on activating transcription factor 4 (ATF4), which controls cell-intrinsic transcriptional programs that are involved in metabolic adaptation, survival and growth^‍. However, whether the ISR–ATF4 axis influences anti-tumour immune responses remains mostly unknown. Here we show that loss of ATF4 decreases tumour progression considerably in immunocompetent mice, but not in immunocompromised ones, by enhancing T cell-dependent anti-cancer immune responses. An unbiased genetic screen of ATF4-regulated genes identifies lipocalin 2 (LCN2) as the principal ATF4-dependent effector that impairs anti-tumour immunity by favouring infiltration with immunosuppressive interstitial macrophages. Furthermore, we find that LCN2 promotes T cell exclusion and immune evasion in preclinical mouse models, and correlates with decreased T cell infiltration in patients with lung and pancreatic adenocarcinomas. Anti-LCN2 antibodies promote robust anti-tumour T cell responses in mouse models of aggressive solid tumours. Our study shows that the ATF4–LCN2 axis has a cell-extrinsic role in suppressing anti-cancer immunity, and could pave the way for an immunotherapy approach that targets LCN2.

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Unlike conventional drugs, whose activity is shaped by binding to a single target, CAR T-cell (CART) engagement with tumor cells involves multiple receptors, adhesion molecules, and signaling proteins. These components are partially organized within cholesterol-rich membrane microdomains called lipid rafts, forming the CAR immune synapse (CARIS)—the interface through which a CART recognizes, attaches to, and kills a tumor cell.

To evaluate CARIS as a pharmacological interface, we compared CD28- and 4-1BB-CARTs, which exhibit distinct therapeutic profiles. The rapid antitumor activity of CD28-CARTs was associated with brief, dynamic synapses supporting rapid signaling and serial killing. Conversely, 4-1BB-CARTs formed longer, mechanically stable synapses consistent with their slower but sustained activity. Synapse strength was measured using z-Movi acoustic force microscopy.

Lipid rafts were enriched at CARIS. Attenuating membrane lipid raft cholesterol preferentially compromised 4-1BB CARIS mechanical stability, while predominantly impairing CD28 CARIS activation and cytolytic output. The molecular networks underlying these differences were mapped by integrating lipid-raft lipidomics, differential SILAC proteomics to distinguish CART-derived from tumor-derived components, and T-cell transcriptomics.

CD28-associated lipid rafts were enriched in diacylglycerol signaling components, PKCθ, small GTPases, Src-family kinases, and AKT/PI3K-proximal modules, supporting rapid signal-to-function coupling. Stomatin-family and flotillin scaffolds favored dynamic remodeling, while CD8-dominant transcriptional programs supported cytolytic degranulation. Engaged tumor cells displayed membrane-stress signatures consistent with late-stage senescence and apoptotic commitment.

In contrast, 4-1BB-associated lipid rafts were enriched in lysophosphatidic acid, an inhibitory lipid known to suppress CD8 effector functions while promoting integrin-dependent adhesion, consistent with the marked enrichment of integrins at CARIS, NF-κB and cytokine signaling components, and ERM scaffolds that couple lipid rafts to cortical actin, sustaining CARIS stability. Engaged tumor cells exhibited signatures of early apoptotic resistance, including damage-response and repair markers.

Together, these findings establish CARIS as a central determinant of CART function and therapeutic behavior. Mapping and manipulating its lipid–protein networks provide a platform for understanding CART pharmacodynamics and developing strategies to improve therapeutic efficacy.

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CD19-directed CAR-T therapy has achieved remarkable clinical outcomes in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Nevertheless, a significant proportion of patients develop primary resistance or relapse, often associated with CD19 antigen loss or downregulation. Given the co-expression of CD19 and BCMA in NHL, we hypothesized that dual antigen targeting could improve therapeutic durability and mitigate antigen escape. To address this, we developed a panel of CD19/BCMA dual-targeting CAR-T cells building on our academic platforms targeting CD19 (varnimcabtagene autoleucel, ARI0001) and BCMA (cesnicabtagene autoleucel, ARI0002h). Multiple strategies were explored, including pooled mono-targeted products, co-transduction with two lentiviral vectors, bicistronic constructs, and tandem/loop CAR designs incorporating dual binding domains within a single receptor. Functional activity and avidity were assessed across varying antigen expression conditions. We found that dual CAR-T cells generated through co-transduction consistently showed superior performance relative to single-target CD19 CAR-T cells and other dual-targeting formats, particularly in models with low CD19 expression. A first-in-human phase I clinical trial (CARTDBG-01; NCT06097455) is currently underway to evaluate the safety and efficacy of ARI0003 in NHL.

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Related publication: Bachiller, M., Barceló-Genestar, N., Rodriguez-Garcia, A., Alserawan, L., Dobaño-López, C., Giménez-Alejandre, M., ... & Guedan, S. (2025). ARI0003: Co-transduced CD19/BCMA dual-targeting CAR-T cells for the treatment of non-Hodgkin lymphoma. Molecular Therapy, 33(1), 317-335. https://doi.org/10.1016/j.ymthe.2024.11.028

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