Abstract Metabolic dysregulation in obesity reshapes immune function, but how lipid signals drive immune suppression remains unclear. Herein, we identify a FABP4–PD-L1 axis that links lipid metabolism to immune checkpoint regulation in myeloid cells. Single-cell transcriptomics revealed a distinct FABP4high immunosuppressive splenic macrophage subset enriched under high-fat diet (HFD) conditions, characterized by impaired antigen presentation and elevated PD-L1 expression. Mechanistically, palmitic acid (PA) was shown to induce FABP4 and promote PD-L1 palmitoylation, leading to its stabilization on the cell surface independent of transcriptional regulation. FABP4 was essential for this process, which enabled PD-L1 surface stabilization, immunosuppression and mammary tumor progression. In humans, a conserved CD14intCD16⁺ monocyte population exhibited elevated FABP4–PD-L1 signaling and correlated with obesity and invasive breast cancer. These findings establish PD-L1 as a metabolically regulated protein and reveal a mechanism by which lipid excess drives immune evasion, suggesting FABP4 as a potential therapeutic target in cancer immunotherapy.
Malignant ascites provides a window into cancer-immune interactions in the peritoneal cavity. Using Cytometry by Time of Flight (CyTOF), we analyzed ascitic immune cells collected from patients with advanced gastrointestinal cancers, nonmalignant forms of hepatitis (NMH), and healthy control peripheral blood mononuclear cells (HC PBMCs). Compositionally, ascitic immune cells shared a myeloid-rich, B and naïve T cell-low signature relative to HC PBMCs. Underneath the shared ascitic compositional signature, three major functional immunophenotypes emerged in response to exogenous stimulation along a suppressive to cytotoxic axis, aligning with Luminex measurements of ascitic cytokine levels: IL-10 enriched, Nonpolarized, and Granzyme B (GZMB) enriched. In our cohort, the IL-10 enriched functional immunophenotype was exclusive to samples from patients with cancer while the GZMB enriched response was dominated by NMH samples. Ascites from patients with pancreatic ductal adenocarcinoma (PDAC) contained a persistent anti-cancer humoral response characterized by IgG that bound to autologous ascitic PDAC cells and multiple allogeneic PDAC cell lines.
Abstract Immune checkpoint inhibitors (ICIs) have transformed the treatment of advanced non-small cell lung cancer (NSCLC), yet a large proportion of patients do not respond. The lung microbiome is a key modulator of antitumor immunity, however, the functional contribution of active microbial communities and their interaction with host transcriptional programs in shaping ICI outcomes remain poorly defined. Here, we prospectively analyzed bronchoalveolar lavage (BAL) samples from 71 NSCLC patients prior to ICI initiation. We profiled the microbiome and host transcriptome in the BAL to look for microbial and host signatures in the lower airways associated with treatment response. We found that enrichment of the lower airway microbiome with oral commensals was associated with disease progression and worse prognosis of patients treated with ICI. Metatranscriptomics-derived microbial clustering revealed distinct functional profiles. The microbial cluster enriched with oral commensals, which was only present among patients with poor response to ICI, had differential regulation of pathways involved in the metabolism of short-chain fatty acids, methane, and pyruvate. This cluster also correlated with host immune pathways including upregulation of interferon signaling, neutrophil degranulation, and mitophagy, all of which could contribute to the poor response to immunotherapy. In a preclinical murine model of lung cancer with dysbiosis, inoculating lung tumor-bearing mice with oral commensals increased intratumoral neutrophils and Th17 responses and impaired ICI efficacy. Our findings support the concept that microbial communities in the lower airways influence tumor–immune interactions via defined metabolic–immune axes.
Abstract Engineered T-cell therapies are limited by progressive dysfunction under chronic tumor antigen exposure, and genetic perturbation of negative regulators has emerged as a promising strategy to sustain effector activity. We reanalyzed a genome-wide CRISPR knockout screen in primary human T cells under repetitive tumor stimulation; protein–protein interaction mapping revealed seven members of the Cullin–RING Ligase 5 (CRL5) complex as an enriched hit cluster. Knockout of any of these members (CUL5, ARIH2, RNF7, UBE2F, UBE2L3, CISH, DCUN1D3) enhanced tumor serial killing, and CUL5, the complex scaffold, emerged as the strongest target. CUL5 knockout T cells exhibited sustained cytotoxicity, elevated effector cytokine production, and fewer exhaustion marker–positive cells while retaining antigen specificity and cytokine dependence. Quantitative ubiquitylation proteomics in resting, and acutely and chronically stimulated T cells mapped CRL5-dependent candidate substrates beyond the canonical JAK/STAT axis, including nutrient transporters, receptor trafficking proteins, and TCR signaling components. The benefit of CUL5 knockout extended across CARs targeting CD19, BCMA, and HER2 with different costimulatory domains, and to 1XX-modified CAR and HLA-independent TCR architectures. Combining CUL5 ablation with a tumor-induced IL-2 circuit produced tumor control across nine rounds of repetitive stimulation, whereas either modification alone eventually failed. In vivo, CUL5 knockout BCMA CAR T cells were enriched in the bone marrow following repeated tumor rechallenge and exhibited enhanced ex vivo cytotoxicity after nearly 90 days in tumor-bearing mice. These findings position CUL5 perturbation as a strategy to improve T-cell persistence and the efficacy of adoptive cell therapies, potentially with engineered cytokine support.
Abstract T-cell exhaustion limits durable antitumor responses and remains a barrier to adoptive cell therapies (ACT) in solid tumors. To identify regulators of T-cell persistence, we performed genome-wide CRISPR knockout screens in primary human CD8⁺ T cells expressing an NY-ESO-1–specific T-cell receptor and subjected them to repeated tumor cell rechallenge. These screens revealed enrichment of single-guide RNAs targeting components of Cullin–RING E3 ligases, particularly Cullin-5 (CRL5) assemblies comprising CUL5, RNF7, and ARIH2. Disruption of CRL5 genes in T cells preserved cytotoxic activity and enhanced cytokine production while sustaining effector-associated transcriptional programs and suppressing exhaustion-related signatures. Pharmacologic inhibition of cullin neddylation with the NEDD8-activating enzyme inhibitor MLN4924 recapitulated CRL5 loss and enhanced T-cell effector function in vitro. In a human melanoma xenograft model, ACT with CRL5-deficient T cells improved tumor control. Together, these findings identify CRL5 as a negative regulator of T-cell fitness and a potential therapeutic target to enhance ACT durability in solid tumors.
