�����ƽ��

CD8+ T cell exhaustion (Tex) limits immune control of cancer, but the underlying molecular drivers are unclear. In the present study, we identified the prostaglandin I2 (prostacyclin) receptor PTGIR as a cell-intrinsic regulator of T cell exhaustion. Transcriptomic profiling of terminally exhausted (Ttex) CD8+ T cells revealed increased activation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response pathway. Enhancing NRF2 activity (by conditional deletion of Kelch-like ECH-associated protein 1 (KEAP1)) boosts glutathione production in CD8+ T cells but accelerates terminal exhaustion. NRF2 upregulates PTGIR expression in CD8+ T cells. Silencing PTGIR expression enhances T cell effector function (that is, interferon-γ and granzyme production) and limits Ttex cell development in chronic infection and cancer models. Mechanistically, PTGIR signaling impairs T cell metabolism and cytokine production while inducing transcriptional features of Tex cells. These findings identify PTGIR as a NRF2-dependent immune checkpoint that regulates balance between effector and exhausted CD8+ T cell states. Targeting CD8+ T cell exhaustion is a strategy to enhance immune checkpoint inhibition and to fight cancer. Here the authors show a NRF2-dependent role for the prostaglandin I2 receptor PTGIR in controlling T cell exhaustion.

Abstract Background Virotherapy eradicates tumors by directly killing cancer cells and causing adjuvant effects. However, the mechanism by which non-replicating virotherapy exerts anti-tumor effects is unclear. Methods In this study, we investigated the genes that mediate the anti-tumor effects of ultraviolet (UV)-irradiated Hemagglutinating Virus of Japan envelope (HVJ-E) using RNA sequencing, gene knockout, and a drug-inducible gene expression system. We examined the antitumor effects of Apolipoprotein d (Apod) using genome-wide CRISPR library screening, in situ biotinylation combined with mass spectrometry, flow cytometry, biochemistry, and tumor-bearing mouse models. Results Here, we show that HVJ-E represses tumor growth via Irf7-induced Apod expression in tumor cells in vivo. Irf7 in B16F10 cells is a pivotal transcription factor for HVJ-E-induced anti-tumor effects. Apod substantially suppresses tumor growth even in HVJ-E-insensitive tumors. Apod is required to increase NKG2D-ligand genes in HVJ-E-treated tumors. Genome-wide CRISPR library screening and in situ biotinylation of Apod reveal an association of Apod with ERK2. Mechanistically, Apod prevents the nuclear translocation of ERK2 and Importin7, increasing NKG2D-ligands expression in B16F10 cells and attenuating tumor growth. Treating a local tumor with a combination therapy of Apod with the anti-OX40, T cell costimulatory molecule, antibody substantially repressed tumor growth in target and non-target lesions alongside T cell activation. Conclusion Our findings provide insights into the molecular mechanisms of how HVJ-E induces anti-tumor effects and can aid the development of therapeutic strategies for eliciting anti-tumor immunity.

Iron is an irreplaceable co-factor for metabolism. Iron deficiency affects >1 billion people and decreased iron availability impairs immunity. Nevertheless, how iron deprivation impacts immune cell function remains poorly characterised. We interrogate how physiologically low iron availability affects CD8+ T cell metabolism and function, using multi-omic and metabolic labelling approaches. Iron limitation does not substantially alter initial post-activation increases in cell size and CD25 upregulation. However, low iron profoundly stalls proliferation (without influencing cell viability), alters histone methylation status, gene expression, and disrupts mitochondrial membrane potential. Glucose and glutamine metabolism in the TCA cycle is limited and partially reverses to a reductive trajectory. Previous studies identified mitochondria-derived aspartate as crucial for proliferation of transformed cells. Despite aberrant TCA cycling, aspartate is increased in stalled iron deficient CD8+ T cells but is not utilised for nucleotide synthesis, likely due to trapping within depolarised mitochondria. Exogenous aspartate markedly rescues expansion and some functions of severely iron-deficient CD8+ T cells. Overall, iron scarcity creates a mitochondrial-located metabolic bottleneck, which is bypassed by supplying inhibited biochemical processes with aspartate. These findings reveal molecular consequences of iron deficiency for CD8+ T cell function, providing mechanistic insight into the basis for immune impairment during iron deficiency. Iron has been shown to be necessary for the activation and differentiation of CD8+ T cells. Here the authors investigate changes in CD8+ T cell metabolism in iron limiting conditions and find that aspartate is increased yet downstream nucleotide synthesis is suppressed and addition of exogenous aspartate partially rescues T cell function.

ABSTRACTCytotoxicity is a cornerstone of immune defense, critical for combating tumors and infections. This process relies on the coordinated action of granzymes and pore‐forming proteins, with granzyme B (GZMB) and perforin (PRF1) being key markers and the most widely studied molecules pertaining to cytotoxicity. However, other human granzymes and cytotoxic components remain underexplored, despite growing evidence of their distinct, context‐dependent roles. Natural killer cell granule protein 7 (NKG7) has recently emerged as a crucial cytotoxicity regulator, yet its expression patterns and function are poorly understood. Using large publicly available single‐cell RNA sequencing atlases, we performed a comprehensive profiling of cytotoxicity across immune subsets and tissues. Our analysis highlights NKG7 expression as a strong marker of cytotoxicity, exhibiting a strong correlation with overall cytotoxic activity (r = 0.97) and surpassing traditional markers such as granzyme B and perforin in reliability. Furthermore, NKG7 expression is notably consistent across diverse immune subsets and tissues, reinforcing its versatility and robustness as a cytotoxicity marker. These findings position NKG7 as an invaluable tool for evaluating immune responses and a reliable indicator of cytotoxic functionality across biological and clinical contexts. Cytotoxicity‐associated genes exhibit heterogeneity at the cellular and tissue levels (left). NKG7 gene expression is strongly associated with a cytotoxic gene signature (middle). NKG7 expression is stable and consistently detected in cells across immunologically relevant tissues and within tumor‐infiltrating immune cells (right). Figure generated in collaboration with Susan Stone (https://www.sue‐stone.com).

BackgroundLymphatics provide a route for breast cancer cells to metastasize. Lymphatic endothelial cells (LECs), which form the structure of lymphatic vessels, play a key role in this process. Although LECs are pivotal in cancer progression, studies often rely on commercially available cell lines that may not accurately reflect the tumor microenvironment. Therefore, there is a pressing need to directly study patient-derived LECs to better understand their role in breast cancer.MethodsThis study developed a method to isolate and characterize LECs directly from human breast-to-axilla adipose tissue. We used magnetic cell separation to remove CD45 + leukocytes and fluorescence-activated cell sorting to isolate cells expressing CD31 and podoplanin. Isolated cells were cultured under conditions promoting endothelial cell growth and were characterized through various assays assessing proliferation, tube formation, and gene expression patterns.ResultsThe sorted CD31 + /PDPN + /CD45 − cell populations exhibited marked increases in proliferation upon VEGF-C stimulation and formed tubule structures on BME-coated dishes, confirming their LEC properties. Notably, isolated LECs showed distinct gene expression patterns depending on the presence of lymph node metastasis and lymphatic invasion.ConclusionsThe ability to isolate and characterize patient-derived LECs from mammary adipose tissue offers new insights into the cellular mechanisms underlying breast cancer metastasis. Significant gene expression variability related to disease state highlights the potential of these cells as biomarkers and therapeutic targets. This study emphasizes the importance of using patient-derived cells to accurately assess the tumor microenvironment, potentially leading to more personalized therapeutic approaches.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13058-025-02067-w.

Despite the success of chimeric antigen receptor T (CART) cell therapy in hematological malignancies, durable remissions remain low. Here, we report CART senescence as a potential resistance mechanism in 41BB-costimulated CART cell therapy. To mimic cancer relapse, we utilized an in vitro model with repeated CART cell activation cycles followed by rest periods. Using CD19-targeted CART cells with costimulation via 4-1BB-CD3ζ (BBζ) or CD28-CD3ζ (28ζ), we showed that CART cells undergo functional, phenotypical, and transcriptomic changes of senescence, which is more prominent in BBζ. We then utilized two additional independent strategies to induce senescence through MYC activation and irradiation. Induction of senescence impaired BBζ activity but improved 28ζ activity in preclinical studies. These findings were supported by analyses of independent patient data sets; senescence signatures in CART cell products were associated with non-response to BBζ but with improved clinical outcomes in 28ζ treatment. In summary, our study identifies senescence as a potential mechanism of failure predominantly in 41BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1. SignificanceWe identified senescence as a cause of failure in CART cell therapy, predominantly in 4-1BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1.

ObjectiveThis study investigated the impact of innate lymphoid cell type 2 (ILC2s) on the function of regulatory T cells (Treg) and CD8+ T cells in chronic lymphocytic leukemia (CLL) through IL-9.MethodsPeripheral blood samples were collected from CLL patients (n = 52) and healthy controls (n = 30). ILC2 proportions and IL-9 levels were assessed using flow cytometry and ELISA. Immunofluorescence staining was performed to stain GATA3, CRTH2, and IL-9 in cervical lymph nodes from CLL patients (n = 10) and control subjects with reactive lymphadenitis (n = 10). Correlation analysis between ILC2s and IL-9 was conducted using the Spearman test. ILC2s were sorted and cultured from CLL patients, followed by co-culture experiments with PBMCs of healthy controls and MEC-1 cells, with or without anti-IL-9 antibody intervention. Flow cytometry was used to measure the proportions of ILC2s, Treg cells, PD-1+/TIGIT+/CTLA-4+ Treg subsets, and granzyme B+/perforin+ CD8+ T cells, along with MEC-1 cell apoptosis.ResultsThe proportions of ILC2s and Treg, along with serum IL-9 levels, were significantly elevated in CLL patients (P < 0.05). Peripheral blood ILC2s were positively correlated with IL-9 (r = 0.609, P < 0.001). The average fluorescence intensity of GATA3, CRTH2, and IL-9 in the cervical lymph nodes of CLL patients increased significantly (P < 0.001), and IL-9 showed colocalization with GATA3 and CRTH2. In vitro, IL-9 levels in the supernatant of sorted ILC2s from CLL patients increased. Treatment with anti-IL-9 antibody significantly reduced the PD-1+ Treg and TIGIT+ Treg cells while increasing granzyme B+ CD8+ T cells (P < 0.05). However, there was no significant effect on Treg, CTLA-4+ Treg, and perforin+ CD8+ T cells (P > 0.05). Additionally, anti-IL-9 antibody significantly increased early apoptosis (P < 0.05).ConclusionILC2s affect CD8+ T cells and Treg cells through IL-9, weakening the anti-tumor effects of CD8+ T cells and enhancing the immunosuppressive effects of Treg cells, thereby contributing to CLL pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04082-4.