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Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30 + γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated T H 17 but decreased CD8 + T, γδT, T FH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8 + , as well as reduced CD4 + T cells with an elevated T H 17 over Treg/T FH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets. Crohn’s disease results from transmural inflammation in the gut, but analyses of local immune populations are still lacking. Here, the authors show, by combining multiple single-cell approaches, that intraepithelial and lamina propria T cells are heterogenous, show unique phenotypes, and exhibit altered subsets upon inflammation.

The distinction between malignant mesothelioma and reactive mesothelial proliferation can be challenging both on histology and cytology. Recently, variants of the BRCA1 - associated protein 1 ( BAP1 ) gene resulting in nuclear protein loss were reported in hereditary and sporadic mesothelioma. Using immunohistochemistry, we evaluated the utility of BAP1 expression in the differential diagnosis between mesothelioma and other mesothelial proliferations on a large series of biopsies that included 212 mesotheliomas, 12 benign mesothelial tumors, and 42 reactive mesothelial proliferations. BAP1 stain was also performed in 70 cytological samples (45 mesotheliomas and 25 reactive mesothelial proliferations). BAP1 was expressed in all benign mesothelial tumors, whereas 139/212 (66%) mesotheliomas were BAP1 negative, especially in epithelioid/biphasic compared with sarcomatoid/desmoplastic subtypes (69% vs 15%). BAP1 loss was homogeneous in neoplastic cells except for two epithelioid mesotheliomas showing tumor heterogeneity. By fluorescence in situ hybridization, BAP1 protein loss was paralleled by homozygous deletion of the BAP1 locus in the vast majority of BAP1-negative tumors (31/41, 76%), whereas 9/10 BAP1-positive mesotheliomas were normal. In biopsies interpreted as reactive mesothelial proliferation BAP1 loss was 100% predictive of malignancy, as all 6 cases subsequently developed BAP1-negative mesothelioma, whereas only 3/36 (8%) BAP1-positive cases progressed to mesothelioma. On cytology/cell blocks, benign mesothelial cells were invariably positive for BAP1, whereas 64% of mesotheliomas showed loss of protein; all 6 cases showing BAP1 negativity were associated with histological diagnosis of BAP1-negative mesothelioma. BAP1 stain also showed utility in the differential of mesothelioma from most common pleural and peritoneal mimickers, such as lung and ovary carcinomas, with specificity and sensitivity of 99/70% and 100/70%, respectively. Our results show that BAP1 protein is frequently lost in mesothelioma, especially of epithelioid/biphasic subtype and is commonly associated with homozygous BAP1 deletion. BAP1 immunostain represents an excellent biomarker with an unprecedented specificity (100%) in the distinction between benign and malignant mesothelial proliferations. Finding BAP1 loss in mesothelial cells should prompt to immediately reevaluate the patient; moreover, it might be useful in mapping tumor extent and planning surgical resection.

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8 + T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8 + T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors. Acquisition of dying tumor cell-associated antigens is an essential step for the initiation of anti-tumor immune response by conventional type 1 dendritic cells (cDC1). Here the authors show that the loss of TIM4 expression in lung tumor associated cDC1 is associated with less efficient uptake of cell associated antigens and reduction of CD8 + T cell activation in advanced lung tumors.

Plasmacytoid dendritic cells (pDCs) are multifaceted immune cells executing various innate immunological functions. Their first line of defence consists in type I interferons (I-IFN) production upon nucleic acids sensing through endosomal Toll-like receptor (TLR) 7- and 9-dependent signalling pathways. Type I IFNs are a class of proinflammatory cytokines that have context-dependent functions on cancer immunosurveillance and immunoediting. In the last few years, different studies have reported that pDCs are also able to sense cytosolic DNA through cGAS–STING (stimulator of interferon genes) pathway eliciting a potent I-IFN production independently of TLR7/9. Human pDCs are also endowed with direct effector functions via the upregulation of TRAIL and production of granzyme B, the latter modulated by cytokines abundant in cancer tissues. pDCs have been detected in a wide variety of human malignant neoplasms, including virus-associated cancers, recruited by chemotactic stimuli. Although the role of pDCs in cancer immune surveillance is still uncompletely understood, their spontaneous activation has been rarely documented; moreover, their presence in the tumor microenvironment (TME) has been associated with a tolerogenic phenotype induced by immunosuppressive cytokines or oncometabolites. Currently tested treatment options can lead to pDCs activation and disruption of the immunosuppressive TME, providing a relevant clinical benefit. On the contrary, the antibody–drug conjugates targeting BDCA-2 on immunosuppressive tumor-associated pDCs (TA-pDCs) could be proposed as novel immunomodulatory therapies to achieve disease control in patients with advance stage hematologic malignancies or solid tumors. This Review integrate recent evidence on the biology of pDCs and their pharmacological modulation, suggesting their relevant role at the forefront of cancer immunity.

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B’s necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis. UNC5B is a Netrin-1 receptor expressed in endothelial cells that in the absence of ligand induces apoptosis. Here the authors identify an UNC5B splicing isoform that is insensitive to the pro-survival ligand Netrin-1 and is required for apoptosis-dependent blood vessel development.

Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3 + T-cells and CD163 + tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS 30 ) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1 + pSTAT1Y701 + ) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10 + IRF1 + STAT1 + M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10 + IRF1 + STAT1 + macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.

BackgroundColorectal cancer (CRC) progression is shaped by the tumor microenvironment, particularly tumor-associated macrophages (TAMs), which often adopt immunosuppressive functions. CD300e, a myeloid receptor involved in immune regulation, has an uncharacterized role in CRC.MethodsFunctional studies were conducted in azoxymethane/dextran sodium sulfate and MC38 murine CRC models using CD300e systemic and myeloid-specific CD300e knockout mice, and adoptive transfer experiments assessed macrophage-intrinsic effects. Human studies included analysis of CD300e expression in matched tumor and normal tissue from patients with CRC and in vitro co-culture of patient-derived colon tumor organoids with monocytes to study CD300e induction and TAM polarization.ResultsIn vivo, CD300e deficiency led to reduced tumor burden, enhanced major histocompatibility complex expression on TAMs, and improved T-cell responses. CD300e-deficient macrophages exhibited increased phagocytic activity, antigen presentation, and support for T-cell proliferation and cytotoxicity. Adoptive transfer confirmed that macrophage-intrinsic CD300e expression is sufficient to suppress T-cell function and promote tumor growth. In patients with CRC, CD300e is selectively upregulated in tumor-infiltrating monocytes and macrophages, driving a suppressive phenotype marked by impaired antigen presentation. Tumor-derived signals in vitro induce CD300e expression and promote a protumorigenic macrophage profile.ConclusionsOur findings identify CD300e as a critical regulator of macrophage-mediated immune suppression in CRC and a potential target for reprogramming TAMs to enhance immunotherapy.

Evaluation of B-cell clonality can be challenging in the interpretation of lymphoid infiltrates on tissue sections. Clonality testing based on IG gene rearrangements analysis by PCR (IG-PCR) is the gold standard. Alternatively, B-cell clonality can be assessed by the recognition of immunoglobulin light chain (IgLC) restriction, by immunohistochemistry (IHC), chromogenic in situ hybridization (ISH) or flow cytometry (FC). IG-PCR requires molecular facilities, and FC requires cell suspensions, both not widely available in routine pathology units. This study evaluates the performance of B-cell clonality detection by IgLC-RNAscope® (RNAsc) in a group of 216 formalin-fixed, paraffin-embedded samples including 185 non-Hodgkin B-cell lymphomas, 11 Hodgkin lymphomas (HL) and 20 reactive samples. IgLC-RNAsc, performed in parallel with FC in 53 cases, demonstrated better performances (93% vs 83%), particularly in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%) diagnosis. IgLC-RNAsc was also superior to IHC and ISH especially in samples with limited tumor cell content, where IG-PCR was not informative. Performed for the first time on mediastinal lymphomas, IgLC-RNAsc identified monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL). IGK/L double-negative cells were detected in 1 PMBCL, 2 MGZL, and all classical HL, while monotypic IgLC expression appeared to be a hallmark in nodular lymphocyte-predominant HL. IgLC-RNAsc demonstrates to be a powerful tool in B-cell lymphoma diagnosis, above all in challenging cases with limited tumor cell content, ensuring in situ investigations on mechanisms of Ig regulation across lymphoma entities.

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.

Plasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production. The activation of tumor-associated pDCs was evaluated by and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer. Based on a set of microscopic, functional and analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape. These findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM).