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Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3–5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial–mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions. Single-cell RNA sequencing and imaging of macrophages in human non-small cell lung cancer and in a mouse model of lung adenocarcinoma show that tissue-resident macrophages have a key role in early tumour progression.

International audience; Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.

Summary: Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA)contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion)in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies., SCOPUS: re.j, info:eu-repo/semantics/published

In order to evaluate the immune infiltrate of AT/RT, we conducted a combined immunohistochemical, multiparametric flow cytometry and transcriptomic analysis of a series of 49 human AT/RT. This revealed substantial heterogeneity, with some subgroups of AT/RT showing a prominent immune infiltrate. In details, our analyses indicated that: i) myeloid cells were the most abundant immune population, including both microglial cells and non-resident pro-tumoral M2-polarized macrophages, ii) tumor-infiltrating lymphocytes consisted in equal proportions of CD4+ and CD8+ T cells with few regulatory T cells, and iii) immune modulatory molecules PD-L1 and TIM-3 were expressed at high levels. Intratumoral cytolytic activity of CD8+ T cells highly correlated with both interferon gamma and alpha signatures, confirming the involvement of both adaptive and innate immune cells in the anti-tumor response. A genetically engineered mouse model of AT/RT recently established in our laboratory recapitulates many of these traits, including important infiltration by both lymphoid and myeloid cells sharing similar phenotypic characteristics. Using this model, we showed that blockade of the PD-1/PD-L1 pathway significantly impaired tumor growth and induced memory against a second engraftment, confirming an anti-tumoral T cell memory response. Combination with TIM-3 blockade showed synergistic effects. Additionally, targeting the myeloid infiltrate by TLR3 activation with poly(I:C) induced a potent anti-tumor effect which, combined with PD-1 blockade, led to complete tumor regression in over 85% of treated mice. In conclusion, we demonstrate that immune infiltration is a recurrent property of AT/RT and that immunotherapy, particularly combination regimens, have promising therapeutic potential.

Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection., Author Summary Exposure of cells to environmental stresses, such as heat shock and viral infection, induces a cellular response leading to the formation of Stress Granules (SGs) composed of stalled translation initiation complexes (RNA-binding proteins and mRNA). The subsequent inhibition of host translation participates to cell survival. Viruses modulate or interfere with SG formation to control viral replication and antiviral responses, but differences exist in the dynamics and outcome of the stress responses induced by various viruses. Our study shows that Rabies virus (RABV) induces the formation of SGs in infected cells. We combined different methods of advanced imaging techniques (live-cell imaging, 3D analysis, FISH experiments) to characterize for the first time these structures. SGs are highly dynamic structures that increase in size by fusion events, exhibit transient assembly or persist throughout infection. They localize close to viral factories, cytoplasmic structures characteristic of RABV infection involved in viral replication and transcription. Viral messenger RNAs, but not viral genomic RNA, are transported from the factories to SGs, indicating the communication between both compartments. In addition, we provide some evidence that RABV-induced cellular stress is dependent on double-stranded RNA-activated protein kinase (PKR). Our data indicate that PKR also participates in innate immune responses through the induction of Interferon-B gene. Taken together, our results give an insight on new and important aspects of RABV infection and host antiviral stress responses.

The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers., JOURNAL ARTICLE, SCOPUS: re.j, info:eu-repo/semantics/published