Vijayanand and colleagues use genome-wide RNA sequencing for transcriptional profiling of CD8+ T cells from tumors and adjacent uninvolved lung tissue from patients with early-stage lung cancer. A tissue-resident memory signature is associated with enhanced cytotoxicity and improved survival. Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103hi tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

Background: Intestinal macrophages are key immune cells in the maintenance of intestinal immune homeostasis and have a role in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms by which macrophages exert a pathological influence in both ulcerative colitis (UC) and Crohn disease (CD) are not yet well understood. Methods: We purified intestinal macrophages from gastrointestinal mucosal biopsies (patients with UC, patients with CD, and healthy donors) and analyzed their transcriptome by RNA sequencing and bioinformatics, confirming results with quantitative polymerase chain reaction and immunohistochemistry. Results: Compared with those of healthy donors, intestinal macrophages in patients with UC and with CD showed cellular reprograming of 1287 and 840 dysregulated genes, respectively (false discovery rate ≤ 0.1). The UC and CD intestinal macrophages showed an activated M1 inflammatory phenotype and the downregulation of genes engaged in drug/xenobiotic metabolism. Only macrophages from CD showed, concomitant to an M1 phenotype, a significant enrichment in the expression of M2 and fibrotic and granuloma-related genes. For the first time, we showed (and validated by quantitative polymerase chain reaction and immunohistochemistry) that intestinal macrophages in patients with IBD present both M1 and M2 features, as recently described for tumor-associated macrophages, that affect key pathways for IBD pathology, represented by key markers such as MMP12 (fibrosis), CXCL9 (T-cell attraction), and CD40 (T-cell activation). Conclusions: Our data support the therapeutic targeting of macrophages to maintain remission in IBD but also indicate that a shift toward an M2 program—as proposed by some reports—may not limit the recruitment and activation of T cells because M2 features do not preclude M1 activation in patients with UC or CD and could exacerbate M2-related CD-specific features such as fibrosis and the formation of granulomas.

Gene expression analysis of TIL rich HPV-driven head and neck tumors reveals a distinct B-cell signature when compared to HPV independent tumors

Objective—: Hereditary hemorrhagic telangiectasia is a genetic disorder characterized by visceral and mucocutaneous arteriovenous malformations (AVMs). Clinically indistinguishable hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 are caused by mutations in ENG and ALK1 , respectively. In this study, we have compared the development of visceral and mucocutaneous AVMs in adult stages between Eng - and Alk1 -inducible knockout (iKO) models. Approach and Results—: Eng or Alk1 were deleted from either vascular endothelial cells (ECs) or smooth muscle cells in adult stages using Scl-CreER and Myh11-CreER lines, respectively. Latex perfusion and intravital spectral imaging in a dorsal skinfold window chamber system were used to visualize remodeling vasculature during AVM formation. Global Eng deletion resulted in lethality with visceral AVMs and wound-induced skin AVMs. Deletion of Alk1 or Eng in ECs, but not in smooth muscle cells, resulted in wound-induced skin AVMs. Visceral AVMs were observed in EC-specific Alk1 -iKO but not in Eng -iKO. Intravital spectral imaging revealed that Eng -iKO model exhibited more dynamic processes for AVM development when compared with Alk1 -iKO model. Conclusions—: Both Alk1 - and Eng -deficient models require a secondary insult, such as wounding, and ECs are the primary cell type responsible for the pathogenesis. However, Alk1 but not Eng deletion in ECs results in visceral AVMs.

Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-β type I membrane receptor restricted almost entirely to endothelial cells (ECs) and involved in vascular remodelling and angiogenesis. Previous reports have shown that the ubiquitous TGF-β type I receptor ALK5 and the type II receptor are located in cholesterol-rich membrane microdomains named caveolae. The aim of this work was to assess the location of ALK1 in endothelial caveolae as well as to study the role of caveolin-1 on the TGF-β/ALK1 signalling pathway. The subcellular distribution of ALK1 was analysed by confocal microscopy and co-fractionation experiments in human ECs. The association between human ALK1 and caveolin-1 was studied in caveolin-1-deficient human epithelial cells by co-immunoprecipitation. The functional role of caveolin-1 on the ALK1-mediated TGF-β signalling was elucidated using ALK1-specific luciferase reporters in human ECs, caveolin-1^−/−mouse embryonic fibroblasts, and rat myoblasts. Confocal microscopy analyses, as well as cholesterol depletion experiments in the presence of cholesterol-depleting agents such as nystatin or methyl-β-cyclodextrin, demonstrated that ALK1 is located in endothelial caveolae. Also, co-immunoprecipitation assays showed that ALK1 associates with the main caveolae component caveolin-1. Mapping of the ALK1/caveolin-1 interaction revealed that the caveolin-1 scaffolding domain and the caveolin-1 binding motif in ALK1 are responsible for this association. Moreover, this hitherto not reported interaction had a functional consequence for the ALK1-dependent signalling. In contrast with the previously published ALK5/caveolin-1 interaction, caveolin-1 enhances the TGF-β/ALK1 signalling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signalling pathway. ALK1 is located in endothelial caveolae where it functionally interacts with caveolin-1 through its scaffolding domain, suggesting a joint contribution of ALK1 and caveolin-1 as key mediators of the TGF-β pathway in angiogenesis.

Hereditary hemorrhagic telangiectasia (HHT) or Osler–Weber–Rendu syndrome is an autosomal dominant vascular disorder characterized by telangiectases and internal arteriovenous malformations. It is caused by mutations in elements of the transforming growth factor-β (TGF-β) receptor complex: endoglin , a co-receptor, responsible for HHT1, or ALK1 (activin receptor-like kinase 1), a type I receptor leading to HHT2. Recently, we have established cultures of HHT endothelial cells, primary targets of the disease. These cells showed deficient TGF-β signaling and angiogenesis, representing a useful human model to study the molecular mechanism of this disease. To understand the pathogenic mechanism underlying HHT, we have used total RNA probes to compare HHT versus non-HHT cells by expression microarrays. This work represents a systematic study to identify target genes affected in HHT cells. Given the similarity of symptoms in HHT1 and HHT2, special interest has been put on the identification of common targets for both HHT types. As a result, 277 downregulated and 63 upregulated genes were identified in HHT versus control cells. These genes are involved in biological processes relevant to the HHT pathology, such as angiogenesis, cytoskeleton, cell migration, proliferation and NO synthesis. The type of misregulated genes found in HHT endothelial cells lead us to propose a model of HHT pathogenesis, opening new perspectives to understand this disorder. Moreover, as the disease is originated by mutations in proteins of the TGF-β receptor complex, these results may be useful to find out targets of the TGF-β pathway in endothelium.