Rationale: Extracellular matrix (ECM) is a dynamic tissue that contributes to organ integrity and function, and its regulation of cell phenotype is a major aspect of cell biology. However, standard in vitro culture approaches are of unclear physiologic relevance because they do not mimic the compositional, architectural, or distensible nature of a living organ. In the lung, fibroblasts exist in ECM-rich interstitial spaces and are key effectors of lung fibrogenesis. Objectives: To better address how ECM influences fibroblast phenotype in a disease-specific manner, we developed a culture system using acellular human normal and fibrotic lungs. Methods: Decellularization was achieved using treatment with detergents, salts, and DNase. The resultant matrices can be sectioned as uniform slices within which cells were cultured. Measurements and Main Results: We report that the decellularization process effectively removes cellular and nuclear material while retaining native dimensionality and stiffness of lung tissue. We demonstrate that lung fibroblasts reseeded into acellular lung matrices can be subsequently assayed using conventional protocols; in this manner we show that fibrotic matrices clearly promote transforming growth factor-b-independent myofibroblast differentiation compared with normal matrices. Furthermore, comprehensive analysis of acellular matrix ECM details significant compositional differences between normal and fibrotic lungs, paving the way for further study of novel hypotheses. Conclusions: This methodology is expected to allow investigation of important ECM-based hypotheses in human tissues and permits future scientific exploration in an organ-and disease-specific manner.

Highlights d cDC2 initiate activation but not differentiation of antitumor CD4 + T conv d T reg depletion relieves cDC2 suppression driving antitumor CD4 + T conv differentiation d Human equivalent of mouse cDC2 are present in the tumor and draining lymph node d The balance of human cDC2/T reg in the TME dictates T cell quality and prognosis

Rationale: Tissue fibrosis is considered a dysregulated wound-healing response. Fibronectin containing extra type III domain A (EDA) is implicated in the regulation of wound healing. EDA-containing fibronectin is deposited during wound repair, and its presence precedes that of collagen. Objectives: To investigate the role of EDA-containing fibronectin in lung fibrogenesis. Methods: Primary lung fibroblasts from patients with idiopathic pulmonary fibrosis or from patients undergoing resection for lung cancer were assessed for EDA-containing fibronectin and a-smooth muscle actin (a-SMA) expression. Mice lacking the EDA domain of fibronectin and their wild-type littermates were challenged with the bleomycin model of lung fibrosis. Primary lung fibroblasts from these mice were assayed in vitro to determine the contribution of EDA-containing fibronectin to fibroblast phenotypes. Measurements and Main Results: Idiopathic pulmonary fibrosis lung fibroblasts produced markedly more EDA-containing fibronectin and a-SMA than control fibroblasts. EDA-null mice failed to develop significant fibrosis 21 days after bleomycin challenge, whereas wildtype controls developed the expected increase in total lung collagen. Histologic analysis of EDA-null lungs after bleomycin showed less collagen and fewer a-SMA-expressing myofibroblasts compared with that observed in wild-type mice. Failure to develop lung fibrosis in EDA-null mice correlated with diminished activation of latent transforming growth factor (TGF)-b and decreased lung fibroblast responsiveness to active TGF-b in vitro. Conclusions: The data show that EDA-containing fibronectin is essential for the fibrotic resolution of lung injury through TGF-b activation and responsiveness, and suggest that EDA-containing fibronectin plays a critical role in tissue fibrogenesis.Keywords: fibrosis; fibronectin; TGF-b; myofibroblast Fibroproliferative disorders, characterized by the increased production and deposition of extracellular matrix (ECM) proteins in tissues, are not well understood despite major efforts to elucidate pathogenic mechanisms. Recent attention has focused on ECM components and mesenchymal cell phenotypes as being critical to the development of fibrosis (1, 2). The ECM is a highly dynamic complex that varies in composition according to its tissue localization and physiologic circumstances. Collagens are the predominant ECM proteins identified in fibrotic lesions and are the hallmark of fibroproliferative diseases, but fibronectins (FNs) are also present in abnormally large quantities and localize to areas of active fibrogenesis (3, 4).FNs are multifunctional glycoproteins found in the ECM of tissues and plasma. Two main forms of FN exist: plasma FN, a dimeric and soluble form produced by hepatocytes that lacks the EDA and EDB sequences, and cellular FN (cFN), a multimeric form synthesized by mesenchymal, epithelial, and inflammatory cells, which is deposited in ECM fibrils and that contains variable proportions of the extra type III domains A and B (EDA and EDB) (5...

Energetic metabolism reprogramming is critical for cancer and immune responses. Current methods to functionally profile the global metabolic capacities and dependencies of cells are performed in bulk. We designed a simple method for complex metabolic profiling called SCENITH, for Single Cell ENergetIc metabolism by profilIng Translation inHibition. SCENITH allows for the study of metabolic responses in multiple cell types in parallel by flow cytometry. SCENITH is designed to perform metabolic studies ex vivo, particularly for rare cells in whole blood samples, avoiding metabolic biases introduced by culture media. We analyzed myeloid cells in solid tumors from patients and identified variable metabolic profiles, in ways that are not linked to their lineage nor their activation phenotype. SCENITH ability to reveal global metabolic functions and determine complex and linked immune-phenotypes in rare cell subpopulations will contribute to the information needed for evaluating therapeutic responses or patient stratification.

The UCSF COMET Consortium, Michael Matthay, David J. E rl e , P re scot t G. Woodruff, Charles Langelier, Kirsten K an ge la ri s, C ar ol yn M.

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The COPA syndrome is a monogenic, autoimmune lung and joint disorder first identified in 2015. This study sought to define the main pulmonary features of the COPA syndrome in an international cohort of patients, analyse patient responses to treatment and highlight when genetic testing should be considered.We established a cohort of subjects (N=14) with COPA syndrome seen at multiple centres including the University of California, San Francisco, CA, USA. All subjects had one of the previously established mutations in the COPA gene, and had clinically apparent lung disease and arthritis. We analysed cohort characteristics using descriptive statistics.All subjects manifested symptoms before the age of 12 years, had a family history of disease, and developed diffuse parenchymal lung disease and arthritis. 50% had diffuse alveolar haemorrhage. The most common pulmonary findings included cysts on chest computed tomography and evidence of follicular bronchiolitis on lung biopsy. All subjects were positive for anti-neutrophil cytoplasmic antibody, anti-nuclear antibody or both and 71% of subjects had rheumatoid factor positivity. All subjects received immunosuppressive therapy.COPA syndrome is an autoimmune disorder defined by diffuse parenchymal lung disease and arthritis. We analysed an international cohort of subjects with genetically confirmed COPA syndrome and found that common pulmonary features included cysts, follicular bronchiolitis and diffuse alveolar haemorrhage. Common extrapulmonary features included early age of onset, family history of disease, autoantibody positivity and arthritis. Longitudinal data demonstrated improvement on chest radiology but an overall decline in pulmonary function despite chronic treatment.

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