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Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease characterised by aberrant fibroblast/myofibroblast accumulation and excessive collagen matrix deposition in the alveolar areas of lungs. As the first approved IPF medication, pirfenidone (PFD) significantly decelerates lung function decline while its underlying anti-fibrotic mechanism remains elusive. METHODS: We performed transcriptomic and immunofluorescence analyses of primary human IPF tissues. RESULTS: We showed that myocardin-related transcription factor (MRTF) signalling is activated in myofibroblasts accumulated in IPF lungs. Furthermore, we showed that PFD inhibits MRTF activation in primary human lung fibroblasts at clinically achievable concentrations (half-maximal inhibitory concentration 50-150 µM, maximal inhibition >90%, maximal concentration of PFD in patients

Transforming growth factor-β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2-anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade.

Progressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD.

BackgroundThe genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses.ResultsTo help address this problem, we propose a standardized file format for representing V(D)J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D)J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format.ConclusionsThe VDJML suite will allow users to streamline their V(D)J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://vdjserver.org/vdjml/ . We welcome participation from the community in developing the file format standard, as well as code contributions.

The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

Increased interest in the immune system's involvement in pathophysiological phenomena coupled with decreased DNA sequencing costs have led to an explosion of antibody and T cell receptor sequencing data collectively termed "adaptive immune receptor repertoire sequencing" (AIRR-seq or Rep-Seq). The AIRR Community has been actively working to standardize protocols, metadata, formats, APIs, and other guidelines to promote open and reproducible studies of the immune repertoire. In this paper, we describe the work of the AIRR Community's Data Representation Working Group to develop standardized data representations for storing and sharing annotated antibody and T cell receptor data. Our file format emphasizes ease-of-use, accessibility, scalability to large data sets, and a commitment to open and transparent science. It is composed of a tab-delimited format with a specific schema. Several popular repertoire analysis tools and data repositories already utilize this AIRR-seq data format. We hope that others will follow suit in the interest of promoting interoperable standards. [ABSTRACT FROM AUTHOR]

CD4+ follicular regulatory T (Tfr) cells suppress B cell responses through modulation of follicular helper T (Tfh) cells and germinal center (GC) development. We found that Tfr cells can also promote the GC response through provision of interleukin-10 (IL-10) after acute infection with lymphocytic choriomeningitis virus (LCMV). Sensing of IL-10 by B cells was necessary for optimal development of the GC response. GC B cells formed in the absence of Treg cell– derived IL-10 displayed an altered dark zone state and decreased expression of the transcription factor Forkhead box protein 1 (FOXO1). IL-10 promoted nuclear translocation of FOXO1 in activated B cells. These data indicate that Tfr cells play a multifaceted role in the fine-tuning of the GC response and identify IL-10 as an important mediator by which Tfr cells support the GC reaction. [ABSTRACT FROM AUTHOR]