InVivoPlus mouse IgG1 isotype control, unknown specificity

Although pancreatic cancer is generally refractory to immune checkpoint blockade, recent studies of tumor-infiltrating T cells in human tumor samples demonstrated the presence of in vivo expanded, tumor-reactive T cell receptor (TCR) clonotypes. Here, we explored the T cell repertoire in a murine pancreatic cancer model by combining single-cell transcriptomics with functional TCR characterization. This uncovered a substantial diversity of tumor-reactive TCR clonotypes. Whereas some of these were exclusively reactive against the autologous tumor, most TCRs reacted against syngeneic tumor cells of diverse tissue origin. Immunopeptidome analyses revealed three T cell epitopes reflecting distinct tumor antigen classes also found in human cancers: a mutanome-encoded neoantigen, an epitope encoded by an ectopically expressed endogenous retroviral provirus, and an epitope derived from a cell stress-induced autoantigen. These findings underline the importance of uncovering the antigen specificity of the natural tumor-reactive TCR repertoire to assess its therapeutic potential and safety with regard to personalized immunotherapy.

The stromal cell compartment plays a central part in the maintenance of tissue homeostasis by coordinating with the immune system throughout inception, amplification and resolution of inflammation1. Chronic inflammation can impede the phased regulation of tissue restitution, resulting in the scarring complication of fibrosis. In inflammatory bowel disease, stromal fibroblasts have been implicated in treatment-refractory disease and fibrosis2,3; however, their mechanisms of activation have remained undefined. Through integrative single-cell and spatial profiling of intestinal tissues from patients with inflammatory bowel disease, we uncovered a pathological cell nexus centred on inflammation-associated fibroblasts. These fibroblasts were induced by proinflammatory macrophages (FCN1+IL1B+) and, in turn, produced profibrotic cytokine IL-11. We investigated the inflammation-associated fibroblast activation program at a mechanistic level using genome-wide CRISPR knockout and activation screens and identified the transcription factor GLIS3 as a key regulator of a gene regulatory network governing expression of inflammatory and fibrotic genes. We further demonstrated that the magnitude of the GLIS3 gene expression program in intestinal biopsies could be used to stratify patients with ulcerative colitis by disease severity, and that fibroblast-specific deletion of Glis3 in mice alleviated pathological features of chronic colitis. Taken together, our findings identify a critical immune-stromal cell circuit that functions as a central node in the inflammation-fibrosis cycle.

The mechanisms that govern the transition from acute to chronic pain remain poorly defined. Emerging evidence suggests that immune cells and acute inflammatory responses are not merely pathological but actively contribute to pain resolution and the prevention of chronic pain. Using a mouse model of postoperative pain induced by plantar incision, we demonstrate that inhibition of tumor necrosis factor (TNFα) signaling prolongs pain hypersensitivity. Intraplantar administration of either monoclonal or polyclonal neutralizing anti-TNFα antibodies or Etanercept, a TNF receptor decoy, significantly delayed the resolution of pain in both female and male mice. Unexpectedly, early blockade of TNFα signaling did not reduce pain hypersensitivity but instead extended its duration. These findings underscore a paradoxical yet critical role for TNFα and immune signaling in promoting the resolution of acute pain and preventing its persistence. Together it supports the concept that acute inflammation and immune cells are essential for initiating the resolution of pain.

Calciphylaxis is a rare but life-threatening disorder characterized by ectopic calcification affecting the subcutaneous tissues and blood vessels of the skin. Survival rates are less than a year after diagnosis, and yet despite the severity of the condition, the pathobiology of calciphylaxis is ill understood. Here, we created animal models of calciphylaxis that recapitulated many characteristics of the human phenotype. We demonstrate that cutaneous calcification is preceded by inflammatory cell infiltration. We show that increased local skin inflammation, regardless of the inciting cause, in the presence of hypercalcemia and hyperphosphatemia contributes to cutaneous ectopic calcification. Genetically modified rodents lacking immune activation of T and B cells or NK cells are resistant to developing cutaneous calcification. Consistent with this, administration of the immunosuppressive cyclophosphamide reduced calcific deposits, as did T cell suppression with cyclosporine. We demonstrate that IL-17 is upregulated in calcific skin and neutrophils are the predominant cell type expressing IL-17 and tissue-nonspecific alkaline phosphatase (TNAP) that are necessary for ectopic calcification. Targeting IL-17 with a monoclonal antibody or using a myeloperoxidase inhibitor to blunt neutrophil activation notably attenuated calcific deposits in vivo. Taken together, these observations provide fresh insight into the role of the immune system and the IL-17/neutrophil axis in mediating ectopic calcification in rodent models of calciphylaxis.