Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

Although blood–brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt–β-catenin signaling. Constitutive activation of Wnt–β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Here we find that CD8 + T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49 + CD8 + regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8 + T cells efficiently eliminated pathogenic gliadin-specific CD4 + T cells from celiac disease patients’ leukocytes in vitro. We also find elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 patients, which correlated with disease severity and vasculitis. Selective ablation of Ly49 + CD8 + T cells in virus-infected mice led to autoimmunity post infection. Our results indicate that in both species, these regulatory CD8 + T cells act uniquely to suppress pathogenic T cells in autoimmune and infectious diseases.

Previous reports show a small subset of CD8+ T cells expressing Ly49 proteins in mice can suppress autoimmunity in a model of demyelinating disease. Here we find a markedly increased frequency of CD8+ T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various human autoimmune diseases. Increased KIR+ CD8+ T cells in the gut also correlate with disease activity in Celiac disease (CeD) patients. Moreover, KIR+ CD8+ T cells can efficiently eliminate pathogenic gliadin-specific CD4+ T cells from CeD patients’ leukocytes in vitro. Together with gene expression data, this shows that these cells are the likely equivalent of the mouse Ly49+ CD8+ T cells. Furthermore, we observe elevated levels of KIR+ CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Single-cell RNA and parallelized TCR sequencing reveals that expanded KIR+ CD8+ T cells from these different diseases and healthy subjects display shared phenotypes and similar T cell receptor sequences. Selective ablation of the murine counterpart in virus-infected mice leads to exacerbated autoimmunity developed after infection. These results characterize a regulatory CD8+ T cell subset in humans which we hypothesize functions to control pathogenic cells in autoimmune and infectious diseases, with important implications for the cellular dynamics and possible therapeutic approaches to suppress unwanted autoimmunity. Supported by National Institutes of Health U19-AI057229 Howard Hughes Medical Institute Stanford Diabetes Research Center (P30DK116074)

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