An autophagy program that promotes T cell egress from the lymph node controls responses to immune checkpoint blockade.

Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.

S100A8-enriched microglia populate the brain of tau-seeded and accelerated aging mice.

Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.

A bispecific Clec9A-PD-L1 targeted type I interferon profoundly reshapes the tumor microenvironment towards an antitumor state.

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2. Moreover, we report empowered diversity in the CD8+ T cell repertoire and induction of a shift from naive, dysfunctional CD8+ T cells towards effector, plastic cytotoxic T lymphocytes together with increased presence of NK and NKT cells as well as decreased regulatory T cell levels. These dynamic changes were associated with potent antitumor activity. Tumor clearance and immunological memory, therapeutic immunity on large established tumors and blunted tumor growth at distant sites were obtained upon co-administration of a non-curative dose of chemotherapy.Overall, this study illuminates further application of type I interferon as a safe and efficient way to reshape the suppressive tumor microenvironment and induce potent antitumor immunity; features which are of major importance in overcoming the development of metastases and tumor cell resistance to immune attack. The strategy described here has potential for application across to a broad range of cancer types.

Autophagy critically controls skin inflammation and apoptosis-induced stem cell activation.

Macroautophagy/autophagy is a cellular recycling program regulating cell survival and controlling inflammatory responses in a context-dependent manner. Here, we demonstrate that keratinocyte-selective ablation of Atg16l1, an essential autophagy mediator, results in exacerbated inflammatory and neoplastic skin responses. In addition, mice lacking keratinocyte autophagy exhibit precocious onset of hair follicle growth, indicating altered activation kinetics of hair follicle stem cells (HFSCs). These HFSCs also exhibit expanded potencies in an autophagy-deficient context as shown by de novo hair follicle formation and improved healing of abrasion wounds. ATG16L1-deficient keratinocytes are markedly sensitized to apoptosis. Compound deletion of RIPK3-dependent necroptotic and CASP8-dependent apoptotic responses or of TNFRSF1A/TNFR1 reveals that the enhanced sensitivity of autophagy-deficient keratinocytes to TNF-dependent cell death is driving altered activation of HFSCs. Together, our data demonstrate that keratinocyte autophagy dampens skin inflammation and tumorigenesis but curtails HFSC activation by restraining apoptotic responses.Abbreviations: ATG16L1: autophagy related 16 like 1; DMBA: 2,4-dimethoxybenzaldehyde; DP: dermal papilla; EpdSCs: epidermal stem cells; Gas6: growth arrest specific 6; HF: hair follicle; HFSC: hair follicle stem cell; IFE: interfollicular epidermis; KRT5: keratin 5; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PMK: primary mouse keratinocyte; RIPK3: receptor-interacting serine-threonine kinase 3; scRNAseq: single-cell RNA-sequencing; SG: sebaceous gland; TEWL: transepidermal water loss; TPA: 12-O-tetradecanoylphorbol-13-acetate; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a; UMAP: uniform manifold approximation and projection.

Single-cell profiling identifies a novel human polyclonal unconventional T cell lineage.

In the human thymus, a CD10+ PD-1+ TCRαß+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8ß expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C.

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRß repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity.

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1ß response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.

Fibrotic enzymes modulate wound-induced skin tumorigenesis.

Fibroblasts are a major component of the microenvironment of most solid tumours. Recent research elucidated a large heterogeneity and plasticity of activated fibroblasts, indicating that their role in cancer initiation, growth and metastasis is complex and context-dependent. Here, we performed genome-wide expression analysis comparing fibroblasts in normal, inflammatory and tumour-associated skin. Cancer-associated fibroblasts (CAFs) exhibit a fibrotic gene signature in wound-induced tumours, demonstrating persistent extracellular matrix (ECM) remodelling within these tumours. A top upregulated gene in mouse CAFs encodes for PRSS35, a protease capable of collagen remodelling. In human skin, we observed PRSS35 expression uniquely in the stroma of high-grade squamous cell carcinomas. Ablation of PRSS35 in mouse models of wound- or chemically-induced tumorigenesis resulted in aberrant collagen composition in the ECM and increased tumour incidence. Our results indicate that fibrotic enzymes expressed by CAFs can regulate squamous tumour initiation by remodelling the ECM.

OTULIN Prevents Liver Inflammation and Hepatocellular Carcinoma by Inhibiting FADD- and RIPK1 Kinase-Mediated Hepatocyte Apoptosis.

Inflammatory signaling pathways are tightly regulated to avoid chronic inflammation and the development of disease. OTULIN is a deubiquitinating enzyme that controls inflammation by cleaving linear ubiquitin chains generated by the linear ubiquitin chain assembly complex. Here, we show that ablation of OTULIN in liver parenchymal cells in mice causes severe liver disease which is characterized by liver inflammation, hepatocyte apoptosis, and compensatory hepatocyte proliferation, leading to steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). Genetic ablation of Fas-associated death domain (FADD) completely rescues and knockin expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) significantly protects mice from developing liver disease, demonstrating that apoptosis of OTULIN-deficient hepatocytes triggers disease pathogenesis in this model. Finally, we demonstrate that type I interferons contribute to disease in hepatocyte-specific OTULIN-deficient mice. Our study reveals the critical importance of OTULIN in protecting hepatocytes from death, thereby preventing the development of chronic liver inflammation and HCC.