The linear ubiquitin chain assembly complex regulates TRAIL-induced gene activation and cell death.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ubiquitin ligase which catalyses the generation of linear ubiquitin linkages de novo LUBAC is a crucial component of various immune receptor signalling pathways. Here, we show that LUBAC forms part of the TRAIL-R-associated complex I as well as of the cytoplasmic TRAIL-induced complex II In both of these complexes, HOIP limits caspase-8 activity and, consequently, apoptosis whilst being itself cleaved in a caspase-8-dependent manner. Yet, by limiting the formation of a RIPK1/RIPK3/MLKL-containing complex, LUBAC also restricts TRAIL-induced necroptosis. We identify RIPK1 and caspase-8 as linearly ubiquitinated targets of LUBAC following TRAIL stimulation. Contrary to its role in preventing TRAIL-induced RIPK1-independent apoptosis, HOIP presence, but not its activity, is required for preventing necroptosis. By promoting recruitment of the IKK complex to complex I, LUBAC also promotes TRAIL-induced activation of NF-κB and, consequently, the production of cytokines, downstream of FADD, caspase-8 and cIAP1/2. Hence, LUBAC controls the TRAIL signalling outcome from complex I and II, two platforms which both trigger cell death and gene activation.

Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment.

In a large proportion of cancer patients, CD8 T cells are excluded from the vicinity of cancer cells. The inability of CD8 T cells to reach tumor cells is considered an important mechanism of resistance to cancer immunotherapy. We show that, in human lung squamous-cell carcinomas, exclusion of CD8 T cells from tumor islets is correlated with a poor clinical outcome and with a low lymphocyte motility, as assessed by dynamic imaging on fresh tumor slices. In the tumor stroma, macrophages mediate lymphocyte trapping by forming long-lasting interactions with CD8 T cells. Using a mouse tumor model with well-defined stromal and tumor cell areas, macrophages were depleted with PLX3397, an inhibitor of colony-stimulating factor-1 receptor (CSF-1R). Our results reveal that a CSF-1R blockade enhances CD8 T cell migration and infiltration into tumor islets. Although this treatment alone has minor effects on tumor growth, its combination with anti-PD-1 therapy further increases the accumulation of CD8 T cells in close contact with malignant cells and delays tumor progression. These data suggest that the reduction of macrophage-mediated T cell exclusion increases tumor surveillance by CD8 T cells and renders tumors more responsive to anti-PD-1 treatment.

Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.

Resolution of liver fibrosis requires myeloid cell-driven sinusoidal angiogenesis.

UNLABELLED: Angiogenesis is a key feature of liver fibrosis. Although sinusoidal remodeling is believed to contribute to fibrogenesis, the impact of sinusoidal angiogenesis on the resolution of liver fibrosis remains undefined. Myeloid cells, particularly macrophages, constantly infiltrate the fibrotic liver and can profoundly contribute to remodeling of liver sinusoids. We observe that the development of fibrosis is associated with decreased hepatic vascular endothelial growth factor (VEGF) expression as well as sinusoidal rarefication of the fibrotic scar. In contrast, the resolution of fibrosis is characterized by a rise in hepatic VEGF levels and revascularization of the fibrotic tissue. Genetic ablation of VEGF in myeloid cells or pharmacological inhibition of VEGF receptor 2 signaling prevents this angiogenic response and the resolution of liver fibrosis. We observe increased expression of matrix metalloproteases as well as decreased expression of tissue inhibitor of metalloproteases confined to sinusoidal endothelial cells in response to myeloid cell VEGF. Remarkably, reintroduction of myeloid cell-derived VEGF upon recovery restores collagenolytic acitivity and the resolution of fibrosis. CONCLUSION: We identify myeloid cell-derived VEGF as a critical regulator of extracellular matrix degradation by liver endothelial cells, thereby unmasking an unanticipated link between angiogenesis and the resolution of fibrosis.

Time 2EVOLVE: predicting efficacy of engineered T-cells - how far is the bench from the bedside?

Immunotherapy with gene engineered CAR and TCR transgenic T-cells is a transformative treatment in cancer medicine. There is a rich pipeline with target antigens and sophisticated technologies that will enable establishing this novel treatment not only in rare hematological malignancies, but also in common solid tumors. The T2EVOLVE consortium is a public private partnership directed at accelerating the preclinical development of and increasing access to engineered T-cell immunotherapies for cancer patients. A key ambition in T2EVOLVE is to assess the currently available preclinical models for evaluating safety and efficacy of engineered T cell therapy and developing new models and test parameters with higher predictive value for clinical safety and efficacy in order to improve and accelerate the selection of lead T-cell products for clinical translation. Here, we review existing and emerging preclinical models that permit assessing CAR and TCR signaling and antigen binding, the access and function of engineered T-cells to primary and metastatic tumor ligands, as well as the impact of endogenous factors such as the host immune system and microbiome. Collectively, this review article presents a perspective on an accelerated translational development path that is based on innovative standardized preclinical test systems for CAR and TCR transgenic T-cell products.

Time to evolve: predicting engineered T cell-associated toxicity with next-generation models.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment.

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis.

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.

Boosting the hypoxic response in myeloid cells accelerates resolution of fibrosis and regeneration of the liver in mice.

We have recently shown that targeting Vascular Endothelial Growth Factor (VEGF) specifically in scar-infiltrating myeloid cells prevented remodeling of the sinusoidal vasculature and abrogated the resolution of murine liver fibrosis, thereby unmasking an unanticipated link between angiogenesis and resolution of fibrosis. In a gain of function approach, we wanted to test the impact of VEGF overexpression in myeloid cells on fibrolysis. We observe that genetic inactivation of the von Hippel Lindau protein (VHL), a negative regulator of Hypoxia-inducible factors (HIF) in myeloid cells, leads to increased VEGF expression and most importantly, accelerated matrix degradation and reduced myofibroblast numbers after CCl4 challenge. This is associated with enhanced expression of MMP-2 and -14 as well as lower expression of TIMP-2 in liver endothelial cells. In addition, we report increased expression of MMP-13 in scar-associated macrophages as well as improved liver regeneration upon ablation of VHL in myeloid cells. Finally, therapeutic infusion of macrophages nulli-zygous for VHL or treated with the pharmacologic hydroxylase inhibitor and HIF-inducer Dimethyloxalylglycine (DMOG) accelerates resolution of fibrosis. Hence, boosting the HIF-VEGF signaling axis in macrophages represents a promising therapeutic avenue for the treatment of liver fibrosis.

Targeting VEGF-A in myeloid cells enhances natural killer cell responses to chemotherapy and ameliorates cachexia.

Chemotherapy remains a mainstay of cancer treatment but its use is often limited by the development of adverse reactions. Severe loss of body weight (cachexia) is a frequent cause of death in cancer patients and is exacerbated by chemotherapy. We show that genetic inactivation of vascular endothelial growth factor (VEGF)-A in myeloid cells prevents chemotherapy-induced cachexia by inhibiting skeletal muscle loss and the lipolysis of white adipose tissue. It also improves clearance of senescent tumour cells by natural killer cells and inhibits tumour regrowth after chemotherapy. The effects depend on the chemoattractant chemerin, which is released by the tumour endothelium in response to chemotherapy. The findings define chemerin as a critical mediator of the immune response, as well as an important inhibitor of cancer cachexia. Targeting myeloid cell-derived VEGF signalling should impede the lipolysis and weight loss that is frequently associated with chemotherapy, thereby substantially improving the therapeutic outcome.