BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1ß, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies.

Natural killer (NK) cells belong to the innate immune system and contribute to protecting the host through killing of infected, foreign, stressed or transformed cells. Additionally, via cellular cross-talk, NK cells orchestrate antitumor immune responses. Hence, significant efforts have been undertaken to exploit the therapeutic properties of NK cells in cancer. Current strategies in preclinical and clinical development include adoptive transfer therapies, direct stimulation, recruitment of NK cells into the tumor microenvironment (TME), blockade of inhibitory receptors that limit NK cell functions, and therapeutic modulation of the TME to enhance antitumor NK cell function. In this Review, we introduce the NK cell-cancer cycle to highlight recent advances in NK cell biology and to discuss the progress and problems of NK cell-based cancer immunotherapies.

The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation.

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis.

Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.

Tumor immunoevasion by the conversion of effector NK cells into type 1 innate lymphoid cells.

Avoiding destruction by immune cells is a hallmark of cancer, yet how tumors ultimately evade control by natural killer (NK) cells remains incompletely defined. Using global transcriptomic and flow-cytometry analyses and genetically engineered mouse models, we identified the cytokine-TGF-ß-signaling-dependent conversion of NK cells (CD49a-CD49b+Eomes+) into intermediate type 1 innate lymphoid cell (intILC1) (CD49a+CD49b+Eomes+) populations and ILC1 (CD49a+CD49b-Eomesint) populations in the tumor microenvironment. Strikingly, intILC1s and ILC1s were unable to control local tumor growth and metastasis, whereas NK cells favored tumor immunosurveillance. Experiments with an antibody that neutralizes the cytokine TNF suggested that escape from the innate immune system was partially mediated by TNF-producing ILC1s. Our findings provide new insight into the plasticity of group 1 ILCs in the tumor microenvironment and suggest that the TGF-ß-driven conversion of NK cells into ILC1s is a previously unknown mechanism by which tumors escape surveillance by the innate immune system.

Adoptive T Cell Therapy Targeting Different Gene Products Reveals Diverse and Context-Dependent Immune Evasion in Melanoma.

Tumor immune escape limits durable responses to T cell therapy. Here, we examined how regulation and function of gene products that provide the target epitopes for CD8+ T cell anti-tumor immunity influence therapeutic efficacy and resistance. We used a CRISPR-Cas9-based method (CRISPitope) in syngeneic melanoma models to fuse the same model CD8+ T cell epitope to the C-termini of different endogenous gene products. Targeting melanosomal proteins or oncogenic CDK4R24C (Cyclin-dependent kinase 4) by adoptive cell transfer (ACT) of the same epitope-specific CD8+ T cells revealed diverse genetic and non-genetic immune escape mechanisms. ACT directed against melanosomal proteins, but not CDK4R24C, promoted melanoma dedifferentiation, and increased myeloid cell infiltration. CDK4R24C antigen persistence was associated with an interferon-high and T-cell-rich tumor microenvironment, allowing for immune checkpoint inhibition as salvage therapy. Thus, the choice of target antigen determines the phenotype and immune contexture of recurrent melanomas, with implications to the design of cancer immunotherapies.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8+ T Cells.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.

A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

IFN-λ Diminishes the Severity of Viral Bronchiolitis in Neonatal Mice by Limiting NADPH Oxidase-Induced PAD4-Independent NETosis.

Infants with attenuated type III IFN (IFN-λ) responses are at increased risk of severe lower respiratory tract infection (sLRI). The IL-28Rα-chain and IL-10Rß-chain form a heterodimeric receptor complex, necessary for IFN-λ signaling. Therefore, to better understand the immunopathogenic mechanisms through which an IFN-λlo microenvironment predisposes to a sLRI, we inoculated neonatal wild-type and IL-28R-deficient (IL-28R -/-) mice with pneumonia virus of mice, a rodent-specific pneumovirus. Infected IL-28R -/- neonates displayed an early, pronounced, and persistent neutrophilia that was associated with enhanced reactive oxygen species (ROS) production, NETosis, and mucus hypersecretion. Targeted deletion of the IL-28R in neutrophils was sufficient to increase neutrophil activation, ROS production, NET formation, and mucus production in the airways. Inhibition of protein-arginine deiminase type 4 (PAD4), a regulator of NETosis, had no effect on myeloperoxidase expression, citrullinated histones, and the magnitude of the inflammatory response in the lungs of infected IL-28R -/- mice. In contrast, inhibition of ROS production decreased NET formation, cellular inflammation, and mucus hypersecretion. These data suggest that IFN-λ signaling in neutrophils dampens ROS-induced NETosis, limiting the magnitude of the inflammatory response and mucus production. Therapeutics that promote IFN-λ signaling may confer protection against sLRI.

Targeting inflamed and non-inflamed melanomas: biological background and clinical challenges.

Immune checkpoint inhibitors (ICIs) have demonstrated impressive antitumor activity in patients with advanced and early stage melanoma, thus improving long-term survival outcomes. However, most patients derive limited benefit from immunotherapy, due to the development of primary, adaptive, or acquired resistance mechanisms. Immunotherapy resistance is a complex phenomenon that depends on genetic and epigenetic mechanisms which, in turn, drive the interplay between cancer cells and the tumor microenvironment (TME). Immunologically "cold" (i.e. non-inflamed) tumors lack or have few tumor infiltrating lymphocytes (TILs) as a result of low tumor mutational burden (TMB), defective antigen presentation, or physical barriers to lymphocyte migration, resulting in a minimal benefit from immunotherapy. In contrast, in most cases immunologically "hot" (i.e. inflamed) tumors display high TMB, implying a higher load of neoantigens and increased programmed cell death ligand 1 (PD-L1) expression, with a consequently higher rate of TILs. However, the presence of TILs does not necessarily denote the tumor as immunologically "hot", since the presence of tumor-specific CD8+ T cells persistently exposed to antigenic stimulation induces a dysfunctional state called "exhaustion", which leads to a reduced response to immunotherapy. In recent years, efforts have been made to characterize mechanisms of resistance to immunotherapy, and to investigate strategies to overcome treatment resistance. Indeed, predictors of response and toxicity to immunotherapy are still lacking and, to date, there are no reliable predictive biomarkers to select patients according to baseline clinical, histological, or genomic characteristics. In this review, we will focus on the morphologic and immunohistochemical characteristics of the TME, and on the molecular determinants of resistance to immunotherapy, differentiating between inflamed and non-inflamed melanomas. Then, we will provide a thorough overview of preclinical data on genetic and epigenetic mechanisms with a potential impact on the immune response and patient outcome. Finally, we will focus our attention on the role of potential biomarkers in determining disease response to immunotherapy, in the adjuvant and metastatic setting, providing an insight into current and future research in this field.

Stressed out: NKp46 binds ecto-calreticulin.

In a recent article, Sen Santara et al. demonstrated that the activating natural killer (NK) cell receptor NKp46 binds to externalized calreticulin (ecto-CRT), leading to NK cell degranulation and target cell killing. They show that endoplasmic reticulum stress-induced ecto-CRT serves as a danger-associated molecular pattern, helping NK cells identify and eliminate infected, malignant, stressed or senescent cells.

Genome-wide in vivo screen identifies novel host regulators of metastatic colonization.

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

Hide and seek: Plasticity of innate lymphoid cells in cancer.

The advance of immunotherapies has revolutionized the treatment of cancer patients. Mostly agents modulating the adaptive immune system are currently used. More recently, attempts to stimulate the innate immune system are being promoted for clinical evaluation. Innate lymphoid cells (ILCs) are a highly plastic population of immune cells crucial for tissue homeostasis and the regulation of immune responses and maybe a promising target to improve current cancer immunotherapies. Although we have made significant progress in understanding ILC biology, their impact on tumor development, progression and therapy is controversial. In this review, we discuss the recent advances of ILC function and plasticity in the context of cancer.

Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing.

Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.

Cancer-killing, decoy-resistant interleukin-18.

A recent paper by Zhou et al. describes a new engineered decoy-resistant interleukin-18 with potent antitumor activity and translational potential for cancer immunotherapy.

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma.

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.