Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

The cell stress and immunity cycle in cancer: Toward next generation of cancer immunotherapy.

The cellular stress and immunity cycle is a cornerstone of organismal homeostasis. Stress activates intracellular and intercellular communications within a tissue or organ to initiate adaptive responses aiming to resolve the origin of this stress. If such local measures are unable to ameliorate this stress, then intercellular communications expand toward immune activation with the aim of recruiting immune cells to effectively resolve the situation while executing tissue repair to ameliorate any damage and facilitate homeostasis. This cellular stress-immunity cycle is severely dysregulated in diseased contexts like cancer. On one hand, cancer cells dysregulate the normal cellular stress responses to reorient them toward upholding growth at all costs, even at the expense of organismal integrity and homeostasis. On the other hand, the tumors severely dysregulate or inhibit various components of organismal immunity, for example, by facilitating immunosuppressive tumor landscape, lowering antigenicity, and increasing T-cell dysfunction. In this review we aim to comprehensively discuss the basis behind tumoral dysregulation of cellular stress-immunity cycle. We also offer insights into current understanding of the regulators and deregulators of this cycle and how they can be targeted for conceptualizing successful cancer immunotherapy regimen.

Synthetic biology in mammalian cells: next generation research tools and therapeutics.

Recent progress in DNA manipulation and gene circuit engineering has greatly improved our ability to programme and probe mammalian cell behaviour. These advances have led to a new generation of synthetic biology research tools and potential therapeutic applications. Programmable DNA-binding domains and RNA regulators are leading to unprecedented control of gene expression and elucidation of gene function. Rebuilding complex biological circuits such as T cell receptor signalling in isolation from their natural context has deepened our understanding of network motifs and signalling pathways. Synthetic biology is also leading to innovative therapeutic interventions based on cell-based therapies, protein drugs, vaccines and gene therapies.

BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis.

BNIP3 is a mitophagy receptor with context-dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient's survival and depletion of BNIP3 in B16-F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2-mediated downregulation of HIF-1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3-deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4-mediated ferritinophagy, which fostered PHD2-mediated HIF-1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF-1α levels in BNIP3-depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF-1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro-tumorigenic HIF-1α glycolytic program in melanoma cells.

MCPIP1 Endoribonuclease Activity Negatively Regulates Interleukin-17-Mediated Signaling and Inflammation.

Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore, constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1's endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra(-/-) background. Conversely, IL-17-dependent pathology in Zc3h12a(+/-) mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3' UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ, and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling.

Circulating biomarkers at diagnosis correlate with distant metastases of early luminal-like breast cancer.

There is an urgent need for new and better biomarker modalities to estimate the risk of recurrence within the luminal-like breast cancer (BC) population. Molecular diagnostic tests used in the clinic lack accuracy in identifying patients with early luminal BC who are likely to develop metastases. This study provides proof of concept that various liquid biopsy read-outs could serve as valuable candidates to build a multi-modal biomarker model distinguishing, already at diagnosis, between early metastasizing and non-metastasizing patients. All these blood biomarkers (chemokines, microRNAs, leukemia inhibitory factor, osteopontin, and serum-induced functional myeloid signaling responses) can be measured in baseline plasma/serum samples and could be added to the existing prognostic factors to improve risk stratification and more patient-tailored treatment in early luminal BC.

Regulatory T-cell stability and functional plasticity in health and disease.

FOXP3-expressing regulatory T cells (Treg ) are indispensable for immune homeostasis and tolerance, and in addition tissue-resident Treg have been found to perform noncanonical, tissue-specific functions. For optimal tolerogenic function during inflammatory disease, Treg are equipped with mechanisms that assure lineage stability. Treg lineage stability is closely linked to the installation and maintenance of a lineage-specific epigenetic landscape, specifically a Treg -specific DNA demethylation pattern. At the same time, for local and directed immune regulation Treg must possess a level of functional plasticity that requires them to partially acquire T helper cell (TH ) transcriptional programs-then referred to as TH -like Treg . Unleashing TH programs in Treg , however, is not without risk and may threaten the epigenetic stability of Treg with consequently pathogenic ex-Treg contributing to (auto-) inflammatory conditions. Here, we review how the Treg -stabilizing epigenetic landscape is installed and maintained, and further discuss the development, necessity and lineage instability risks of TH 1-, TH 2-, TH 17-like Treg and follicular Treg .

Immunogenic cell death and its therapeutic or prognostic potential in high-grade glioma.

Immunogenic cell death (ICD) has emerged as a key component of therapy-induced anti-tumor immunity. Over the past few years, ICD was found to play a pivotal role in a wide variety of novel and existing treatment modalities. The clinical application of these techniques in cancer treatment is still in its infancy. Glioblastoma (GBM) is the most lethal primary brain tumor with a dismal prognosis despite maximal therapy. The development of new therapies in this aggressive type of tumors remains highly challenging partially due to the cold tumor immune environment. GBM could therefore benefit from ICD-based therapies stimulating the anti-tumor immune response. In what follows, we will describe the mechanisms behind ICD and the ICD-based (pre)clinical advances in anticancer therapies focusing on GBM.

Early memory differentiation and cell death resistance in T cells predicts melanoma response to sequential anti-CTLA4 and anti-PD1 immunotherapy.

Immune checkpoint blockers (ICBs)-based immunotherapy has revolutionised oncology. However, the benefits of ICBs are limited to only a subset of patients. Herein, the biomarkers-driven application of ICBs promises to increase their efficacy. Such biomarkers include lymphocytic IFNγ-signalling and/or cytolytic activity (granzymes and perforin-1) footprints, whose levels in pre-treatment tumours can predict favourable patient survival following ICB-treatment. However, it is not clear whether such biomarkers have the same value in predicting survival of patients receiving first-line anti-CTLA4 ICB-therapy, and subsequently anti-PD1 ICB-therapy (i.e., sequential ICB-immunotherapy regimen). To address this, we applied highly integrated systems/computational immunology approaches to existing melanoma bulk-tumour transcriptomic and single-cell (sc)RNAseq data originating from immuno-oncology clinical studies applying ICB-treatment. Interestingly, we observed that CD8+/CD4+T cell-associated IFNγ-signalling or cytolytic activity signatures fail to predict tumour response in patients treated with anti-CTLA4 ICB-therapy as a first-line and anti-PD1 ICB-therapy in the second-line setting. On the contrary, signatures associated with early memory CD8+/CD4+T cells (integrating TCF1-driven stem-like transcriptional programme), capable of resisting cell death/apoptosis, better predicted objective response rates to ICB-immunotherapy, and favourable survival in the setting of sequential ICB-immunotherapy. These observations suggest that sequencing of ICB-therapy might have a specific impact on the T cell-repertoire and may influence the predictive value of tumoural immune biomarkers.

Identification and characterization of Poly(ADP-ribose) polymerase-1 interacting proteins during development of Dictyostelium discoideum.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a multifunctional protein that is associated with various biological processes like chromatin remodeling, DNA damage, cell death etc. In Dictyostelium discoideum, PARP-1 has also been implicated in cellular differentiation and development. However, its interacting proteins during multicellular development are not yet explored. Hence, the present study aims to identify PARP-1 interacting proteins during multicellular development of D. discoideum. BRCA1 C-terminus (BRCT) domain of PARP-1, which is mainly involved in protein-protein interactions was cloned in pGEX4T1 vector and developmental interactome of PARP-1 were analyzed by affinity purification-mass spectrometry. These interactions were further confirmed by in-silico protein-protein docking analysis, which led to identification of the proteins that show high affinity for BRCT domain. Initially, the protein structures were modeled on SWISS MODEL and PHYRE2 servers, refined by 3Drefine and validated by PROCHECK. Further, interaction sites of BRCT and the conserved regions in all interacting proteins were predicted using cons-PPISP and ConSurf, respectively. Finally, protein-protein docking analysis was done by HADDOCK. Our results identified 19 possible BRCT interacting proteins during D. discoideum development. Furthermore, interacting residues involved in the interactions and functional regions were explored. This is the first report where PARP-1's developmental interactome in D. discoideum is well established. The current findings demonstrate PARP-1's developmental interactome in D. discoideum and provide the groundwork to understand its regulated functions in developmental biology which would undoubtedly extend our perception towards developmental diseases in higher complex organisms and their treatment.

Insights into the functional aspects of poly(ADP-ribose) polymerase-1 (PARP-1) in mitochondrial homeostasis in Dictyostelium discoideum.

BACKGROUND INFORMATION: Poly(ADP-ribose) Polymerase-1 (PARP-1) is predominantly a nuclear protein and involved in various cellular processes like DNA repair, cell death, development, chromatin modulation etc. PARP-1 utilizes NAD+ and adds negatively charged PAR moieties on the target proteins. Over-activation of PARP-1 has been shown to cause energy crisis mediated cell death in which mitochondrial homeostasis is also affected. Moreover, the presence of mitochondrial NAD+ pools highlights the role of PARP-1 in mitochondria. The aim of present study is to understand the physiological role of PARP-1 in regulating mitochondrial functioning by varying the levels of PARP-1 in Dictyostelium discoideum. Intra-mitochondrial PARylation was analyzed by indirect immunofluorescence. Further, the effect of altered levels of PARP-1 i.e. overexpression, downregulation, knockout and its chemical inhibition was studied on mitochondrial respiration, reactive oxygen species (ROS) levels, ATP production, mitochondrial fission-fusion, mitochondrial morphology and mitochondrial DNA (mtDNA) content of D. discoideum. RESULTS: Our results show intra-mitochondrial PARylation under oxidative stress. Altered levels of PARP-1 caused impairment in the mitochondrial respiratory capacity, leading to elevated ROS levels and reduced ATP production. Moreover, PARP-1 affects the mitochondrial morphology and mtDNA content, alters the mitochondrial fission-fusion processes in lieu of preventing cell death under physiological conditions. CONCLUSION: The current study highlights the physiological role of PARP-1 in mitochondrial respiration, its morphology, fission-fusion processes and mtDNA maintenance in D. discoideum. SIGNIFICANCE: This study would provide new clues on the PARP-1's crucial role in mitochondrial homeostasis, exploring the therapeutic potential of PARP-1 in various mitochondrial diseases.

Cell death and immunity in cancer: From danger signals to mimicry of pathogen defense responses.

The immunogenicity of cancer cells is an emerging determinant of anti-cancer immunotherapy. Beyond developing immunostimulatory regimens like dendritic cell-based vaccines, immune-checkpoint blockers, and adoptive T-cell transfer, investigators are beginning to focus on the immunobiology of dying cancer cells and its relevance for the success of anticancer immunotherapies. It is currently accepted that cancer cells may die in response to anti-cancer therapies through regulated cell death programs, which may either repress or increase their immunogenic potential. In particular, the induction of immunogenic cancer cell death (ICD), which is hallmarked by the emission of damage-associated molecular patterns (DAMPs); molecules analogous to pathogen-associated molecular patterns (PAMPs) acting as danger signals/alarmins, is of great relevance in cancer therapy. These ICD-associated danger signals favor immunomodulatory responses that lead to tumor-associated antigens (TAAs)-directed T-cell immunity, which paves way for the removal of residual, treatment-resistant cancer cells. It is also emerging that cancer cells succumbing to ICD can orchestrate "altered-self mimicry" i.e. mimicry of pathogen defense responses, on the levels of nucleic acids and/or chemokines (resulting in type I interferon/IFN responses or pathogen response-like neutrophil activity). In this review, we exhaustively describe the main molecular, immunological, preclinical, and clinical aspects of immunosuppressive cell death or ICD (with respect to apoptosis, necrosis and necroptosis). We also provide an extensive historical background of these fields, with special attention to the self/non-self and danger models, which have shaped the field of cell death immunology.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

IL-17 Signaling: The Yin and the Yang.

Interleukin (IL)-17 is the founding member of a novel family of inflammatory cytokines. While the proinflammatory properties of IL-17 are key to its host-protective capacity, unrestrained IL-17 signaling is associated with immunopathology, autoimmune disease, and cancer progression. In this review we discuss both the activators and the inhibitors of IL-17 signal transduction, and also the physiological implications of these events. We highlight the surprisingly diverse means by which these regulators control expression of IL-17-dependent inflammatory genes, as well as the major target cells that respond to IL-17 signaling.

Integrating Next-Generation Dendritic Cell Vaccines into the Current Cancer Immunotherapy Landscape.

Cancer immunotherapy is experiencing a renaissance spearheaded by immune checkpoint inhibitors (ICIs). This has spurred interest in 'upgrading' existing immunotherapies that previously experienced only sporadic success, such as dendritic cells (DCs) vaccines. In this review, we discuss the major molecular, immunological, and clinical determinants of existing first- and second-generation DC vaccines. We also outline the future trends for next-generation DC vaccines and describe their major hallmarks and prerequisites necessary for high anticancer efficacy. In addition, using existing data we compare DC vaccines with ICIs targeting CTLA4, PD1, and PD-L1, and argue that in various contexts next-generation DC vaccines are ready to meet some challenges currently confronting ICIs, thereby raising the need to integrate DC vaccines in future combinatorial immunotherapy regimens.

MCPIP1/Regnase-1 Restricts IL-17A- and IL-17C-Dependent Skin Inflammation.

The IL-17 family cytokines IL-17A and IL-17C drive the pathogenesis of psoriatic skin inflammation, and anti-IL-17A Abs were recently approved to treat human psoriasis. Little is known about mechanisms that restrain IL-17 cytokine-mediated signaling, particularly IL-17C. In this article, we show that the endoribonuclease MCP-1-induced protein 1 (MCPIP1; also known as regnase-1) is markedly upregulated in human psoriatic skin lesions. Similarly, MCPIP1 was overexpressed in the imiquimod (IMQ)-driven mouse model of cutaneous inflammation. Mice with an MCPIP1 deficiency (Zc3h12a+/-) displayed no baseline skin inflammation, but they showed exacerbated pathology following IMQ treatment. Pathology in Zc3h12a+/- mice was associated with elevated expression of IL-17A- and IL-17C-dependent genes, as well as with increased accumulation of neutrophils in skin. However, IL-17A and IL-17C expression was unaltered, suggesting that the increased inflammation in Zc3h12a+/- mice was due to enhanced downstream IL-17R signaling. Radiation chimeras demonstrated that MCPIP1 in nonhematopoietic cells is responsible for controlling skin pathology. Moreover, Zc3h12a+/-Il17ra-/- mice given IMQ showed almost no disease. To identify which IL-17RA ligand was essential, Zc3h12a+/-Il17a-/- and Zc3h12a+/-Il17c-/- mice were given IMQ; these mice had reduced but not fully abrogated pathology, indicating that MCPIP1 inhibits IL-17A and IL-17C signaling. Confirming this hypothesis, Zc3h12a-/- keratinocytes showed increased responsiveness to IL-17A and IL-17C stimulation. Thus, MCPIP1 is a potent negative regulator of psoriatic skin inflammation through IL-17A and IL-17C. Moreover, to our knowledge, MCPIP1 is the first described negative regulator of IL-17C signaling.

Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism.

BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS: Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS: GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY: GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

Author Correction: Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin.

This correction refers to our Short Communication published in Cancer Immunology Immunotherapy in the year 2012 [1]. It has come to our attention that some errors resulting from accidental oversight concerning incorrect deletion/replacement of temporary placeholder images during figure assembly and mounting occurred during the assembly of the "Intracellular Proteins" immunoblots presented in Fig. 1A and Fig. 1D.