PD-L1+ macrophages suppress T cell-mediated anticancer immunity.

Recently, we showed that an autologous DC-based vaccine induces an increase in immunosuppressive PD-L1+ tumor-associated macrophages (TAM) both in the tumor and the tumor draining lymph nodes, thereby blunting the efficacy of therapeutic immunization. Only the combination of the DC vaccine with anti-PD-L1 immune checkpoint inhibition, but not the use of antibodies targeting PD-1 alone, was able to set off CD8+ cytotoxic T lymphocyte (CTL)-mediated tumor suppression in mice. In sum, we delineated a PD-L1 checkpoint blockade-based strategy to avoid TAM-induced T cell exhaustion during DC vaccine therapy.

Trial watch: local anesthetics in cancer therapy.

Preclinical evidence indicates potent antitumor properties of local anesthetics. Numerous underlying mechanisms explaining such anticancer effects have been identified, suggesting direct cytotoxic as well as indirect immunemediated effects that together reduce the proliferative, invasive and migratory potential of malignant cells. Although some retrospective and correlative studies support these findings, prospective randomized controlled trials have not yet fully confirmed the antineoplastic activity of local anesthetics, likely due to the intricate methodology required for mitigating confounding factors. This trial watch aims at compiling all published preclinical and clinical research, along with completed and ongoing trials, that explore the potential antitumor effects of local anesthetics.

Trial watch: dexmedetomidine in cancer therapy.

Dexmedetomidine (DEX) is a highly selective α2-adrenoceptor agonist that is widely used in intensive and anesthetic care for its sedative and anxiolytic properties. DEX has the capacity to alleviate inflammatory pain while limiting immunosuppressive glucocorticoid stress during major surgery, thus harboring therapeutic benefits for oncological procedures. Recently, the molecular mechanisms of DEX-mediated anticancer effects have been partially deciphered. Together with additional preclinical data, these mechanistic insights support the hypothesis that DEX-induced therapeutic benefits are mediated via the stimulation of adaptive anti-tumor immune responses. Similarly, published clinical trials including ancillary studies described an immunostimulatory role of DEX during the perioperative period of cancer surgery. The impact of DEX on long-term patient survival remains elusive. Nevertheless, DEX-mediated immunostimulation offers an interesting therapeutic option for onco-anesthesia. Our present review comprehensively summarizes data from preclinical and clinical studies as well as from ongoing trials with a distinct focus on the role of DEX in overcoming (tumor microenvironment (TME)-imposed) cancer therapy resistance. The objective of this update is to guide clinicians in their choice toward immunostimulatory onco-anesthetic agents that have the capacity to improve disease outcome.

High-throughput assessment of cellular senescence.

Cellular senescence is a molecular process that is activated in response to a large variety of distinct stress signals. Mechanistically, cellular senescence is characterized by an arrest in cell cycle accompanied by phenotypic adaptations and physiological alterations including changes in the secretory profile of senescent cells termed the senescence-associated secretory phenotype (SASP). Here we describe a detailed, automation- compatible method for the detection of senescence-associated beta galactosidase (SA-ß-gal) activity as a hallmark of cellular senescence using a conventional fluorescent microscope equipped with a transmitted light module. Moreover, we outline a protocol for the automated analysis of cellular senescence using convolutional neural networks (CNNs) and mathematical morphology. In sum, we provide a toolset for the high throughput assessment of cellular senescence based on light microscopy and automated image analysis.

Mechanistic exploration of autophagy and aging by RNA interference.

Cellular senescence is a cellular process with organismal impact that is mechanistically counterbalanced to a certain extent by frequent episodes of autophagy. Here we describe a detailed, automation-compatible method for the use of RNA-interference (RNAi; also called post-transcriptional gene silencing (PTGS))-mediated silencing of autophagy related protein-coding gene expression. RNAi is a conserved biological response to double-stranded RNA that mediates resistance to endogenous parasites and exogenous pathogenic nucleic acids. RNAi mediated by short interfering RNA (siRNA) is widely used for gene function analysis. The accurate use of RNAi for the inference of gene function necessitates that both specificity and efficacy of the siRNA-mediated knockdown are monitored. In this manuscript, we exemplify these crucial steps employing siRNAs targeting the autophagy and lysosomal biogenesis associated transcription factor TFE3 and validate their specificity on protein and mRNA level.

Lymph node and tumor-associated PD-L1+ macrophages antagonize dendritic cell vaccines by suppressing CD8+ T cells.

Current immunotherapies provide limited benefits against T cell-depleted tumors, calling for therapeutic innovation. Using multi-omics integration of cancer patient data, we predict a type I interferon (IFN) responseHIGH state of dendritic cell (DC) vaccines, with efficacious clinical impact. However, preclinical DC vaccines recapitulating this state by combining immunogenic cancer cell death with induction of type I IFN responses fail to regress mouse tumors lacking T cell infiltrates. Here, in lymph nodes (LNs), instead of activating CD4+/CD8+ T cells, DCs stimulate immunosuppressive programmed death-ligand 1-positive (PD-L1+) LN-associated macrophages (LAMs). Moreover, DC vaccines also stimulate PD-L1+ tumor-associated macrophages (TAMs). This creates two anatomically distinct niches of PD-L1+ macrophages that suppress CD8+ T cells. Accordingly, a combination of PD-L1 blockade with DC vaccines achieves significant tumor regression by depleting PD-L1+ macrophages, suppressing myeloid inflammation, and de-inhibiting effector/stem-like memory T cells. Importantly, clinical DC vaccines also potentiate T cell-suppressive PD-L1+ TAMs in glioblastoma patients. We propose that a multimodal immunotherapy and vaccination regimen is mandatory to overcome T cell-depleted tumors.

BCL2 inhibition stimulates dendritic cell function for improved anticancer immunotherapy.

Recently we developed a dendritic cell (DC) genotype-phenotype screening platform that is based on CRISPR/Cas9-mediated gene editing of immortalized DC precursors. Whole genome screening for gain-of-function phenotypes led to the identification of BCL2 as a DC-specific immune checkpoint. Genetic or pharmacological inhibition of BCL2 similarly enhanced the antigen presentation capacity of conventional type-1 dendritic cells (cDC1) and mediated T cell-dependent anticancer immunity. The therapeutic anticancer efficacy of the BCL2 inhibitor venetoclax in mice was further increased when combined with a PD-1-targeted immune checkpoint inhibitor. In sum, we delineated a novel strategy of dual checkpoint blockade for cancer immunotherapy in which improvement of DC antigen presentation and avoidance of T cell exhaustion can be advantageously combined.

Preconditioning with immunogenic cell death-inducing treatments for subsequent immunotherapy.

Since the dawn of anticancer immunotherapy, the clinical use of immune checkpoint inhibitors (ICI) has increased exponentially. Monoclonal antibodies targeting CTLA-4 and the PD-1/PD-L1 interaction were first introduced for the treatment of patients with unresectable melanoma. In melanoma, ICI lead to durable regression in a significant number of patients and have thus been clinically approved as a first-line treatment of advanced disease. Over the past years an increasing number of regulatory approvals have been granted for the use of ICI in patients affected by a large range of distinct carcinomas. In retrospect surprisingly, it has been discovered that particularly successful chemotherapeutic treatments are able to trigger anticancer immune responses because they induce immunogenic cell death (ICD), hence killing cancer cells in a way that they elicit an immune response against tumor-associated antigens. Logically, preclinical studies as well as clinical trials are currently exploring the possibility to combine ICD inducers with ICI to obtain optimal therapeutic effects. Here, we provide a broad overview of current strategies for the implementation of combinatorial approaches involving ICD induction followed by ICI in anticancer therapy.

Immunogenic cell death (ICD) enhancers-Drugs that enhance the perception of ICD by dendritic cells.

The search for immunostimulatory drugs applicable to cancer immunotherapy may profit from target-agnostic methods in which agents are screened for their functional impact on immune cells cultured in vitro without any preconceived idea on their mode of action. We have built a synthetic mini-immune system in which stressed and dying cancer cells (derived from standardized cell lines) are confronted with dendritic cells (DCs, derived from immortalized precursors) and CD8+ T-cell hybridoma cells expressing a defined T-cell receptor. Using this system, we can identify three types of immunostimulatory drugs: (i) pharmacological agents that stimulate immunogenic cell death (ICD) of malignant cells; (ii) drugs that act on DCs to enhance their response to ICD; and (iii) drugs that act on T cells to increase their effector function. Here, we focus on strategies to develop drugs that enhance the perception of ICD by DCs and to which we refer as "ICD enhancers." We discuss examples of ICD enhancers, including ligands of pattern recognition receptors (exemplified by TLR3 ligands that correct the deficient function of DCs lacking FPR1) and immunometabolic modifiers (exemplified by hexokinase-2 inhibitors), as well as methods for target deconvolution applicable to the mechanistic characterization of ICD enhancers.

Immunogenic cell stress and death in the treatment of cancer.

The successful treatment of oncological malignancies which results in long-term disease control or the complete eradication of cancerous cells necessitates the onset of adaptive immune responses targeting tumor-specific antigens. Such desirable anticancer immunity can be triggered via the induction of immunogenic cell death (ICD) of cancer cells, thus converting malignant cells into an in situ vaccine that elicits T cell mediated adaptive immune responses and establishes durable immunological memory. The exploration of ICD for cancer treatment has been subject to extensive research. However, functional heterogeneity among ICD activating therapies in many cases requires specific co-medications to achieve full-blown efficacy. Here, we described the hallmarks of ICD and classify ICD activators into three distinct functional categories namely, according to their mode of action: (i) ICD inducers, which increase the immunogenicity of malignant cells, (ii) ICD sensitizers, which prime cellular circuitries for ICD induction by conventional cytotoxic agents, and (iii) ICD enhancers, which improve the perception of ICD signals by antigen presenting dendritic cells. Altogether, ICD induction, sensitization and enhancement offer the possibility to convert well-established conventional anticancer therapies into immunotherapeutic approaches that activate T cell-mediated anticancer immunity.

Trial watch: beta-blockers in cancer therapy.

Compelling evidence supports the hypothesis that stress negatively impacts cancer development and prognosis. Irrespective of its physical, biological or psychological source, stress triggers a physiological response that is mediated by the hypothalamic-pituitary-adrenal axis and the sympathetic adrenal medullary axis. The resulting release of glucocorticoids and catecholamines into the systemic circulation leads to neuroendocrine and metabolic adaptations that can affect immune homeostasis and immunosurveillance, thus impairing the detection and eradication of malignant cells. Moreover, catecholamines directly act on ß-adrenoreceptors present on tumor cells, thereby stimulating survival, proliferation, and migration of nascent neoplasms. Numerous preclinical studies have shown that blocking adrenergic receptors slows tumor growth, suggesting potential clinical benefits of using ß-blockers in cancer therapy. Much of these positive effects of ß-blockade are mediated by improved immunosurveillance. The present trial watch summarizes current knowledge from preclinical and clinical studies investigating the anticancer effects of ß-blockers either as standalone agents or in combination with conventional antineoplastic treatments or immunotherapy.

Immunogenic chemotherapy sensitizes RAS-mutated colorectal cancers to immune checkpoint inhibitors.

Recent clinical trials have compared the use of different chemotherapeutic regimens as "immune induction therapies" to sensitize cancers to immune checkpoint inhibitors (ICI). Cytotoxic drugs reputed to be inducers of immunogenic cell death (ICD) appeared to be particularly efficient for this purpose. A trial published in Nature Medicine by Thibaudin et al. reveals the capacity of oxaliplatin-based chemotherapy to sensitize RAS-mutant unresectable metastatic colorectal cancer to ICIs blocking CTLA-4 and PD-L1.

Immunogenic Cell Stress and Death Sensitize Tumors to Immunotherapy.

The efficacy of chemotherapy with cytotoxicants and that of targeted therapies with more sophisticated agents is limited due to the plasticity of malignant cells, which leads to the inevitable development of resistance [...].

Conventional type 1 dendritic cells (cDC1) in cancer immunity.

Cancer immunotherapy, alone or in combination with conventional therapies, has revolutionized the landscape of antineoplastic treatments, with dendritic cells (DC) emerging as key orchestrators of anti-tumor immune responses. Among the distinct DC subsets, conventional type 1 dendritic cells (cDC1) have gained prominence due to their unique ability to cross-present antigens and activate cytotoxic T lymphocytes. This review summarizes the distinctive characteristics of cDC1, their pivotal role in anticancer immunity, and the potential applications of cDC1-based strategies in immunotherapy.

3,4-dimethoxychalcone induces autophagy and reduces neointimal hyperplasia and aortic lesions in mouse models of atherosclerosis.

Autophagy inducers can prevent cardiovascular aging and age-associated diseases including atherosclerosis. Therefore, we hypothesized that autophagy-inducing compounds that act on atherosclerosis-relevant cells might have a protective role in the development of atherosclerosis. Here we identified 3,4-dimethoxychalcone (3,4-DC) as an inducer of autophagy in several cell lines from endothelial, myocardial and myeloid/macrophagic origin, as demonstrated by the aggregation of the autophagosome marker GFP-LC3 in the cytoplasm of cells, as well as the downregulation of its nuclear pool indicative of autophagic flux. In this respect, 3,4-DC showed a broader autophagy-inducing activity than another chalcone (4,4- dimethoxychalcone), spermidine and triethylene tetramine. Thus, we characterized the potential antiatherogenic activity of 3,4-DC in two different mouse models, namely, (i) neointima formation with smooth muscle expansion of vein segments grafted to the carotid artery and (ii) genetically predisposed ApoE-/- mice fed an atherogenic diet. In the vein graft model, local application of 3,4-DC was able to maintain the lumen of vessels and to reduce neointima lesions. In the diet-induced model, intraperitoneal injections of 3,4-DC significantly reduced the number of atherosclerotic lesions in the aorta. In conclusion, 3,4-DC stands out as an autophagy inducer with potent antiatherogenic activity.

The BCL2 inhibitor venetoclax mediates anticancer effects through dendritic cell activation.

BCL2 is an apoptosis-inhibitory oncoprotein that also possesses apoptosis-unrelated activities. Pharmacological BCL2 inhibitors have been developed with the scope of driving BCL2-dependent cancer cells into apoptosis, and one BCL2 antagonist, venetoclax, has been clinically approved for the treatment of specific leukemias and lymphomas. Nonetheless, it appears that venetoclax, as well as genetic BCL2 inhibition, can mediate anticancer effects through an indirect action. Such an indirect effect relies on the enhancement of the immunostimulatory function of dendritic cells, hence increasing tumor immunosurveillance. Mechanistically, BCL2 inhibition involves improved antigen presentation by conventional type-1 dendritic cells (cDC1s) due to the activation of an interferon response, leading to a T cell-mediated anticancer immune response that can be further enhanced by PD-1 blockade. These findings support the emerging hypothesis that successful antineoplastic drugs generally mediate their effects indirectly, through the immune system, rather via merely cell-autonomous effects on malignant cells.

AMPK activation induces immunogenic cell death in AML.

Survival of patients with acute myeloid leukemia (AML) can be improved by allogeneic hematopoietic stem cell transplantation (allo-HSCT) because of the antileukemic activity of T and natural killer cells from the donor. However, the use of allo-HSCT is limited by donor availability, recipient age, and potential severe side effects. Similarly, the efficacy of immunotherapies directing autologous T cells against tumor cells, including T-cell recruiting antibodies, chimeric antigen receptor T-cell therapy, and immune checkpoint inhibitors are limited in AML because of multiple mechanisms of leukemia immune escape. This has prompted a search for novel immunostimulatory approaches. Here, we show that activation of adenosine 5'-monophosphate-activated protein kinase (AMPK), a master regulator of cellular energy balance, by the small molecule GSK621 induces calreticulin (CALR) membrane exposure in murine and human AML cells. When CALR is exposed on the cell surface, it serves as a damage-associated molecular pattern that stimulates immune responses. We found that GSK621-treated murine leukemia cells promote the activation and maturation of bone marrow-derived dendritic cells. Moreover, vaccination with GSK621-treated leukemia cells had a protective effect in syngeneic immunocompetent recipients bearing transplanted AMLs. This effect was lost in recipients depleted of CD4/CD8 T cells. Together, these results demonstrate that AMPK activation by GSK621 elicits traits of immunogenic cell death and promotes a robust immune response against leukemia. Pharmacologic AMPK activation thus represents a new potential target for improving the activity of immunotherapy in AML.

A mitochondrial checkpoint to adaptive anticancer immunity.

BCL2 robustly preserves mitochondrial integrity, hence inhibiting innate immune signaling and apoptotic cell death in several cell types. Here, we comment on our recent data demonstrating that BCL2 also limits the ability of dendritic cells to elicit adaptive immune responses, lending support to a universal immunosuppressive function for the mitochondrial immune checkpoint.

The Chlamydia effector CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab35.

The obligate intracellular bacterium Chlamydia trachomatis inserts a family of inclusion membrane (Inc) proteins into the membrane of its vacuole (the inclusion). The Inc CpoS is a critical suppressor of host cellular immune surveillance, but the underlying mechanism remained elusive. By complementing a cpoS mutant with various natural orthologs and variants of CpoS, we linked distinct molecular interactions of CpoS to distinct functions. Unexpectedly, we found CpoS to be essential for the formation of inclusion membrane microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. While the function of CpoS in microdomains was uncoupled from its role in the suppression of host cellular defenses, we found the ability of CpoS to interact with Rab GTPases to be required not only for the manipulation of membrane trafficking, such as to mediate transport of ceramide-derived lipids (sphingolipids) to the inclusion, but also for the inhibition of Stimulator of interferon genes (STING)-dependent type I interferon responses. Indeed, depletion of Rab35 phenocopied the exacerbated interferon responses observed during infection with CpoS-deficient mutants. Overall, our findings highlight the role of Inc-Inc interactions in shaping the inclusion microenvironment and the modulation of membrane trafficking as a pathogenic immune evasion strategy. IMPORTANCE Chlamydia trachomatis is a prevalent bacterial pathogen that causes blinding ocular scarring and urogenital infections that can lead to infertility and pregnancy complications. Because Chlamydia can only grow within its host cell, boosting the intrinsic defenses of human cells may represent a novel strategy to fight pathogen replication and survival. Hence, CpoS, a Chlamydia protein known to block host cellular defenses, or processes regulated by CpoS, could provide new opportunities for therapeutic intervention. By revealing CpoS as a multifunctional virulence factor and by linking its ability to block host cellular immune signaling to the modulation of membrane trafficking, the present work may provide a foundation for such rationale targeting and advances our understanding of how intracellular bacteria can shape and protect their growth niche.