Dead Cell-associated Antigens Research Articles

Abstract 1540: The dendritic cell receptor DNGR-1 shapes immunity to cancer

Abstract Background: Central to the success of CD8+ T cell-mediated anti-cancer response are type 1 conventional dendritic cells (cDC1s), acting as professional antigen presenting cells. In both mouse and human, DNGR-1 (a.k.a. CLEC9A) is a dedicated cDC1 receptor that, upon binding to its physiological ligand filamentous-actin (F-actin) exposed upon cell death, prime cytotoxic CD8+ T cells specifically against dead cell-associated antigens. Here, we interrogate the role of DNGR-1 in shaping the tumor microenvironment during carcinogenesis. Methods: We utilized two models of chemical carcinogenesis: 3-methylcholanthrene (MCA)-induced sarcoma, and azoxymethane (AOM) and dextran sodium sulfate colitis (DSS)-associated carcinoma. Rag1−/- (no B and T cells), Clec9agfp/gfp, Clec9acre/cre and WT (wildtype) mice on C57BL/6 background were used. Primary MCA-sarcoma cell lines were generated and subsequently tested in a series of transplantation experiments in naïve syngeneic WT mice. Whole exome and RNA sequencing were performed to elucidate the mutational landscape and decipher the immunopeptidome of these primary cell lines. Results: Upon challenge with MCA, there is shorter latency for tumorigenesis in DNGR-1-deficient mice (n=46), like in Rag1−/− mice (n=43), in contrast to immunocompetent WT hosts (n=59) (median: 95 vs 113 days, hazard ratio: 1.72, 95% CI 1.11-2.68, p=0.008). DNGR-1-deficient mice also display increased susceptibility to AOM-DSS carcinogenesis compared to co-housed WT mice. Majority (n=16/24, 68%) of MCA-induced primary sarcoma cell lines derived from DNGR-1-deficient hosts are highly immunogenic and were controlled/rejected at a similar rate, upon challenge in secondary naïve WT recipients, like those derived from Rag1−/− mice (n=13/19, 68%). Whole-exome sequencing of cell lines derived from DNGR-1-deficient hosts revealed that they harbor high mutational burden, including mutations in F-actin-binding proteins that have been previously associated with better survival outcomes in patients with cancer (Giampazolias et al. Cell, 2021). Collectively, these data argue that DNGR-1 protects against carcinogenesis and selects for less immunogenic tumor cells. To evaluate the degree of immunoediting which occurred in the respective primary host mice, we applied the evolutionary metric based on the ratio of nonsynonymous to synonymous mutations (dN/dS), and found that they are less immunoedited compared to cell lines derived from WT hosts. Conclusion: Our findings highlight DNGR-1 as a novel immune checkpoint that couples cell death sensing and (neo-)antigen cross-presentation to functional tumor-specific CD8+ T cell responses during cancer immunoediting. Understanding the mechanism by which DNGR-1 sculpts the mutational landscape of tumors may further inform the dynamics of cancer evolution and may have translational therapeutic implications in directing effective immunotherapy strategies. Citation Format: Kok Haw Lim, Evangelos Giampazolias, Bruno Frederico, Neil C. Rogers, Probir Chakravarty, Gavin Kelly, Ana Cardoso, Emma Nye, Alejandro Suárez-Bonnet, Simon Priestnall, Robert Goldstone, Cell Services Science Technology Platform, Sonia Lee, Oliver Schulz, Jessica Strid, Luis Zapata, Caetano Reis e Sousa. The dendritic cell receptor DNGR-1 shapes immunity to cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1540.

SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cells

Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ Tcells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ Tcells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity.

Molecular determinants of immunogenic cell death elicited by radiation therapy.

Cancer cells undergoing immunogenic cell death (ICD) can initiate adaptive immune responses against dead cell-associated antigens, provided that (1) said antigens are not perfectly covered by central tolerance (antigenicity), (2) cell death occurs along with the emission of immunostimulatory cytokines and damage-associated molecular patterns (DAMPs) that actively engage immune effector mechanisms (adjuvanticity), and (3) the microenvironment of dying cells is permissive for the initiation of adaptive immunity. Finally, ICD-driven immune responses can only operate and exert cytotoxic effector functions if the microenvironment of target cancer cells enablesimmune cell infiltration and activity. Multiple forms of radiation, including non-ionizing (ultraviolet) and ionizing radiation, elicit bona fide ICD as they increase both the antigenicity and adjuvanticity of dying cancer cells. Here, we review the molecular determinants of ICD as elicited by radiation as we critically discuss strategies to reinforce the immunogenicity of cancer cells succumbing to clinically available radiation strategies.

IRE1α activation in Bone Marrow-Derived Dendritic Cells Modulates Innate Recognition of Melanoma Cells and Favors CD8+ T cell Priming

The IRE1α/XBP1s signaling pathway is an arm of the unfolded protein response (UPR) that safe guards the fidelity of the cellular proteome during endoplasmic reticulum (ER) stress. IRE1α has also emerged as a key regulator of dendritic cell (DC) homeostasis where it controls key features including the cross-presentation of dead cell-associated antigens. This aspect is highly applicable to the field of antitumor immunity, as representative tumor antigens are likely to be contained within dying cancer cells. Thus, the aim of this work is to elucidate the role of IRE1α signaling in innate recognition and antigen-presentation of melanoma-associated antigens to CD8+ T cells. We found that melanoma cell lysates (MEL) were potent activators of the IRE1α/ XBP1s pathway and additional members of the UPR including CHOP and BiP, in various cultures of bone marrow derived DCs (BMDCs). To assess the role of IRE1α in the recognition of MEL by BMDCs we used the aldehydes 4µ8C and STF-083010, two pharmacological inhibitors of the IRE1α RNAse domain. BMDCs stimulated with MEL in presence of 4µ8C produced lower levels of pro-inflammatory cytokines and showed curtailed abilities to cross-present MEL-associated antigens to CD8+ T cells. Remarkably, IRE1α blockade specifically decreased cross-presentation of MEL-associated antigens without interfering with the phagocytic capacity of DCs, endogenous MHC-I antigen presentation or presentation of tumor antigens via MHC-II. Furthermore, BMDCs expressing a mutant isoform IRE1α that lacks the RNase domain were less efficient at inducing CD8+ T cell proliferation to a melanoma-as-sociated antigen in vivo. Our data indicates that activation of the IRE1α/XBP1s axis in BMDCs promotes CD8+ T cell priming to melanoma antigens. Knowledge derived from this study may be considered in the design of DC-based vaccines for cancer immunotherapy.

Applying nanotechnology to boost cancer immunotherapy by promoting immunogenic cell death

Tumor immunotherapy, especially immune checkpoint blockade (ICB), has revolutionized the cancer field. However, the limited response of tumors to immunotherapy is a major obstacle. Tumor immunogenic cell death (ICD) is a death mode of tumor cells that can promote tumor immunity. ICD can induce strong antitumor immune responses through the ectopic exposure of calreticulin on the plasma membrane surface and the release of the non-histone nuclear protein high-mobility group box 1 (HMGB1), ATP, and interferon (IFN), thus activating an adaptive immune response against dead cell-associated antigens and enhancing the therapeutic effect of tumor immunotherapy. Chemotherapy, radiotherapy, photothermal therapy, magneto-thermodynamics therapy, nanopulse stimulation, and oncolytic virus therapy can all induce a strong antitumor immune response by ICD. In addition, the application of nanotechnology can precisely target drug delivery and improve the efficacy of immunotherapy. Here we introduce the basic concepts and molecular mechanisms underlying the induction of ICD. Then, we summarize and discuss the progress in the application of nanotechnology in immunotherapy to promote ICD. Finally, we attempt to define the challenges and future directions in this area to extend the benefits of ICD to a broader patient population.

Abstract 1636: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering “don't eat me” and “don't find me” signals. Recent literature demonstrates that C-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) and macrophages (MPs) is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from the human tumor microenvironment, in particular, tumor-associated MPs. We evaluated whether CLEC-1 could also be a receptor for DAMPs and influences phagocytosis. We found that CLEC-1 fusion protein binds specifically to secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray), or culture stress conditions suggesting that ligands of CLEC-1 are generated upon stress and programmed cell death. Importantly, further to promising results in KO CLEC-1 mice, we observed in vivo that CLEC-1 deficient mice, in contrast to wild-type littermates, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy. Importantly, disruption of CLEC-1 signaling by Fc-CLEC-1 fusion protein also promotes tumor eradication. We then generated and identified different anti-human CLEC-1 antagonist monoclonal antibodies (mAbs) with the capacity to block CLEC-1/CLEC-1L interaction. We developed innovative antagonist CLEC-1 mAbs which, in contrast to non-antagonist CLEC-1 control mAb, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs or MPs. Indeed, TGFβ-polarized DCs phagocytose more efficiently a NSCLC cell line (A549) as well as Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) when CLEC-1 is antagonized by Abs. Similarly, macrophages significantly more efficiently engulfed human tumors in the presence of CLEC-1 antagonist Abs, in particular when tumor cells were opsonized such as Rituximab-opsonized Raji cells, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) or Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+). Importantly, we observed both in vitro and in vivo that DCs from Clec1a deficient mice cross-present more efficiently dead cell-associated antigens to CD8+ T cells (OT-1). We generated hCLEC-1 knock-in mice and in vivo preclinical evaluation of CLEC-1 blocking mAbs is ongoing. Altogether, these data illustrate that CLEC-1 broadly notably inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors, and hampers DC antigen cross-presentation. Citation Format: Vanessa Gauttier, Sabrina Pengam, Marion Drouin, Javier Saenz, Bérangère Evrard, Kevin Biteau, Caroline Mary, Géraldine Teppaz, Ariane Desselle, Virginie Thépénier, Emmanuelle Wilhelm, Elise Chiffoleau, Nicolas Poirier. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1636.

Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity

SummaryCross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8+ T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8+ T cell responses.

DNGR-1 limits Flt3L-mediated antitumor immunity by restraining tumor-infiltrating type I conventional dendritic cells

BackgroundConventional type 1 dendritic cells (cDC1s) are central to antitumor immunity and their presence in the tumor microenvironment associates with improved outcomes in patients with cancer. DNGR-1 (CLEC9A) is a...

Abstract 3423: CLEC-1 suppress dendritic cell antigen presentation and is a novel myeloid immune checkpoint target for cancer immunotherapy

Abstract Recent literature demonstrates that tumor hijacks physiological mechanisms of C-type lectin receptors (CLRs) that normally restrain immune cell-mediated tissue damage to suppress myeloid cell activation and promotes immune escape. We previously demonstrated that the orphan CLR CLEC-1 expressed by myeloid cells, is enhanced by the immunosuppressive cytokine TGFβ and tempers downstream CD4+ Th1 and Th17 responses following sterile inflammation. Interestingly, we observed a high CLEC-1 expression by human CD11b+ myeloid cells from ovarian ascites and from in-vitro generated pro-tumoral M2 macrophages. Moreover, open-source data show that Clec1a is highly expressed by XCR1-expressing mouse dendritic cells (DC). As these cDC1 are specialized in cross-presentation of dead cell-associated antigens, we evaluated whether CLEC-1 could be a receptor of damaged cells and regulate anti-tumor immunity. Interestingly, we observed that both human and mouse CLEC-1 bind specifically to secondary necrotic cells. No binding was observed with viable, primary necrotic or with early apoptotic cells suggesting that the ligand(s) of CLEC-1 correspond(s) to Damage-Associated Molecular Pattern(s) induced by the cell death process. Then, to test if CLEC-1 in DCs influences cross-presentation of dead cell-associated antigens, we immunized WT and Clec1a KO mice with OVA-loaded dead cells and co-injected OVA-specific TCR transgenic CD8+ OT-I T cells. Impressively, we observed an increased proliferation of CD8+ T cells in the absence of CLEC-1, demonstrating that CLEC-1 in DCs tempers cross-presentation of dead cell-associated antigens. Interestingly, we observed in a model of subcutaneous MC38 colon carcinoma that the combination of the absence of CLEC-1 with cytotoxic and immunogenic chemotherapy (cyclophosphamide), reduces significantly the tumor growth and cures most of the mice. Similarly, in orthotopic models of mesothelioma (AK7) or hepatocarcinoma (Hepa1.6), we observed a prolongation of survival of Clec1a KO mice. Mechanistically, this was associated with an increase of effector CD69+ and of central memory CD44hi CD62Lhi CCR7hi CD4+ and CD8+ T cells in both liver and spleen. Furthermore, we observed a decrease in myeloid-derived suppressor cells. Besides, macrophages display a more mature phenotype relative to MHC class II expression. In addition, transcriptomics analysis performed in tumor-bearing liver of Clec1a KO mice revealed a profound decrease in CSF1 expression suggesting a defect in immunosuppressive myeloid cell recruitment. Moreover, we observed an increase in CCL17 and CCL7 expression, both known to activate CD8+ T cells through DCs and to enhance anti-tumor immunity. Taken together, these results suggest that CLEC-1 in DCs by acting as sensor of cell damage tempers immune response and represents a novel pharmacological target for cancer immunotherapy. Citation Format: Marion Drouin, Javier Saenz, Bérangère Evrard, Vanessa Gauttier, Géraldine Teppaz, Maria-Dolores Lopez-Robles, Cédric Louvet, Nicolas Poirier, Elise Chiffoleau. CLEC-1 suppress dendritic cell antigen presentation and is a novel myeloid immune checkpoint target for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3423.

DNGR-1, a Dendritic Cell-Specific Sensor of Tissue Damage That Dually Modulates Immunity and Inflammation.

DNGR-1 (encoded by CLEC9A) is a C-type lectin receptor (CLR) with an expression profile that is mainly restricted to type 1 conventional dendritic cells (cDC1s) both in mice and humans. This delimited expression pattern makes it appropriate for defining a cDC1 signature and for therapeutic targeting of this population, promoting immunity in mouse models. Functionally, DNGR-1 binds F-actin, which is confined within the intracellular space in healthy cells, but is exposed when plasma membrane integrity is compromised, as happens in necrosis. Upon F-actin binding, DNGR-1 signals through SYK and mediates cross-presentation of dead cell-associated antigens. Cross-presentation to CD8+ T cells promoted by DNGR-1 during viral infections is key for cross-priming tissue-resident memory precursors in the lymph node. However, in contrast to other closely related CLRs such as Dectin-1, DNGR-1 does not activate NFκB. Instead, recent findings show that DNGR-1 can activate SHP-1 to limit inflammation triggered by heterologous receptors, which results in reduced production of inflammatory chemokines and neutrophil recruitment into damaged tissues in both sterile and infectious processes. Hence, DNGR-1 reduces immunopathology associated with tissue damage, promoting disease tolerance to safeguard tissue integrity. How DNGR-1 signals are conditioned by the microenvironment and the detailed molecular mechanisms underlying DNGR-1 function have not been elucidated. Here, we review the expression pattern and structural features of DNGR-1, and the biological relevance of the detection of tissue damage through this CLR.

Methods to Detect Immunogenic Cell Death In Vivo.

In response to selected stressors, cancer cells can undergo a form of regulated cell death that-in immunocompetent syngeneic hosts-is capable of eliciting an adaptive immune response specific for dead cell-associated antigens. Thus, such variant of regulated cell death manifests with robust antigenicity and adjuvanticity. As compared to their normal counterparts, malignant cells are highly antigenic per se, implying that they express a variety of antigens that are not covered by central tolerance. However, the precise modality through which cancer cells die in response to stress has a major influence on adjuvanticity. Moreover, the adjuvanticity threshold to productively drive anticancer immune responses is considerably lower in tumor-naïve hosts as compared to their tumor-bearing counterparts, largely reflecting the establishment of peripheral tolerance to malignant lesions in the latter (but not in the former). So far, no cellular biomarker or combination thereof has been found to reliably predict the ability of cancer cell death to initiate antitumor immunity. Thus, although some surrogate biomarkers of adjuvanticity can be used for screening purposes, the occurrence of bona fide immunogenic cell death (ICD) can only be ascertained in vivo. Here, we describe two methods that can be harnessed to straightforwardly determine the immunogenicity of mouse cancer cells succumbing to stress in both tumor-naïve and tumor-bearing hosts.

Myosin II Synergizes with F-Actin to Promote DNGR-1-Dependent Cross-Presentation of Dead Cell-Associated Antigens

SummaryConventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1+ cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8+ T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8+ T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design.

Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster.

Damage-associated molecular patterns (DAMPs) are molecules released by dead cells that trigger sterile inflammation and, in vertebrates, adaptive immunity. Actin is a DAMP detected in mammals by the receptor, DNGR-1, expressed by dendritic cells (DCs). DNGR-1 is phosphorylated by Src-family kinases and recruits the tyrosine kinase Syk to promote DC cross-presentation of dead cell-associated antigens. Here we report that actin is also a DAMP in invertebrates that lack DCs and adaptive immunity. Administration of actin to Drosophila melanogaster triggers a response characterised by selective induction of STAT target genes in the fat body through the cytokine Upd3 and its JAK/STAT-coupled receptor, Domeless. Notably, this response requires signalling via Shark, the Drosophila orthologue of Syk, and Src42A, a Drosophila Src-family kinase, and is dependent on Nox activity. Thus, extracellular actin detection via a Src-family kinase-dependent cascade is an ancient means of detecting cell injury that precedes the evolution of adaptive immunity.

ID: 9: Dendritic cells in immunity to infection and cancer

Direct sensing of pathogen components is a major trigger of dendritic cell (DC) activation, leading to innate and adaptive immunity. Over the years, we have studied multiple pattern-recognition pathways that mediate DC activation. One pathway for sensing infection by RNA viruses involves recognition of viral genomes or virallyinfected cells in endosomal compartments and utilises members of the toll-like receptor (TLRs) family, including TLR9, 7, or 3. Viral genomes can additionally be recognised in the cytosol by DExD/H-box helicases such as RIG-I, which are activated by RNAs bearing 5’ tri- and di-phosphates. Finally, a distinct pathway involves cell surface and phagosomal recognition of fungi by C-type lectins, which signal via Syk kinase. Notably, we have recently found that some Syk-coupled C-type lectins are involved in functions other than microbial recognition by DC. One, CLEC-2, allows DCs to relax lymph node stromal cells, permitting expansion of lymph nodes upon inflammation. Another, DNGR-1, allows DCs to detect dead cells by binding exposed F-actin and facilitates cross-presentation of dead cell-associated antigens. Interestingly, DNGR-1 marks CD8 α + DCs, a specialised subtype of DCs in mice and their human equivalents. DNGR-1 additionally can be used to fate map DCcommitted precursors in mouse to define DCs by ontogeny. These studies help build a global picture of the receptors and signalling pathways that regulate DC activation and have applications in immunotherapy of cancer and infectious diseases.

Structure of the Complex of F-Actin and DNGR-1, a C-Type Lectin Receptor Involved in Dendritic Cell Cross-Presentation of Dead Cell-Associated Antigens

DNGR-1 is a C-type lectin receptor that binds F-actin exposed by dying cells and facilitates cross-presentation of dead cell-associated antigens by dendritic cells. Here we present the structure of DNGR-1 bound to F-actin at 7.7 Å resolution. Unusually for F-actin binding proteins, the DNGR-1 ligand binding domain contacts three actin subunits helically arranged in the actin filament, bridging over two protofilaments, as well as two neighboring actin subunits along one protofilament. Mutation of residues predicted to mediate ligand binding led to loss of DNGR-1-dependent cross-presentation of dead cell-associated antigens, formally demonstrating that the latter depends on F-actin recognition. Notably, DNGR-1 has relatively modest affinity for F-actin but multivalent interactions allow a marked increase in binding strength. Our findings shed light on modes of actin binding by cellular proteins and reveal how extracellular detection of cytoskeletal components by dedicated receptors allows immune monitoring of loss of cellular integrity.

Isolation of Splenic Dendritic Cells Using Fluorescence-activated Cell Sorting.

The spleen is a vastly vasculated organ and consists of a complex organized network of innate and adaptive immune cells. This permits the specialized functions of the spleen such as antibacterial and antifungal immunity and iron metabolism among others (Mebius and Kraal, 2005). Different dendritic cell (DC) subsets reside in the spleen and can be defined by the expression of unique surface markers. These DC subsets are recognized to perform non-redundant functions in the immune system (Merad et al., 2013). In our recent study, we found that Inositol Requiring Enzyme (IRE)-1 is specifically activated in splenic CD8a+ DCs. Furthermore, loss of X-box binding protein (XBP)-1 - the transcription factor regulated by IRE-1 - resulted in defective cross-presentation of dead cell associated antigens by splenic CD8a+ DCs (Osorio et al., 2014). This protocol allows the isolation of specific DC subsets for experimental use ex-vivo.

Tumor lysates elicit activation of the unfolded protein response sensor IRE-1α and the transcription factor XBP-1s in murine dendritic cells

Event Abstract Back to Event Tumor lysates elicit activation of the unfolded protein response sensor IRE-1α and the transcription factor XBP-1s in murine dendritic cells Cristóbal Costoya1, 2, Flavio Salazar-Onfray1, 2 and Fabiola Osorio1* 1 Universidad de Chile, Institute of Biomedical Sciences, Faculty of Medicine, Chile 2 Universidad de Chile, Millennium Institute on Immunology and Immunotherapy, Chile Preserving protein homeostasis is essential for several cellular processes including survival and adaptation to environmental cues. Noxious stimuli such as hypoxia, nutrient deprivation, pathogen recognition, low pH and toxins modify the rate of protein synthesis in the endoplasmic reticulum (ER). The imbalance between the burden of newly synthesized proteins and the capacity of the ER to handle this load causes ER stress. To prevent the detrimental effects of ER stress, eukaryotic cells have evolved an integrated signal transduction mechanism collectively referred to as the unfolded protein response (UPR), which aims to restore protein folding, increase the ER biosynthetic machinery and maintain protein homeostasis. The most conserved branch of the UPR is the signaling pathway controlled by the sensor IRE-1α and one of its substrates, the transcription factor XBP-1s. In the immune system, the IRE-1α/XBP-1s axis has emerged as a key regulator of DC function. We have recently reported that IRE-1α regulates the process of cross-presentation of dead cell-associated antigens, a key feature of DCs from the CD8α+ lineage. In addition XBP-1s, a transcription factor activated in response to IRE-1α-dependent splicing, maintains the homeostasis of the ER in these cells. So far, whereas IRE-1α and XBP-1s play a prominent role in DC biology, it has not been explored the role of this pathway in sensing innate stimuli in DCs. In this work, we have screened various compounds of immunological relevance as potential activators of the UPR in murine cultures of DCs generated in presence of FLT3-L, which is an in-vitro system that replicates the DC subtypes found in steady state mice. The stimuli tested are the allergen extracted from the house dust mite (HDM), lipopolysaccharide (LPS) derived from gram-negative bacteria and tumor lysates obtained from an allogenic mix of melanoma lines named TRIMEL. We found that of all of the stimuli, TRIMEL preferentially induced the activation of XBP-1s and the transcription of a reported target gene Erp44 in DCs. Furthermore; we observed that TRIMEL was also competent to activate additional UPR members including the ER-stress responsive chaperone BiP and the apoptosis regulator CHOP. Altogether, these results indicate that detection of tumor lysates by DCs results in activation of the IRE-1α/XBP-1s axis and the UPR. Ongoing work is currently carried out to elucidate the contribution of this pathway to DC activation, which may be of relevance in the context of cancer immunotherapy. Acknowledgements Financed by grant CONICYT, PAI, No 82130031 /016-F. Keywords: mouse model, tumor immunology, Dendritic Cells, XBP-1, IRE1α, er stress, UPR signaling pathways Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Tumor immunology Citation: Costoya C, Salazar-Onfray F and Osorio F (2015). Tumor lysates elicit activation of the unfolded protein response sensor IRE-1α and the transcription factor XBP-1s in murine dendritic cells. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00058 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 14 May 2015; Published Online: 14 Sep 2015. * Correspondence: PhD. Fabiola Osorio, Universidad de Chile, Institute of Biomedical Sciences, Faculty of Medicine, Santiago, - Other -, 8380453, Chile, fabiolaosorio@med.uchile.cl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Cristóbal Costoya Flavio Salazar-Onfray Fabiola Osorio Google Cristóbal Costoya Flavio Salazar-Onfray Fabiola Osorio Google Scholar Cristóbal Costoya Flavio Salazar-Onfray Fabiola Osorio PubMed Cristóbal Costoya Flavio Salazar-Onfray Fabiola Osorio Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Screening of novel immunogenic cell death inducers within the NCI Mechanistic Diversity Set

Immunogenic cell death (ICD) inducers can be defined as agents that exert cytotoxic effects while stimulating an immune response against dead cell-associated antigens. When initiated by anthracyclines, ICD is accompanied by stereotyped molecular changes, including the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, the lysosomal secretion of ATP during the blebbing phase of apoptosis, and the release of high mobility group box 1 (HMGB1) from dead cells. By means of genetically engineered human osteosarcoma U2OS cells, we screened the 879 anticancer compounds of the National Cancer Institute (NCI) Mechanistic Diversity Set for their ability to promote all these hallmarks of ICD in vitro. In line with previous findings from our group, several cardiac glycosides exhibit a robust propensity to elicit the major manifestations of ICD in cultured neoplastic cells. This screen pointed to septacidin, an antibiotic produced by Streptomyces fibriatus, as a novel putative inducer of ICD. In low-throughput validation experiments, septacidin promoted CRT exposure, ATP secretion and HGMB1 release from both U2OS cells and murine fibrosarcoma MCA205 cells. Moreover, septacidin-killed MCA205 cells protected immunocompetent mice against a re-challenge with living cancer cells of the same type. Finally, the antineoplastic effects of septacidin on established murine tumors were entirely dependent on T lymphocytes. Altogether, these results underscore the suitability of the high-throughput screening system described here for the identification of novel ICD inducers.

F-Actin Is an Evolutionarily Conserved Damage-Associated Molecular Pattern Recognized by DNGR-1, a Receptor for Dead Cells

Sterile inflammation can be initiated by innate immune recognition of markers of tissue injury termed damage-associated molecular patterns (DAMPs). DAMP recognition by dendritic cells (DCs) has also been postulated to lead to T cell responses to foreign antigens in tumors or allografts. Many DAMPs represent intracellular contents that are released upon cell damage, notably after necrosis. In this regard, we have previously described DNGR-1 (CLEC9A) as a DC-restricted receptor specific for an unidentified DAMP that is exposed by necrotic cells and is necessary for efficient priming of cytotoxic T cells against dead cell-associated antigens. Here, we have shown that the DNGR-1 ligand is preserved from yeast to man and corresponds to the F-actin component of the cellular cytoskeleton. The identification of F-actin as a DNGR-1 ligand suggests that cytoskeletal exposure is a universal sign of cell damage that can be targeted by the innate immune system to initiate immunity.

Correction: CpG Promotes Cross-Presentation of Dead Cell-Associated Antigens by Pre-CD8α+ Dendritic Dells

de Brito, C., M. Tomkowiak, R. Ghittoni, C. Caux, Y. Leverrier, and J. Marvel. 2011. CpG promotes cross-presentation of dead cell-associated antigens by pre-CD8α+ dendritic dells. J. Immunol . 186: [1503–1511][1]. In the title, the phrase “Dendritic Dells” should be “Dendritic Cells”.