cDC1 Dendritic Cells Research Articles

Abstract 6342: Anti-tumor in vivo efficacy of different classes of anti-CLEC-1 myeloid checkpoint antibodies in monotherapy and in combination with chemotherapeutic agents

Abstract The c-type lectin receptor CLEC-1 is a pattern recognition receptor [1] expressed by endothelial and myeloid cells in mice, non-human primates, and humans. While genetic deletion of CLEC-1 in mice does not lead to any developmental defect, CLEC-1 deletion or CLEC-1 targeting promotes anti-tumor response by increasing cross-presentation of necrotic or damaged cells by cDC1 dendritic cells and by enhancing T-cell activation [2]. To date, the identification of CLEC-1 endogenous ligand(s) and their biological activity into myeloid or endothelial cells remained to be fully investigated. We aimed to further characterize ligands that are involved into CLEC-1 activation and develop monoclonal antagonist anti CLEC-1 antibody anti-tumor therapy that are able fully counteract inhibitory signaling induced into myeloid cells. Using LC/MS campaign, we confirmed CLEC-1 specific binding to the E3 ubiquitin ligase TRIM21and to the secreted histidine rich glycoprotein (HRG) as previously described [2] and also identify several novel intra-cellular and cell surface proteins that bind to CLEC-1 receptor in protein specific-manner. To block interaction of CLEC-1 ligands, we developed different class of antagonist monoclonal antibodies and evaluated their anti-tumor efficacy in humanized CLEC-1 KI orthotopic and ectopic tumor mouse models fully resistant or partially resistant to ICI therapy. While blocking of CLEC-1 binding to its secreted ligand HRG moderately increases anti-tumor responses, inhibition of CLEC-1 binding to its cytoplasmic and membrane ligands significantly impairs MC38 tumor growth (n=12, p=0.04). Importantly, high anti-tumor efficacy was observed in combination therapy with cyclophosphamide in MC38 mouse model. Additionally, antagonist anti-CLEC-1 antibodies significantly increase overall survival of Hepa1.6-bearing mice (n=32 for each treatment, p<0.002), as compared to corresponding isotype control treatment in 3 independent experiments. Altogether, our results further dissect the mechanism of action of the myeloid checkpoint CLEC-1 in its ability to impair anti-tumor immunity and support its use as a novel and highly promising anti-CLEC antagonist antibody for cancer immunotherapy. Citation Format: Aurore Morello, Mylène Deramé, Marion Drouin, Emmanuelle Wilhelm, Stéphanie Neyton, Irène Baccelli, Caroline Mary, Vanessa Gauttier, Isabelle Girault, Kévin Biteau, Cécile Batty, Géraldine Teppaz, Ariane Desselle, Marion Colonello, Marine Malloci, Elise Chiffoleau, Nicolas Poirier. Anti-tumor in vivo efficacy of different classes of anti-CLEC-1 myeloid checkpoint antibodies in monotherapy and in combination with chemotherapeutic agents [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 6342.

Lung cDC1 and cDC2 dendritic cells priming naive CD8+ T cells in situ prior to migration to draining lymph nodes

The current paradigm indicates that naive Tcells are primed in secondary lymphoid organs. Here, we present evidence that intranasal administration of peptide antigens appended to nanofibers primes naive CD8+ Tcells in the lung independently and prior to priming in the draining mediastinal lymph node (MLN). Notably, comparable accumulation and transcriptomic responses of CD8+ Tcells in lung and MLN are observed in both Batf3KO and wild-type (WT) mice, indicating that, while cDC1 dendritic cells (DCs) are the major subset for cross-presentation, cDC2 DCs alone are capable of cross-priming CD8+ Tcells both in the lung and draining MLN. Transcription analyses reveal distinct transcriptional responses in lung cDC1 and cDC2 to intranasal nanofiber immunization. However, both DC subsets acquire shared transcriptional responses upon migration into the lymph node, thus uncovering a stepwise activation process of cDC1 and cDC2 toward their ability to cross-prime effector and functional memory CD8+ Tcell responses.

Intratumoral Gene Transfer of mRNAs Encoding IL12 in Combination with Decoy-Resistant IL18 Improves Local and Systemic Antitumor Immunity.

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Depletion of Conventional Type-1 Dendritic Cells in Established Tumors Suppresses Immunotherapy Efficacy.

These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.

Abstract 6216: Intratumoral B0112 in combination with radiotherapy synergizes to achieve CD8 T cell mediated local tumor control

Abstract Background: Radioimmunotherapy combines irradiation of tumor lesions with immunotherapy to achieve local and abscopal control of cancer. Most immunotherapy agents are given systemically, but strategies for delivering immunotherapy locally are under clinical scrutiny to maximize efficacy and avoid toxicity. Local immunotherapy by injecting various patterns of molecules shows efficacy both preclinical and clinically. BO112 is a viral mimetic based on nanoplexed double stranded RNA (poly IC) which exerts immune-mediated antitumor effects in mice and humans upon intratumoral delivery. BO112 and external beam irradiation were used to make the proof-of-concept local immunotherapy plus radiation therapy combinations. Methods: Murine transplantable tumor cells lines (TSA, MC38 and B16 ova) were used to show increased immunogenic features under irradiation as well as bilateral tumor models in which only one of the lesions could be irradiated. Flow cytometry on cell suspensions from tumor draining lymph nodes and multiplex tissue immunofluorescence were used to determine mechanims of antitumor immunity. Depletions of immune cell populations and knock out mice for IFNAAR and BAFT3 genes were used to delineate the immune system requirements for efficacy. Results: In cultures of TSA breast cancer cells the combination of irradiation and BO112 showed more prominent features of immunogenic tumor-cell death including type I interferon reléase. Injection of BO112 in the tumor lesions under radiation achieved excellent control of the treated tumors whilst modest delays in contralateral tumor progression were observed. Local effects were associated with more prominent presence of antitumor CTLs and cDC1 dendritic cells in the tumor and regional lymph nodes. Importantly, local irradiation plus BO112 in a tumor lesion enhanced the therapeutic effect of radiotherapy on distant uninjected tumor lesion. Conclusions: This study demonstrates that local BO112 immunotherapy and external beam radiation therapy can exert synergies to achieve local tumor control. This beneficial effect can be extended to non-injected lesions as long as they are also irradiated, suggesting strategies to treat oligometastatic patients with lesions susceptible to radiotherapy and at least one of them accessible for BO112 repeated intratumoral injections. Citation Format: Maria.E. Rodriguez-Ruiz, Irantzu serrano, Eneko Garate, Inmaculada Rodriguez, maite alvarez, Carmen Molina, Miguel Fernandez de Sanmamed, Leire Arbea, Marta Moreno, Marisol Quintero, Ignacio Melero. Intratumoral B0112 in combination with radiotherapy synergizes to achieve CD8 T cell mediated local tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6216.

NF-κB-dependent IRF1 activation programs cDC1 dendritic cells to drive antitumor immunity.

Conventional type 1 dendritic cells (cDC1s) are critical for antitumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8+ T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the antitumor functions of cDC1s in immunogenic tumors are poorly understood. Using single-cell transcriptomics to examine the molecular pathways regulating intratumoral cDC1 maturation, we found nuclear factor κB (NF-κB) and interferon (IFN) pathways to be highly enriched in a subset of functionally mature cDC1s. We identified an NF-κB-dependent and IFN-γ-regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. By mapping the trajectory of intratumoral cDC1 maturation, we demonstrated the dynamic reprogramming of tumor-infiltrating cDC1s by NF-κB and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-κB or IFN regulatory factor 1 (IRF1) in cDC1s, resulting in impaired expression of IFN-γ-responsive genes and consequently a failure to efficiently recruit and activate antitumoral CD8+ T cells. Last, we demonstrate the relevance of these findings to patients with melanoma, showing that activation of the NF-κB/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-κB/IRF1 axis in cDC1s may therefore represent an important focal point for the development of new diagnostic and therapeutic approaches to improve cancer immunotherapy.

Abstract 697: Defining anti-tumor immune stimulatory mechanisms of MEM-288, a CD40 ligand and IFN-beta dual-transgene armed oncolytic adenovirus

Abstract MEM-288 is a dual-transgene armed oncolytic adenovirus being developed for cancer treatment as a standalone agent or in combination with immune checkpoint inhibitors (ICI). MEM-288 is armed with two potent immune agonist transgenes: MEM40, a novel chimeric CD40 ligand (CD40L) designed for stable cell surface expression of CD40L that functions as a potent activator of CD40 expressing dendritic cells (DCs), and the DC and T cell activating cytokine IFNβ. We have evaluated MEM-288 activity in multiple in vivo immunocompetent mouse models to determine impact on systemic antitumor T cell activity after intratumoral virus injection. We were able to readily detect both tumor and immune cell infection and transgene expression following MEM-288 intratumoral administration in mouse tumor models and freshly resected human NSCLC tumors. MEM-288 injection in the syngeneic mouse lung metastatic model 344SQ demonstrated anti-metastatic activity that correlated with a significant enhancement in tumor infiltrating lymphocytes (TILs) and an increase in the systemic antitumor CD8 T cell response. MEM-288 also generated a highly efficacious response against both injected and distant non-injected contralateral tumors in the B16-F10 melanoma model, both as a monotherapy and in combination with ICI. These antitumor and immune responses were only found with MEM-288 as compared to the base adenovirus vector encoding only GFP transgene, indicating the observed effects were highly transgene-dependent. Dissecting the immune stimulatory effects further, we found MEM-288 injection in B16 tumors led to a significant increase in DC activation markers CD80 and CD86 suggesting that DCs may represent a key target of MEM-288 encoded transgenes leading to systemic T cell responses. In support of this hypothesis, we found the MEM-288 CD8 T cell response was completely abolished in BATF3 knockout (KO) mice which lack the cDC1 DC subset (CD8α conventional DC deficient). This suggests that cDC1s are a key mediator of MEM-288 activity. Furthermore, MEM-288 activity was also impaired in both CD40 and type 1 IFN receptor (IFNAR1) KO mice suggesting that both transgenes mediate MEM-288 immune stimulation. In summary, these results demonstrate MEM-288's capacity to induce a potent increase in systemic T cell responses through transgene-mediated stimulation of tumor DCs. This immune stimulatory capacity complements the additional mechanistic features of MEM-288, including enhanced tumor-specific viral replication, oncolysis, and tumor antigen release. MEM-288 is currently being prepared for first-in-human clinical testing in solid tumors as a monotherapy and in combination with ICI. Citation Format: Hong Zheng, Chase D. Powell, Scott Antonia, Mark J. Cantwell, Bradford A. Perez, Amer A. Beg. Defining anti-tumor immune stimulatory mechanisms of MEM-288, a CD40 ligand and IFN-beta dual-transgene armed oncolytic adenovirus [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 697.

Abstract 1698: Cellular cytotoxicity is a form of immunogenic cell death

Abstract Background: The immune response to cancer is often conceptualized with the Cancer Immunity Cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or NK lymphocytes, is actually immunogenic currently remains unknown. Methods: In this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated natural killer cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I MHC-multimer stainings. Mice deficient in BATF-3, IFNAR and STING were used to study mechanistic requirements. Results: We observe in co-cultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. OVA-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100 specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+ EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient in BATF3-dependent dendritic cells (cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+ T lymphocytes. Conclusion: These results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death. Citation Format: Luna Minute, Alvaro Teijeira, Alfonso Rodriguez Sánchez-Paulete, Maria del Carmen Ochoa, Maite Alvarez, Itziar Otano, Iñaki Etxeberria, Elixabet Bolaños, Arantza Azpilikueta, Saray Garasa, Noelia Casares, José Luiz Pérez-Gracia, Maria Esperanza Rodríguez-Ruiz, Pedro Berraondo, Ignacio Melero. Cellular cytotoxicity is a form of immunogenic cell death [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 1698.

NCoR1 fine-tunes type-I IFN response in cDC1 dendritic cells by directly regulating Myd88-IRF7 axis under TLR9.

Plasmacytoid dendritic cells (DCs) are reported to induce robust type-I interferon (IFN) response, whereas cDC1 DCs develop moderate type-I IFN response upon TLR9 stimulation. It is very interesting to understand how this signaling under TLR9 is tightly regulated for the induction of type-I IFNs. Here, we report co-repressor protein NCoR1 as the major factor fine-tuning the signaling pathways regulating IFN-β expression under TLR9 in cDC1 DCs. We found that NCoR1 knockdown induced a robust IFN-β-mediated antiviral response upon TLR9 activation in cDC1 DCs. At the molecular level, we showed that NCoR1 directly repressed MyD88-IRF7 signaling axis in cDC1 cells. Therefore, NCoR1 depletion enhanced pIRF7 levels, IFN-β secretion, and downstream pSTAT1-pSTAT2 signaling, leading to sustained induction of IFN stimulatory genes. Integrative genomic analysis depicted strong enrichment of an antiviral gene-module in CpG-activated NCoR1 knockdown DCs upon TLR9 activation. Moreover, we confirmed our findings in primary DCs derived from splenocytes of WT and NCoR1 DC-/- animals, which showed protection from Sendai and Vesicular Stomatitis viruses upon CpG activation. Ultimately, we identified that NCoR1-HDAC3 complex is involved in repressing the type-I IFN response in cDC1 DCs.

Absence of MHC class II on cDC1 dendritic cells triggers fatal autoimmunity to a cross-presented self-antigen.

Conventional dendritic cells expressing the XCR1 chemokine receptor (cDC1s) excel at cross-presentation. Here, we developed and used a mouse model in which a Cre recombinase is expressed under the control of the Xcr1 gene while preserving XCR1 expression. We used it to generate mice with conditional deletion of MHC class II (MHCII) molecules on cDC1s. By preventing cDC1s to receive suppressive regulatory T cell inputs via MHCII-restricted interactions, the objective of the present study was to gauge whether MHCII-deficient cDC1s lose their capacity of tolerizing autoreactive CD8+ T cells. Whereas MHCII+ cDC1 readily cross-tolerized strongly autoreactive CD8+ T cells specific for a keratinocyte-derived self-antigen, MHCII-deficient cDC1s converted them into potent effectors capable of triggering a fast-onset lethal autoimmunity associated with severe skin histopathological manifestations. Preventing egress of such pathogenic self-reactive CD8+ T cell effectors from the cutaneous draining lymph nodes abrogated the autoimmune condition. Therefore, our results revealed that the cross-tolerizing capacity of cDC1s is not a property fully acquired at the time they undergo homeostatic maturation but needs to be enforced via MHCII-restricted, suppressive interactions with regulatory T cells.

Cellular cytotoxicity is a form of immunogenic cell death

BackgroundThe immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic...

Intratumor Adoptive Transfer of IL-12 mRNA Transiently Engineered Antitumor CD8+ T Cells

Retroviral gene transfer of interleukin-12 (IL-12) into Tcells markedly enhances antitumor efficacy upon adoptive transfer but has clinically shown unacceptable severe side effects. To overcome the toxicity, we engineered tumor-specific CD8+ Tcells to transiently express IL-12. Engineered Tcells injected intratumorally, but not intravenously, led to complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced by co-injection with agonist anti-CD137 mAb or by transient co-expression of CD137 ligand. This treatment induced epitope spreading of the endogenous CD8+ Tcell immune response in a manner dependent on cDC1 dendritic cells. Mouse and human tumor-infiltrating Tlymphocyte cultures can be transiently IL-12 engineered to attain marked immunotherapeutic effects.

Abstract 2331: Intratumor adoptive transfer of IL-12 mRNA transiently engineered anti-tumor CD8+ T cells

Abstract Adoptive T-cell therapy with TILs, CAR-T cells, and TCR-engineered T-cells is achieving remarkable clinical efficacy. Retroviral gene transfer of IL-12 into the T cell cultures markedly enhances efficacy but has shown unacceptable severe side effects in the clinic. To ensure transient gene transfer, tumor specific CD8 T cells were engineered by electroporation of mRNA encoding single chain IL-12 into OT1 and Pmel-1 TCR transgenic lymphocytes. Engineered T cells were injected intratumorally and achieved complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced if a small dose of agonist anti-CD137 mAb was co-injected or if CD137-ligand mRNA was co-electroporated. Importantly, intratumor T-cell administration was more efficacious that the intravenous or subcutaneous routes. Therapeutic effects were dependent on IFNγ, cDC1 dendritic cells and endogenous T cells. Interestingly, treatment induced epitope spreading of the immune response to antigens not recognized by the adoptively transferred lymphocytes. Efficacy was also achieved when mouse TILs cultures were used and human TIL cultures can also be transiently IL-12 mRNA engineered. Intratumor release, transient IL-12 mRNA-engineering and repeated administration jointly constitute a safe, feasible and powerful cancer immunotherapy strategy. Note: This abstract was not presented at the meeting. Citation Format: Inaki Etxeberria, Elixabet Bolaños, Alvaro Teijeira, Arantza Azpilicueta, Jose Ignacio Quetglas, Alfonso Rodriguez Sanchez-Paulete, Itziar Otano, Uxua Mancheño, Sandra Hervas-Stubbs, Susana Inoges, Saray Garasa, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Intratumor adoptive transfer of IL-12 mRNA transiently engineered anti-tumor CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2331.