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Dendritic cells (DCs) can process the antigens of cancer vaccine and thus stimulate the CD8+ T cells to recognize and kill the tumor cells that express these antigens. However, lack of promising carriers for presenting the antigens to DCs is one of the main barriers to the development of clinically effective cancer vaccines. Another limitation is the risk of inflammatory side effects induced by the adjuvants. It is still unclear how we can develop ideal adjuvant-free DC vaccine carriers without adjuvants. Methods: A 12-mer peptide carrier (CBP-12) with high affinity for Clec9a expressed on DCs was developed using an in silico rational optimization method. The therapeutic effects of the adjuvant-free vaccine comprising CBP-12 and exogenous or endogenous antigenic peptides were investigated in terms of antigen cross-presentation efficacy, specific cytotoxic T lymphocyte response, and antitumor activity. We also explored the mechanism involved in the antitumor effects of the adjuvant-free CBP-12 vaccine. Finally, we assessed the effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Results: Here, we developed CBP-12 as a vaccine carrier that enhanced the uptake and cross-presentation of the antigens, thus inducing strong CD8+ T cell responses and antitumor effects in both anti-PD-1-responsive (B16-OVA) and -resistant (B16) models, even in adjuvant-free conditions. CBP-12 bound to and activated Clec9a, thereby stimulating Clec9a+ DC to product IL-21, but not IL-12 by activating of Syk. The antitumor effects of the CBP-12 conjugated peptide vaccines could be blocked by an IL-21 neutralizing antibody. We also observed the synergistic antitumor effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Conclusions: CBP-12 could serve as an adjuvant-free peptide vaccine carrier for cancer immunotherapy.

Conventional 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 dead cell-sensing receptor highly restricted to cDC1s. DNGR-1 has been involved in both cross-presentation of dead cell-associated antigens and processes of disease tolerance, but its role in antitumor immunity has not been clarified yet.

Summary Conventional 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., Graphical Abstract, Highlights • Myosin II amplifies the activity of the DNGR-1 ligand F-actin • Lack of myosin II in donor cells reduces DNGR-1-dependent cross-presentation • Beads with F-actin and myosin II can target antigens to cDC1 for CD8 T cell priming, Schulz et al. show that dead cells lacking myosin II have a diminished capacity to serve as antigen donors for dendritic cells that express the DNGR-1 receptor. DNGR-1 binds to F-actin exposed on cell corpses, and myosin II organizes actin filaments into bundles that cross-link the receptor more efficiently.

Summary Cross-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., Graphical abstract, Highlights • Secreted gelsolin (sGSN) inhibits DNGR-1 binding to F-actin • sGSN dampens DNGR-1-dependent cross-presentation of dead cell-associated antigens • sGSN impairs DNGR-1-dependent cDC1-mediated anti-tumor immunity • Low sGSN expression and mutations in FABPs correlate with cancer patient survival, The secreted gelsolin component of the plasma actin-scavenging system impairs the ability of the receptor DNGR-1 to recognize dead cells and selectively dampens cross-presentation of tumor antigens by type 1 dendritic cells, acting as a barrier to anti-tumor immunity.

// Zhongyi Yan 1 , Yahong Wu 1 , Jiangfeng Du 1 , Guodong Li 1 , Shengdian Wang 2 , Wenpeng Cao 1 , Xiuman Zhou 1 , Chunjing Wu 1 , Dan Zhang 1 , Xueli Jing 1 , Yifan Li 1 , Hongfei Wang 1 , Yanfeng Gao 1, 3 , Yuanming Qi 1, 3 1 School of Life Sciences, Zhengzhou University, Zhengzhou, China 2 Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 3 Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou, China Correspondence to: Yanfeng Gao, email: gaoyf@zzu.edu.cn Yuanming Qi, email: qym@zzu.edu.cn Keywords: Clec9a, dendritic cells, cancer immunotherapy, peptide Received: February 11, 2016 Accepted: May 09, 2016 Published: May 26, 2016 ABSTRACT Dendritic cells (DCs) are professional antigen-presenting cells with antigen recognition molecules on the surface. Clec9a is selectively expressed on mouse CD8a + DCs and CD103 + DCs subsets, which are functionally similar to human BDCA3 + DCs. It is reported that Clec9a is responsible for the antigen cross-presentation of these DC subsets. In the present study, by using phage display technique, we discovered a novel peptide WH, which can selectively bind to mouse Flt3L induced Clec9a + DCs or Clec9a over-expressed HEK-293T cells. Furthermore, by using computer-aided docking model and mutation assay, we observed that Asp 248 and Trp 250 are two key residues for Clec9a to bind with peptide WH. When coupled with OVA 257-264 epitope, peptide WH can significantly enhance the ability of Clec9a + DCs to activate OVA-specific CD8 + T cells, which elicit strong ability to secret IFN-γ, express perforin and granzyme B mRNA. In B16-OVA lung metastasis mouse model, WH-OVA 257-264 fusion peptide can also enhance the activation of CD8 + T cells and decrease the lung metastasis loci. All these results suggested that peptide WH could be considered as an antigen delivery carrier targeting Clec9a + DCs for cancer immunotherapy.

International audience; Type 1 conventional DCs (cDC1) excel in the cross-priming of CD8(+) T cells, which is crucial for orchestrating efficient immune responses against viruses or tumors. However, our understanding of their physiological functions and molecular regulation has been limited by the lack of proper mutant mouse models allowing their conditional genetic targeting. Because the Xcr1 and A530099j19rik (Karma/Gpr141b) genes belong to the core transcriptomic fingerprint of mouse cDC1, we used them to engineer two novel Cre-driver lines, the Xcr1(Cre) and Karma(Cre) mice, by knocking in an IRES-(Cre) expression cassette into their 3'-UTR. We used genetic tracing to characterize the specificity and efficiency of these new models in several lymphoid and non-lymphoid tissues, and compared them to the Clec9a(Cre) mouse model, which targets the immediate precursors of cDCs. Amongst the three Cre-driver mouse models examined, the Xcr1(Cre) model was the most efficient and specific for the fate mapping of all cDC1, regardless of the tissues examined. The Karma(Cre) model was rather specific for cDC1 when compared with the Clec9a(Cre) mouse, but less efficient than the Xcr1(Cre) model. Unexpectedly, the Xcr1(Cre) model targeted a small fraction of CD4(+) T cells, and the Karma(Cre) model a significant proportion of mast cells in the skin. Importantly, the targeting specificity of these two mouse models was not changed upon inflammation. A high frequency of germline recombination was observed solely in the Xcr1(Cre) mouse model when both the Cre and the floxed alleles were brought by the same gamete irrespective of its gender. Xcr1, Karma, and Clec9a being differentially expressed within the cDC1 population, the three CRE-driver lines examined showed distinct recombination patterns in cDC1 phenotypic subsets. This advances our understanding of cDC1 subset heterogeneity and the differentiation trajectory of these cells. Therefore, to the best of our knowledge, upon informed use, the Xcr1(Cre) and Karma(Cre) mouse models represent the best tools currently reported to specifically and faithfully target cDC1 in vivo, both at steady state and upon inflammation. Future use of these mutant mouse models will undoubtedly boost our understanding of the biology of cDC1.

Summary The ability to generate large numbers of distinct types of human dendritic cells (DCs) in vitro is critical for accelerating our understanding of DC biology and harnessing them clinically. We developed a DC differentiation method from human CD34+ precursors leading to high yields of plasmacytoid DCs (pDCs) and both types of conventional DCs (cDC1s and cDC2s). The identity of the cells generated in vitro and their strong homology to their blood counterparts were demonstrated by phenotypic, functional, and single-cell RNA-sequencing analyses. This culture system revealed a critical role of Notch signaling and GM-CSF for promoting cDC1 generation. Moreover, we discovered a pre-terminal differentiation state for each DC type, characterized by high expression of cell-cycle genes and lack of XCR1 in the case of cDC1. Our culture system will greatly facilitate the simultaneous and comprehensive study of primary, otherwise rare human DC types, including their mutual interactions., Graphical Abstract, Highlights • A CD34+ cell culture protocol yields large numbers of human pDCs and cDC1/2s • Notch signaling is critical for cDC1 generation and GM-CSF has a synergistic effect • scRNAseq confirms homology of in-vitro-derived DC types to their blood counterparts • CLEC9A-positive XCR1-negative cells were identified as immediate precursors of cDC1s, Balan et al. report a protocol to simultaneously generate large numbers of human pDCs, cDC1s, and cDC2s from cord blood and non-mobilized CD34+ progenitors. This culture system will enable experimental testing of mechanisms controlling the differentiation or functions of human DC types and their translational application to treat cancer.

Type 1 conventional CD103+ dendritic cells (cDC1) contribute significantly to the cytotoxic T lymphocyte (CTL) response during influenza virus infection; however, the mechanisms by which cDC1s promote CTL recruitment and viral clearance are unclear. We demonstrate that cDC1 ablation leads to a deficient influenza-specific primary CD8+ T cell response alongside severe pulmonary inflammation, intensifying susceptibility to infection. The diminished pulmonary CTL population is not only a consequence of reduced priming in the lymph node (LN), but also of dysregulated CD8+ T cell egression from the LN and reduced CD8+ T cell viability in the lungs. cDC1s promote S1PR expression on CTLs, a key chemokine receptor facilitating CTL LN egress, and express high levels of the T cell survival cytokine, IL-15, to support CTL viability at the site of infection. Moreover, cDC1 ablation leads to severe impairment of CD8+ T cell memory recall and cross-reactive protection, suggesting that cDC1 are not only involved in primary T cell activation, but also in supporting the development of effective memory CD8+ T cell precursors. Our findings demonstrate a previously unappreciated and multifaceted role of CD103+ DCs in controlling pulmonary T cell-mediated immune responses. MOE (Min. of Education, S’pore) Published version

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair. In addition, antigens present in necrotic cells can sometimes provoke a specific immune response and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection. In the mouse, the CD8alpha+ subset of dendritic cells phagocytoses dead cell remnants and cross-primes CD8+ T cells against cell-associated antigens. Here we show that CD8alpha+ dendritic cells use CLEC9A (also known as DNGR-1), a recently-characterized C-type lectin, to recognize a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair the uptake of necrotic cell material by CD8+ dendritic cells, but specifically reduces cross-presentation of dead-cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue in its intracellular tail that allows the recruitment and activation of the tyrosine kinase SYK, which is also essential for cross-presentation of dead-cell-associated antigens. Thus, CLEC9A functions as a SYK-coupled C-type lectin receptor to mediate sensing of necrosis by the principal dendritic-cell subset involved in regulating cross-priming to cell-associated antigens.

International audience; Innate immunity is an arsenal of molecules and receptors expressed by professional phagocytes, glial cells and neurons and involved in host defence and clearance of toxic and dangerous cell debris. However, any uncontrolled innate immune responses within the central nervous system (CNS) are widely recognized as playing a major role in the development of autoimmune disorders and neurodegeneration, with multiple sclerosis (MS) and Alzheimer's diseases (AD) being primary examples. Critically, neuroimmune regulatory proteins (NIReg) may control the adverse immune responses in health and diseases. NIRegs are found mainly on neurons, glia, endothelia and ependymal cells and include GPI-anchored molecules (CD24, CD90, complement regulators CD55 and CD59), molecules of the immunoglobulin superfamily (siglec CD22, Siglec 10, CD200, ICAM-5) and others (CD47, fractalkine, TAM receptor tyrosine kinase and complement C3a and factor H). These regulators modulate the innate immune response in the CNS and for instance critically control the level of phagocytosis and inflammation engaged by resident microglia and infiltrating immune cells. Others will sequester and neutralize proinflammatory molecules such as HMGB1 and DNA. Moreover, some NIRegs can instigate the recruitment of stem cells to mediate tissue repair. In the absence of these regulators, when neurons die by apoptosis, become infected or damaged, microglia and infiltrating immune cells are free to cause injury and an adverse inflammatory response in acute and chronic settings. The therapeutic applications of NIRegs should be exploited given their natural and selective healing properties.

DC NK lectin group receptor-1 (DNGR-1, also known as CLEC9A) is a C-type lectin receptor expressed by mouse CD8alpha+ DC and by their putative equivalents in human. DNGR-1 senses necrosis and regulates CD8+ T-cell cross-priming to dead-cell-associated antigens. In addition, DNGR-1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8+ T-cell and Ab responses. In this study, we evaluated whether DNGR-1 targeting can be additionally used to manipulate antigen-specific CD8+ T lymphocytes. Injection of small amounts of antigen-coupled anti-DNGR-1 mAb into mice promoted MHC class II antigen presentation selectively by CD8alpha+ DC. In the steady state, this was sufficient to induce proliferation of antigen-specific naïve CD4+ T cells and to drive their differentiation into Foxp3+ regulatory lymphocytes. Co-administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4+ T-cell behavior: poly I:C induced a strong IL-12-independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR-1 is a versatile approach for inducing functionally distinct CD4+ T-cell responses. Given the restricted pattern of expression of DNGR-1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.

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. FC was a recipient of a PhD La Caixa fellowship (LCF/BQ/ES14/10320011). CF was supported by AECC Foundation (INVES192DELF). Work in the DS laboratory was funded by the CNIC; by the European Research Council (ERC-2016-Consolidator Grant 725091); by the European Commission (635122-PROCROP H2020); by Ministerio de Ciencia, Innovación e Universidades (MICINN), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-79040-R); by Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); by FIS-Instituto de Salud Carlos III, MICINN and FEDER (RD16/0015/0018-REEM); by Acteria Foundation; by Atresmedia (Constantes y Vitales prize); and by Fundació La Marató de TV3 (201723). The CNIC was supported by the Instituto de Salud Carlos III (ISCIII), the MICINN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Sí