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1
Spreu, Jessica, Eike C. Kienle, Birgit Schrage, and Alexander Steinle. "CLEC2A: a novel, alternatively spliced and skin-associated member of the NKC-encoded AICL–CD69–LLT1 family." Immunogenetics 59, no. 12 (November 29, 2007): 903–12. http://dx.doi.org/10.1007/s00251-007-0263-1.
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Gonçalves-Maia, Maria, Yannick Gache, Miguel Basante, Estelle Cosson, Emie Salavagione, Margot Muller, Françoise Bernerd, et al. "NK Cell and Fibroblast-Mediated Regulation of Skin Squamous Cell Carcinoma Invasion by CLEC2A Is Compromised in Xeroderma Pigmentosum." Journal of Investigative Dermatology 140, no. 9 (September 2020): 1723–32. http://dx.doi.org/10.1016/j.jid.2020.01.021.
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Elleisy, Nagi, Sarah Rohde, Astrid Huth, Nicole Gittel, Änne Glass, Steffen Möller, Georg Lamprecht, Holger Schäffler, and Robert Jaster. "Genetic association analysis of CLEC5A and CLEC7A gene single-nucleotide polymorphisms and Crohn’s disease." World Journal of Gastroenterology 26, no. 18 (May 14, 2020): 2194–202. http://dx.doi.org/10.3748/wjg.v26.i18.2194.
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Araúzo-Bravo, Marcos J., Denis Delic, Daniela Gerovska, and Frank Wunderlich. "Protective Vaccination Reshapes Hepatic Response to Blood-Stage Malaria of Genes Preferentially Expressed by NK Cells." Vaccines 8, no. 4 (November 13, 2020): 677. http://dx.doi.org/10.3390/vaccines8040677.
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The role of natural killer (NK) cells in the liver as first-line post infectionem (p.i.) effectors against blood-stage malaria and their responsiveness to protective vaccination is poorly understood. Here, we investigate the effect of vaccination on NK cell-associated genes induced in the liver by blood-stage malaria of Plasmodium chabaudi. Female Balb/c mice were vaccinated at weeks 3 and 1 before being infected with 106P. chabaudi-parasitized erythrocytes. Genes preferentially expressed by NK cells were investigated in livers of vaccination-protected and non-protected mice on days 0, 1, 4, 8, and 11 p.i. using microarrays, qRT-PCR, and chromosome landscape analysis. Blood-stage malaria induces expression of specific genes in the liver at different phases of infection, i.e., Itga1 in expanding liver-resident NK (lrNK) cells, Itga2 in immigrating conventional NK (cNK) cells; Eomes and Tbx21 encoding transcription factors; Ncr1, Tnfsf10, Prf1, Gzma, Gzmb, Gzmc, Gzmm, and Gzmk encoding cytolytic effectors; natural killer gene complex (NKC)-localized genes encoding the NK cell receptors KLRG1, KLRK1, KLRAs1, 2, 5, 7, KLRD1, KLRC1, KLRC3, as well as the three receptors KLRB1A, KLRB1C, KLRB1F and their potential ligands CLEC2D and CLEC2I. Vaccination enhances this malaria-induced expression of genes, but impairs Gzmm expression, accelerates decline of Tnfsf10 and Clec2d expression, whereas it accelerates increased expression of Clec2i, taking a very similar time course as that of genes encoding plasma membrane proteins of erythroblasts, whose malaria-induced extramedullary generation in the liver is known to be accelerated by vaccination. Collectively, vaccination reshapes the response of the liver NK cell compartment to blood-stage malaria. Particularly, the malaria-induced expansion of lrNK cells peaking on day 4 p.i. is highly significantly (p < 0.0001) reduced by enhanced immigration of peripheral cNK cells, and KLRB1F:CLEC2I interactions between NK cells and erythroid cells facilitate extramedullary erythroblastosis in the liver, thus critically contributing to vaccination-induced survival of otherwise lethal blood-stage malaria of P. chabaudi.
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Macri, Christophe, Claire Dumont, Scott Panozza, Mireille H. Lahoud, Irina Caminschi, Jose A. Villadangos, Angus P. R. Johnston, and Justine D. Mintern. "Antibody-mediated targeting of antigen to C-type lectin-like receptors Clec9A and Clec12A elicits different vaccination outcomes." Molecular Immunology 81 (January 2017): 143–50. http://dx.doi.org/10.1016/j.molimm.2016.12.010.
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Vitry, Julien, Guillaume Paré, Andréa Murru, Xavier Charest-Morin, Halim Maaroufi, Kenneth R. McLeish, Paul H. Naccache, and Maria J. Fernandes. "Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region." International Journal of Molecular Sciences 22, no. 19 (September 22, 2021): 10207. http://dx.doi.org/10.3390/ijms221910207.
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CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.
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Kenderian, Saad S., Marco Ruella, Olga Shestova, Michael Klichinsky, Miriam Y. Kim, Craig Soderquist, Adam Bagg, et al. "Leukemia Stem Cells Are Characterized By CLEC12A Expression and Chemotherapy Refractoriness That Can be Overcome By Targeting with Chimeric Antigen Receptor T Cells." Blood 128, no. 22 (December 2, 2016): 766. http://dx.doi.org/10.1182/blood.v128.22.766.766.
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Abstract Chemo-refractory acute myeloid leukemia (AML) is associated with poor prognosis and treatment options are extremely limited. Most of these patients are ineligible for allogeneic stem cell transplantation. Chemo-refractory AML is thought to arise due to selection pressure of resistant clones from prior use of chemotherapy or in some cases pre-exist due to properties of the leukemic stem cells (LSC). CLEC12A (also known as CLL1) has previously been described as being selectively over expressed in LSCs. Successful modalities to target CLEC12A and eradicate the LSC would overcome chemo-refractoriness in AML and would represent a vertical advance in the field. In this study, we confirm that CLEC12A is heterogenously expressed on AML blasts and over-expressed on AML LSC. We also show that CLEC12A is overexpressed on bone marrows from patients with AML that fail to achieve a complete remission after induction chemotherapy, suggesting that it could be a marker for residual disease that is refractory to chemotherapy. We then separated AML blasts into CLEC12A positive or negative cells by magnetic sorting. CLEC12A positive blasts selected from AML patients were more resistant to chemotherapy compared to CLEC12A negative blasts (20% killing of CLEC12A positive AML cells versus 43% of CLEC12A negative AML cells when cultured with cytarabine 10 µg/ml, P=0.01). This finding was confirmed by using the AML MOLM14 cell line engineered to overexpress CLEC12A. CLEC12Ahigh MOLM14 cells were more resistant to chemotherapy compared to wild type MOLM14 cells (P=0.003). We then evaluated CLEC12A resistance to chemotherapy in a patient derived AML xenograft model. We found a relative increase in CLEC12A positive cells post Ara-C induction chemotherapy in AML xenograft models (Figure 1). The observation that CLEC12A positive cells are more resistant to chemotherapy provided a solid rationale to target CLEC12A with chimeric antigen receptor T (CART) cells. We therefore developed a second generation CLEC12A directed CAR construct using CD3z and 41BB costimulatory domains and generated CLEC12A CART cells by lentiviral transduction with this construct. Upon incubation with primary AML samples or AML cell lines, CLEC12A CART cells resulted in modest effector functions, due to the heterogeneity of CLEC12A expression on AML blasts. However when CLEC12A overexpressed MOLM14 cell line or CLEC12Apos selected leukemic cells were used as targets, CLEC12A-CART cells resulted in potent cytotoxicity, proliferation and cytokine production, indicating that CLEC12A-CART cells are more specific for LSC. To test the in vivo anti-leukemic activity of CLEC12A CARTs, we used primary human AML blasts xenografted into NSG-S mice (NOD-SCID-γc-/-, additionally transgenic for human stem cell factor, IL3 and GM-CSF). Treatment with CLEC12A CART (single dose, 1x105 total T cells via tail vein injection) resulted in modest activity against AML when employed as monotherapy. To investigate the potential role of CLEC12A CART cells in eradication of MRD and LSC, mice were treated first with chemotherapy (cytarabine 60 mg/kg intraperitoneal injection daily for 5 days) followed by a single dose (1x105 total T cells via tail vein injection) of either CLEC12A CARTs or control untransduced T cells (UTD). Treatment with CLEC12A CART cells resulted in eradication of leukemia and prolonged survival in these mice (overall survival at 200 days of 100% after CLEC12A CARTs compared to 20% after UTD, p=0.01, Figure 2). In conclusion, our preclinical studies reveal that CLEC12A positive cells in leukemia are resistant to chemotherapy and can be successfully targeted with CART cells. CLEC12A CART cells can potentially be employed as a consolidation regimen after induction chemotherapy to eradicate LSC and MRD in AML. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Singh:Novartis: Employment. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Johnson & Johnson: Research Funding; Pfizer: Honoraria. Gill:Novartis: Patents & Royalties, Research Funding.
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Chen, Po-Ku, Shie-Liang Hsieh, Joung-Liang Lan, Chi-Chen Lin, Shih-Hsin Chang, and Der-Yuan Chen. "Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still’s Disease." Journal of Immunology Research 2020 (April 23, 2020): 1–11. http://dx.doi.org/10.1155/2020/9473497.
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C-type lectin domain family 5-member A (CLEC5A) associates with adaptor DAP12 (DNAX activation protein 12) to form receptor complexes involved in inflammatory responses. We postulated a potential role of CLEC5A in the pathogenesis of adult-onset Still’s disease (AOSD) and aimed to investigate CLEC5A expression and its association with activity parameters and disease course. In 34 AOSD patients and 12 healthy controls (HC), circulating levels of CLEC5A-expressing monocytes or granulocytes were determined by flow cytometry analysis, the mRNA expression of CLEC5A and DAP12 on PBMCs by quantitative PCR, and plasma levels of proinflammatory cytokines by ELISA. AOSD patients had significantly higher percentages and mean fluorescence intensity (MFI) of CLEC5A-expressing monocytes (median 62.1% and 3.20, respectively) or granulocytes (72.6% and 3.22, respectively) compared with HC (in monocytes: 17.0% and 0.65, both p<0.001; in granulocytes: 67.3%, p<0.05 and 0.90, p<0.001; respectively). Patients also had significantly higher levels of CLEC5A mRNA expression on PBMCs compared with HC (median 1.77 vs. 0.68, p<0.05). The levels of CLEC5A-expressing monocytes or granulocytes were positively associated with activity scores and levels of IL-1β and IL-18 in AOSD patients. The patients with a systemic pattern had significantly higher levels of CLEC5A-expressing granulocytes and IL-18 compared to those with a chronic articular pattern of disease course. After 6 months of therapy, levels of CLEC5A-expressing monocytes and granulocytes significantly declined, paralleling the decrease of AOSD activity. Elevated CLEC5A levels and their positive association with activity parameters suggest that CLEC5A is involved in the pathogenesis and may serve as an activity indicator of AOSD.
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Caminschi, Irina, Anna I. Proietto, Fatma Ahmet, Susie Kitsoulis, Joo Shin Teh, Jennifer C. Y. Lo, Alexandra Rizzitelli, et al. "The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement." Blood 112, no. 8 (October 15, 2008): 3264–73. http://dx.doi.org/10.1182/blood-2008-05-155176.
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Abstract A novel dendritic cell (DC)–restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8+ conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.
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Kan, Hung-Wei, Chin-Hong Chang, Ying-Shuang Chang, Yi-Ting Ko, and Yu-Lin Hsieh. "Genetic loss-of-function of activating transcription factor 3 but not C-type lectin member 5A prevents diabetic peripheral neuropathy." Laboratory Investigation 101, no. 10 (June 25, 2021): 1341–52. http://dx.doi.org/10.1038/s41374-021-00630-5.
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AbstractWe investigated the mediating roles of activating transcription factor 3 (ATF3), an injury marker, or C-type lectin member 5A (CLEC5A), an inflammatory response molecule, in the induction of endoplasmic reticulum (ER) stress and neuroinflammation in diabetic peripheral neuropathy in ATF3 and CLEC5A genetic knockout (aft3−/− and clec5a−/−, respectively) mice. ATF3 was expressed intranuclearly and was upregulated in mice with diabetic peripheral neuropathy (DN) and clec5a−/− mice. The DN and clec5a−/− groups also exhibited neuropathic behavior, but not in the aft3−/− group. The upregulation profiles of cytoplasmic polyadenylation element-binding protein, a protein translation–regulating molecule, and the ER stress-related molecules of inositol-requiring enzyme 1α and phosphorylated eukaryotic initiation factor 2α in the DN and clec5a−/− groups were correlated with neuropathic behavior. Ultrastructural evidence confirmed ER stress induction and neuroinflammation, including microglial enlargement and proinflammatory cytokine release, in the DN and clec5a−/− mice. By contrast, the induction of ER stress and neuroinflammation did not occur in the aft3−/− mice. Furthermore, the mRNA of reactive oxygen species–removing enzymes such as superoxide dismutase, heme oxygenase-1, and catalase were downregulated in the DN and clec5a−/− groups but were not changed in the aft3−/− group. Taken together, the results indicate that intraneuronal ATF3, but not CLEC5A, mediates the induction of ER stress and neuroinflammation associated with diabetic neuropathy.
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Nasu, Junta, Tomofumi Uto, Tomohiro Fukaya, Hideaki Takagi, Takehito Fukui, Noriaki Miyanaga, Yotaro Nishikawa, Sho Yamasaki, Yoshihiro Yamashita, and Katsuaki Sato. "Pivotal role of the carbohydrate recognition domain in self-interaction of CLEC4A to elicit the ITIM-mediated inhibitory function in murine conventional dendritic cells in vitro." International Immunology 32, no. 10 (May 16, 2020): 673–82. http://dx.doi.org/10.1093/intimm/dxaa034.
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Abstract C-type lectin receptors (CLRs), pattern recognition receptors (PRRs) with a characteristic carbohydrate recognition domain (CRD) in the extracellular portion, mediate crucial cellular functions upon recognition of glycosylated pathogens and self-glycoproteins. CLEC4A is the only classical CLR that possesses an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM), which possibly transduces negative signals. However, how CLEC4A exerts cellular inhibition remains unclear. Here, we report that the self-interaction of CLEC4A through the CRD is required for the ITIM-mediated suppressive function in conventional dendritic cells (cDCs). Human type 2 cDCs (cDC2) and monocytes display a higher expression of CLEC4A than cDC1 and plasmacytoid DCs (pDCs) as well as B cells. The extracellular portion of CLEC4A specifically binds to a murine cDC cell line expressing CLEC4A, while its extracellular portion lacking the N-glycosylation site or the EPS motif within the CRD reduces their association. Furthermore, the deletion of the EPS motif within the CRD or ITIM in CLEC4A almost completely impairs its suppressive effect on the activation of the murine cDC cell line, whereas the absence of the N-glycosylation site within the CRD exhibits partial inhibition on their activation. On the other hand, antagonistic monoclonal antibody (mAb) to CLEC4A, which inhibits the self-interaction of CLEC4A and its downstream signaling in murine transfectants, enhances the activation of monocytes and monocyte-derived immature DCs upon stimulation with a Toll-like receptor (TLR) ligand. Thus, our findings suggest a pivotal role of the CRD in self-interaction of CLEC4A to elicit the ITIM-mediated inhibitory signal for the control of the function of cDCs.
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Watson, Aleksandra A., Andrey A. Lebedev, Benjamin A. Hall, Angharad E. Fenton-May, Alexei A. Vagin, Wanwisa Dejnirattisai, James Felce, et al. "Structural Flexibility of the Macrophage Dengue Virus Receptor CLEC5A." Journal of Biological Chemistry 286, no. 27 (May 12, 2011): 24208–18. http://dx.doi.org/10.1074/jbc.m111.226142.
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The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1–4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.
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Arvindam, Upasana Sunil, Paulien van Hauten, Caroline Hallstrom, Daniel A. Vallera, Harry Dolstra, Jeffrey S. Miller, and Martin Felices. "CD16-IL15-CLEC12A Trispecific Killer Engager (TriKE) Drives NK Cell Expansion, Activation, and Antigen Specific Killing of Cancer Stem Cells in Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 1454. http://dx.doi.org/10.1182/blood-2018-99-117150.
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Abstract Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen. The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects. The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. Figure 1. Figure 1. Disclosures Vallera: GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.
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Schreibelt, Gerty, Lieke J. J. Klinkenberg, Luis J. Cruz, Paul J. Tacken, Jurjen Tel, Martin Kreutz, Gosse J. Adema, Gordon D. Brown, Carl G. Figdor, and I. Jolanda M. de Vries. "The C-type lectin receptor CLEC9A mediates antigen uptake and (cross-)presentation by human blood BDCA3+ myeloid dendritic cells." Blood 119, no. 10 (March 8, 2012): 2284–92. http://dx.doi.org/10.1182/blood-2011-08-373944.
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Abstract CLEC9A is a recently discovered C-type lectin receptor involved in sensing necrotic cells. In humans, this receptor is selectively expressed by BDCA3+ myeloid dendritic cells (mDCs), which have been proposed to be the main human cross-presenting mDCs and may represent the human homologue of murine CD8+ DCs. In mice, it was demonstrated that antigens delivered with antibodies to CLEC9A are presented by CD8+ DCs to both CD4+ and CD8+ T cells and induce antitumor immunity in a melanoma model. Here we assessed the ability of CLEC9A to mediate antigen presentation by human BDCA3+ mDCs, which represent < 0.05% of peripheral blood leukocytes. We demonstrate that CLEC9A is only expressed on immature BDCA3+ mDCs and that cell surface expression is lost after TLR-mediated maturation. CLEC9A triggering via antibody binding rapidly induces receptor internalization but does not affect TLR-induced cytokine production or expression of costimulatory molecules. More importantly, antigens delivered via CLEC9A antibodies to BDCA3+ mDCs are presented by both MHC class I (cross-presentation) and MHC class II to antigen-specific T cells. We conclude that CLEC9A is a promising target for in vivo antigen delivery in humans to increase the efficiency of vaccines against infectious or malignant diseases.
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Azamor, Tamiris, Andréa Marques Vieira da Silva, Juliana Gil Melgaço, Ana Paula dos Santos, Caroline Xavier-Carvalho, Lucia Elena Alvarado-Arnez, Leonardo Ribeiro Batista-Silva, et al. "Activation of an Effective Immune Response after Yellow Fever Vaccination Is Associated with the Genetic Background and Early Response of IFN-γ and CLEC5A." Viruses 13, no. 1 (January 12, 2021): 96. http://dx.doi.org/10.3390/v13010096.
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The yellow fever vaccine (YF17DD) is highly effective with a single injection conferring protection for at least 10 years. The YF17DD induces polyvalent responses, with a TH1/TH2 CD4+ profile, robust T CD8+ responses, and synthesis of interferon-gamma (IFN-γ), culminating in high titers of neutralizing antibodies. Furthermore, C-type lectin domain containing 5A (CLEC5A) has been implicated in innate outcomes in other flaviviral infections. Here, we conducted a follow-up study in volunteers immunized with YF17DD, investigating the humoral response, cellular phenotypes, gene expression, and single nucleotide polymorphisms (SNPs) of IFNG and CLEC5A, to clarify the role of these factors in early response after vaccination. Activation of CLEC5A+ monocytes occurred five days after vaccination (DAV). Following, seven DAV data showed activation of CD4+ and CD8+T cells together with early positive correlations between type II IFN and genes of innate antiviral response (STAT1, STAT2, IRF7, IRF9, OAS1, and RNASEL) as well as antibody levels. Furthermore, individuals with genotypes rs2430561 AT/AA, rs2069718 AG/AA (IFNG), and rs13237944 AC/AA (CLEC5A), exhibited higher expression of IFNG and CLEC5A, respectively. Together, we demonstrated that early IFN-γ and CLEC5A responses, associated with rs2430561, rs2069718, and rs13237944 genotypes, may be key mechanisms in the long-lasting immunity elicited by YF17DD.
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Li, Kai, Konstantin Neumann, Vikas Duhan, Sukumar Namineni, Anne Louise Hansen, Tim Wartewig, Zsuzsanna Kurgyis, et al. "The uric acid crystal receptor Clec12A potentiates type I interferon responses." Proceedings of the National Academy of Sciences 116, no. 37 (August 26, 2019): 18544–49. http://dx.doi.org/10.1073/pnas.1821351116.
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The detection of microbes and damaged host cells by the innate immune system is essential for host defense against infection and tissue homeostasis. However, how distinct positive and negative regulatory signals from immune receptors are integrated to tailor specific responses in complex scenarios remains largely undefined. Clec12A is a myeloid cell-expressed inhibitory C-type lectin receptor that can sense cell death under sterile conditions. Clec12A detects uric acid crystals and limits proinflammatory pathways by counteracting the cell-activating spleen tyrosine kinase (Syk). Here, we surprisingly find that Clec12A additionally amplifies type I IFN (IFN-I) responses in vivo and in vitro. Using retinoic acid-inducible gene I (RIG-I) signaling as a model, we demonstrate that monosodium urate (MSU) crystal sensing by Clec12A enhances cytosolic RNA-induced IFN-I production and the subsequent induction of IFN-I–stimulated genes. Mechanistically, Clec12A engages Src kinase to positively regulate the TBK1-IRF3 signaling module. Consistently, Clec12A-deficient mice exhibit reduced IFN-I responses upon lymphocytic choriomeningitis virus (LCMV) infection, which affects the outcomes of these animals in acute and chronic virus infection models. Thus, our results uncover a previously unrecognized connection between an MSU crystal-sensing receptor and the IFN-I response, and they illustrate how the sensing of extracellular damage-associated molecular patterns (DAMPs) can shape the immune response.
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Yamasaki, Sho. "Clec12a: Quieting the Dead." Immunity 40, no. 3 (March 2014): 309–11. http://dx.doi.org/10.1016/j.immuni.2014.03.001.
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Noordhuis, Paul, Monique Terwijn, Arjo P. Rutten, Linda Smit, Gert J. Ossenkoppele, and Gerrit J. Schuurhuis. "Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1×CD3 BiTE Antibody." Blood 116, no. 21 (November 19, 2010): 2890. http://dx.doi.org/10.1182/blood.v116.21.2890.2890.
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Abstract Abstract 2890 Response rates of ±80% in Acute Myeloid Leukemia (AML) are observed after conventional therapy but ±30% of patients experience a relapse. In the elderly the outcome is even worse. A small population of therapy resistant leukemia cells, minimal residual disease (MRD), are thought to be responsible for relapse of AML. The leukemic stem cells (LSC) herein have self renewal potential and reside in the CD34+CD38- stem cell compartment and side population (SP) compartment and can be identified via aberrant marker expression and scatter properties. Several markers are identified that show differential expression on AML (stem) cells versus normal hematopoietic stem cells (HSC). Previously we showed that CLEC12A (CLL-1, MICL, KLRL1, DCAL-2) is expressed on blasts of 90% of AML patients with varying expression. Importantly, CLEC12A is expressed on LSC but not on normal HSC (van Rhenen, Blood 110(7), 2007). This unique expression pattern paves the way to develop therapies that potentially eliminate CLEC12A-positive LSC and preserves CLEC12A-negative HSC. Drug-conjugated antibodies (ADCs) targeting CLEC12A and Bispecific T cell Engager (BiTE) scFv-antibodies targeting T-cells to CLEC12A positive cells could be instrumental to achieve this goal. We evaluated the response of AML cells to ADCs conjugated via cleavable and non-cleavable linkers to the maytansine derivates DM1 and DM4 and to the BiTE antibody CLL-1×CD3. ADC activity was assessed by colony formation capacity after 24 hours exposure to 0.1–100 nM ADC in 29 freshly obtained AML samples. The response to the BiTE antibody was tested by flow cytometry in 9 AML samples via induction of apoptosis (Annexin V/7AAD) after 24 hours exposure. To determine the effect of ADC on self-renewal in normal bone marrow (NBM), colony formation capacity was asseses during long term liquid culture after 24 hours exposure to 1–100 nM ADC. Furthermore internalisation of CLEC12A in AML progenitor and stem cells was tested. Several splice variants of CLEC12A are reported (CLL-1, MICLα, MICLβ, MICLγ) that have different intra-cellular signalling motifs or lack the transmembrane motif or the extra-cellular c-type lectin-like domain. Since these variants could not all be distinguished or detected by extra-cellular antibody binding, we evaluated these splice variants by Q-RT-PCR. After 24 hours exposure, a median IC50 value of >100 nM was observed for the unconjugated antibody CR2357. The median IC50 values for ADCs with non-cleavable linkers were 10 nM for CR2357-SMCC-DM1 (4,3 DM1/mAb), 2 nM for CR2357-PEG4-MAL-DM1 (5.9 DM1/mAb) and 0.8 nM for CR2357-PEG4-MAL-DM1 (10 DM1/mAb). For CR2357-SPDB-DM4 (4 DM4/mAb) which has a cleavable linker the median IC50 was 4 nM. The median IC50 of ADCs with non-cleavable linkers were significantly correlated to each other (r=0.730-0.784, p<0.01). CR2357-PEG4-MAL-DM1 (10 DM1/mAb) was significantly correlated to CLEC12A membrane expression (r=0.649, p<0.05). Prelimanary data of colony formation capacity during long term liquid culture of NBM showed that at >5 weeks after exposure, this was reduced to 15–50% for CR2357 and CR2357-PEG4-MAL-DM1 (10 DM1/mAb) relative to the untreated control. Exposure of AML cells to the CLL-1×CD3 BiTE antibody with donor T-cells (E:T=10:1 and 1:1) showed a dose dependent activation of T-cells as measured by increased CD25 and CD69 expression on CD4+ and CD8+ T-cells. Importantly, besides T-cell activation, Annexin V/7AAD staining of AML cells showed a specific decrease of CLEC12A-positive viable cells while in CLEC12A-negative cells viability remained constant. Internalisation of CR2357 antibody in CD34+/CD38+ progenitor cells and in CD34+/CD38- LSC was clearly demonstrated. Q-RT-PCR of CLEC12A splice variant expression showed highest expression for MICLα > MICLβ ∼F MICLγ > CLL-1 indicating that MICLα is the main variant expressed on the cellular membrane. Downstream signalling will therefore mainly be mediated by SHP-1/2 phosphatases. Although expression levels in AML, NBM, and sorted sub-populations varied, the ratio between the splice variants remained almost similar suggesting that the individual splice variants play a similar role in the different cell populations. In conclusion: these result show that targeting of CLEC12A-positive AML cells by ADCs and BiTE antibodies results in specific cell kill and might be a promising approach for the eradication of LSC that survive conventional therapy. Disclosures: No relevant conflicts of interest to declare.
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Sanchez-Canteli, Mario, Francisco Hermida-Prado, Christian Sordo-Bahamonde, Irene Montoro-Jiménez, Esperanza Pozo-Agundo, Eva Allonca, Aitana Vallina-Álvarez, et al. "Lectin-Like Transcript 1 (LLT1) Checkpoint: A Novel Independent Prognostic Factor in HPV-Negative Oropharyngeal Squamous Cell Carcinoma." Biomedicines 8, no. 12 (November 25, 2020): 535. http://dx.doi.org/10.3390/biomedicines8120535.
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Lectin-like transcript 1 (LLT1) expression by tumor cells contributes to immune evasion, thereby emerging as a natural killer (NK) cell-mediated immunotherapeutic target. This study is the first to investigate LLT1 expression (encoded by CLEC2D gene) in head and neck cancers to ascertain its impact on patient prognosis. LLT1 expression was analyzed by immunohistochemistry in a homogeneous cohort of human papillomavirus (HPV)-negative oropharyngeal squamous cell carcinomas (OPSCC), and correlated with clinical data. Results were further validated using transcriptomic data from the TCGA database. Tumoral LLT1 expression was detected in 190/221 (86%) OPSCC specimens, whereas normal pharyngeal epithelium was negative. Patients harboring LLT1-positive tumors showed significantly lower disease-specific (DSS) and overall survival (OS) (p = 0.049 and p = 0.036, respectively, log-rank test). High density of LLT1-positive tumor-infiltrating lymphocytes (TIL) was also frequently detected in 160 (73%) OPSCC samples, and significantly associated with better DSS and OS (p < 0.001 and p = 0.007, respectively). Multivariate Cox analysis further revealed that tumoral LLT1 expression and infiltration of LLT1-positive TIL were independent prognostic factors for DSS and OS. CLEC2D mRNA levels are also significantly increased in primary tumors compared to normal tissue. Strikingly, the prognostic impact of CLEC2D mRNA levels varied depending on HPV status in OPSCC, and among distinct cancer types. CLEC2D expression was significantly correlated with NK cell infiltration using the MCP-counter model. These findings uncover LLT1/CLEC2D as an independent prognostic factor in HPV-negative OPSCC, and a potential novel target for immunotherapy.
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Masterman, Kelly-Anne, Oscar L. Haigh, Kirsteen M. Tullett, Ingrid M. Leal-Rojas, Carina Walpole, Frances E. Pearson, Jonathon Cebon, et al. "Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141+ dendritic cells to activate naïve and memory NY-ESO-1-specific CD8+ T cells." Journal for ImmunoTherapy of Cancer 8, no. 2 (July 2020): e000691. http://dx.doi.org/10.1136/jitc-2020-000691.
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BackgroundDendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs.MethodsHuman anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, tumor necrosis factor and CD107a and by lysis of target tumor cells.ResultsCLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro.ConclusionsThese data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.
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Bell, Elaine. "CLEC9A: linking necrosis and immunity." Nature Reviews Immunology 9, no. 4 (April 2009): 223. http://dx.doi.org/10.1038/nri2531.
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Kirkham, Christina L., Oscar A. Aguilar, Tao Yu, Miho Tanaka, Aruz Mesci, Kuan-Lun Chu, Jason H. Fine, et al. "Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition." Journal of Innate Immunity 9, no. 4 (2017): 343–58. http://dx.doi.org/10.1159/000454926.
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Natural killer (NK) cells are innate lymphocytes that aid in self-nonself discrimination by recognizing cells undergoing pathological alterations. The NKR-P1B inhibitory receptor recognizes Clr-b, a self-encoded marker of cell health downregulated during viral infection. Here, we show that Clr-b loss during mouse cytomegalovirus (MCMV) infection is predicated by a loss of Clr-b (Clec2d) promoter activity and nascent transcripts, driven in part by MCMV ie3 (M122) activity. In contrast, uninfected bystander cells near MCMV-infected fibroblasts reciprocally upregulate Clr-b expression due to paracrine type-I interferon (IFN) signaling. Exposure of fibroblasts to type-I IFN augments Clec2d promoter activity and nascent Clr-b transcripts, dependent upon a cluster of IRF3/7/9 motifs located ∼200 bp upstream of the transcriptional start site. Cells deficient in type-I IFN signaling components revealed IRF9 and STAT1 as key transcription factors involved in Clr-b upregulation. In chromatin immunoprecipitation experiments, the Clec2d IRF cluster recruited STAT2 upon IFN-α exposure, confirming the involvement of ISGF3 (IRF9/STAT1/STAT2) in positively regulating the Clec2d promoter. These findings demonstrate that Clr-b is an IFN-stimulated gene on healthy bystander cells, in addition to a missing-self marker on MCMV-infected cells, and thereby enhances the dynamic range of innate self-nonself discrimination by NK cells.
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Hsu, Amy P., Joie Davis, Alexandria L. Chaput, Daniel A. Powell, Nima Pouladi, Yves Lussier, Joshua Fierer, et al. "42. Common Population Variants Cause Susceptibility to Disseminated Coccidioidomycosis." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S22—S23. http://dx.doi.org/10.1093/ofid/ofaa417.041.
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Abstract Background Coccidioides are endemic, dimorphic fungi found in soils of southwestern United States, Mexico and Central America. Infection occurs via inhalation of arthroconidia which swell, differentiate into spherules and rupture releasing endospores. While the majority of infected individuals will never report illness, roughly 1/3 seek medical attention for fungal pneumonia and ~1% of those present with disseminated coccidioidomycosis (DCM). IL12-IFNγ pathway mutations have been reported in DCM but are exceedingly rare and cannot account for the ~500–600 cases of DCM/year. Methods We performed whole exome sequencing on 66 individuals with DCM, retaining variants predicted damaging (CADD >15) with a population frequency < 10%. Results Homozygous CLEC7A c.714T >G; p.Y238* causing a truncated Dectin-1 receptor was overrepresented (OR=9.8449, 95% CI 3.0841 to 31.4260, P=0.0001). Dectin-1 signaling pathway variants included 3 homozygous and 11 heterozygous CLEC7A p.Y238* individuals, one each CLEC7A p.I223S and MALT1 p.R149Q and five PLCG2 p.R268W. Since Dectin-1 is the receptor for b-glucan, a major Coccidioides cell-wall component, we hypothesized that Dectin-1 pathway variants could affect fungal recognition and cellular response. Healthy control PBMCs stimulated with purified β-glucan or heat-killed Candida albicans induced 6-fold more TNFα than patients with homozygous or heterozygous CLEC7A, PLCG2 or MALT1 variants (P=0.0022, Ordinary one-way ANOVA). Additionally, one patient with a family history of DCM but lacking a defined mutation also failed to up-regulate TNFα after stimulation. Normalized TNF production from healthy control and DCM patient’s peripheral blood mononuclear cells Conclusion These data are consonant with increased dissemination in Clec7a-/- mice as well as in patients receiving anti-TNF biologics. These gene variants accounted for 31% of our DCM cohort (21/66 patients). This is the first demonstration of variants outside the IL12-IFNg pathway impairing fungal recognition and cellular response in coccidioidomycosis. Common heterozygous variants may be sufficient for disease susceptibility to highly pathogenic organisms. Disclosures Michail Lionakis, MD, ScD, Matinas BioPharma (Research Grant or Support)
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Kerscher, Bernhard, Ivy M. Dambuza, Maria Christofi, Delyth M. Reid, Sho Yamasaki, Janet A. Willment, and Gordon D. Brown. "Signalling through MyD88 drives surface expression of the mycobacterial receptors MCL (Clecsf8, Clec4d) and Mincle (Clec4e) following microbial stimulation." Microbes and Infection 18, no. 7-8 (July 2016): 505–9. http://dx.doi.org/10.1016/j.micinf.2016.03.007.
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Masterman, Kelly-Anne, Oscar Haigh, Kirsteen Tullett, Ingrid Leal-Rojas, Carina Walpole, Frances Pearson, Jonathon Cebon, et al. "612 Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141+ dendritic cells to activate naïve and memory NY-ESO-1-specific CD8+ T cells." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A648. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0612.
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BackgroundDendritic cells (DC) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T cell mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DC, the human cDC1 equivalent. CD141+ DC exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 to human CD141+ DC. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1 specific naïve and memory CD8+ T cells was examined and compared to a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DC.MethodsHuman anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1- epitope specific CD8+ T cells and reactivity of T cell responses in melanoma patients was assessed by IFNγ production following incubation of CD141+ DC and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, TNF and CD107a and by lysis of target tumor cells.ResultsCLEC9A-NY-ESO-1 Ab were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DC for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in melanoma patients. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro.ConclusionsThese data advocate human CLEC9A-NY-ESO-1 antibody as an attractive strategy for specific targeting of CD141+ DC to enhance tumour immunogenicity in NY-ESO-1-expressing malignancies.Ethics ApprovalWritten informed consent was obtained for human sample acquisition in line with standards established by the Declaration of Helsinki. Study approval was granted by the Mater Human Research Ethics Committee (HREC13/MHS/83 and HREC13/MHS/86) and The U.S. Army Medical Research and Materiel Command (USAMRMC) Office of Research Protections, Human Research Protection Office (HRPO; A-18738.1, A-18738.2, A-18738.3). All animal experiments were approved by the University of Queensland Animal Ethics Committee and conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes in addition to the laws of the United States and regulations of the Department of Agriculture.
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Swystun, Laura L., Natalia Rydz, Colleen Notley, Jonathan J. Riches, Andrew D. Paterson, Robert R. Montgomery, Paula D. James, and David Lillicrap. "Genetic Variability of the CLEC4M Endothelial Lectin Receptor Modulates Binding and Internalization of Von Willebrand Factor and Contributes to Variance in Plasma VWF Levels." Blood 120, no. 21 (November 16, 2012): 16. http://dx.doi.org/10.1182/blood.v120.21.16.16.
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Abstract Abstract 16 Type 1 von Willebrand's Disease (VWD) can result from decreased synthesis or accelerated clearance of von Willebrand Factor (VWF), resulting in partial quantitative deficiency. Approximately 35% of individuals with Type 1 VWD do not have a putative mutation in their VWF gene, suggesting that genes other than VWF may contribute to the pathophysiology of this disease. Recently, the CHARGE GWAS meta-analysis identified single nucleotide polymorphisms in the gene encoding the C-type lectin domain family 4 member M (CLEC4M) as being significantly associated with plasma VWF levels in normal individuals. CLEC4M is a pathogen recognition receptor with a polymorphic extracellular neck region consisting of a variable number of tandem repeats (VNTR) (3 – 9 repeats). We hypothesize that CLEC4M binds to and clears VWF from the circulation, and that different CLEC4M VNTR alleles may contribute to differences in plasma levels of VWF in normal subjects and patients with Type 1 VWD. Previously, genotyping of 555 subjects (196 cases with type 1 VWD, and 362 family members) for the CLEC4M VNTR number showed that the most frequently documented alleles were VNTR 5 (29%), 6 (15%), and 7 (53%). Family-based association analysis on kindreds with type I VWD has demonstrated a significant excess transmission of VNTR 6 to the type I VWD phenotype (p=0.005) and an association of this VNTR allele with VWF:RCo (p=0.037). In the present studies, we have complemented this genetic association data with experiments to directly evaluate the ability of CLEC4M to bind and internalize VWF. Further, we characterized the ability of different CLEC4M VNTR alleles to facilitate VWF clearance. Binding of VWF to CLEC4M was assessed with a modified ELISA using a recombinant CLEC4M-Fc chimera. CLEC4M-Fc bound to Humate P (plasma-derived VWF-FVIII) in a dose-dependent manner. CLEC4M-Fc also bound to recombinant human VWF, and factor VIII-free plasma-derived VWF. CLEC4M-Fc demonstrated a 70% increase in binding to de-O-glycosylated Humate P (p=0.041), and a 75% decrease in binding to de-N-glycosylated Humate P (p=0.046) relative to controls. Additionally, pre-incubation of CLEC4M-Fc with the polysaccharide mannan attenuated binding to all VWF preparations by approximately 50%. Binding and internalization of VWF by HEK 293 cells stably expressing CLEC4M (VNTR allele 7) was assessed with immunofluorescence and ELISA. Binding of VWF co-localized with CLEC4M expression on HEK 293 cells. CLEC4M and VWF co-localized with early endosomal antigen-1, suggesting that CLEC4M participates in receptor-mediated endocytosis of VWF. CLEC4M-expressing cells bound and internalized VWF in a dose- and time-dependent manner relative to controls. Preincubation of CLEC4M expressing cells with mannan inhibited VWF binding and internalization by 50% (p=0.0088). The contribution of CLEC4M genetic variability to VWF binding and internalization was measured using HEK 293 cells expressing CLEC4M with 4, 6, 7, and 9 tandem repeats. Decreased binding and internalization of VWF was observed with cells expressing CLEC4M 4 and 9 tandem repeat constructs as compared to CLEC4M with 7 tandem repeats (CLEC4M 4 – 60% reduction, p < 0.001; CLEC4M 9 – 45% reduction, p=0.006). Cells expressing the CLEC4M VNTR combination 4 and 9, had a 55% decrease in binding and internalization of VWF relative to cells expressing CLEC4M with 7 VNTRs (p < 0.001). These VNTR associated differences in VWF binding/internalization were not accounted for by variances in the CLEC4M expression levels in the transfected HEK 293 cells. These studies demonstrate that the C-type lectin CLEC4M binds to and internalizes VWF through an N-glycan-dependent mechanism. Additionally, it provides further evidence that polymorphisms in the CLEC4M gene contribute to quantitative VWF deficiency. Disclosures: Montgomery: Gen-Probe/GTI Diagnostics: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.
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LONG, DAVID G., and BARBARA J. CRANDALL-STOTLER. "Taxonomic changes in Cleveaceae (Marchantiidae, Marchantiophyta)—a correction." Phytotaxa 273, no. 4 (September 12, 2016): 299. http://dx.doi.org/10.11646/phytotaxa.273.4.6.
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The recently published combination Clevea nana (Lindenb.) Crand.-Stotl. & D.G.Long, which was intended to replace the name Clevea hyalina (Sommerf.) Lindb. on the basis of nomenclatural priority, is a later homonym of Clevea nana (Shimizu & S.Hatt.) Borovichev & Bakalin published in 2013 for a different taxon. Consequently, the name Clevea hyalina is the correct name for the widespread dioicous member of the genus. The heterotypic name Clevea nana (Shimizu & S.Hatt.) Borovichev & Bakalin refers to a different monoicous species of the genus that had previously been placed in synonymy of Clevea pusilla (Steph.) Rubasinghe & D.G.Long.
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Swystun, Laura L., Colleen Notley, Ilinca Georgescu, Paula D. James, and David Lillicrap. "The Endothelial Lectin Receptor CLEC4M Internalizes Factor VIII and Von Willebrand Factor Via a Clathrin-Coated Pit-Dependent Mechanism." Blood 122, no. 21 (November 15, 2013): 1091. http://dx.doi.org/10.1182/blood.v122.21.1091.1091.
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Abstract Regulation of plasma levels of the coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII) involves a dynamic balance between biosynthesis, secretion, and clearance. Clearance of VWF and FVIII occurs through receptor-mediated endocytosis, with both LDL receptor gene family (eg. LRP1), and lectin receptors (eg. asialoglycoprotein receptor, siglec-5) contributing to this process. We have previously characterized the endothelial lectin CLEC4M as an endocytic receptor for FVIII and VWF. These proteins represent the first endogenous glycoprotein ligands indentified for CLEC4M. Previous studies have characterized CLEC4M as an adhesive receptor capable of facilitating viral infection in trans. Thus, the mechanism by which CLEC4M internalizes VWF and FVIII, and the subsequent fate of these ligands, is uncharacterized. We hypothesize that CLEC4M is able to endocytose VWF and FVIII via a clathrin-coated pit-dependent mechanism. We further hypothesize that endocytosis of FVIII and VWF by CLEC4M-expressing cells targets FVIII and VWF to lysosomes for degradation. As CLEC4M is expressed exclusively on the endothelial cells of the hepatic sinusoids and lymph nodes, and commercially prepared liver sinusoidal endothelial cells do not retain CLEC4M expression, we generated a HEK 293 cell line that stably expresses CLEC4M (>90% positive by flow cytometry). We first inhibited the CLEC4M-mediated endocytic pathways by preincubating cells with methyl-β-cyclodextrin to deplete cell membrane cholesterol, dynasore hydrate to inhibit dynamin GTPase activity, and pitstop-2 to block the N-terminus of clathrin. Cells were then incubated with recombinant human FVIII, or plasma derived VWF-FVIII complex for 1 hour and internalization was visualized by immunofluorescence. A quantitative analysis of VWF or FVIII-positive objects was performed using Image Pro software. CLEC4M-expressing 293 cells internalized both FVIII as well as the VWF-FVIII complex. Binding and internalization of VWF was reduced by methyl-β-cyclodextran (65%, p=0.067), by dynasore hydrate (92%, p=0.055), and by pitstop-2 (83%, p=0.032). Binding and internalization of FVIII was similarly reduced by methyl-β-cyclodextran (50%, p=0.0050), by dynasore hydrate (60%, p=0.0225), and by pitstop-2 (90%, p=0.0086). This suggests that CLEC4M mediates endocytosis of VWF and FVIII via a clathrin-coated pit-dependent mechanism that involves lipid rafts. To visualize the endocytic pathway of VWF and FVIII, CLEC4M-expressing 293 cells were incubated with FVIII or the VWF-FVIII complex for 15, 30, or 60 minutes. Colocalization of FVIII, VWF and/or CLEC4M with markers for early endosomes (early endosomal antigen-1), late endosomes (Rab9), and lysosomes (LAMP1) was observed using immunofluorescence. For all conditions, CLEC4M-negative and isotype controls were performed. We have previously confirmed that FVIII and VWF are internalized by CLEC4M expressing cells, and that VWF colocalizes with a marker for early endosomes. When CLEC4M-expressing cells were exposed to the VWF-FVIII complex, we observed a partial colocalization of VWF and FVIII, confirming that CLEC4M is able to internalize the VWF-FVIII complex. When CLEC4M-expressing cells were exposed to FVIII, we observed colocalization between FVIII and early endosomal antigen 1 within 15 minutes, confirming that upon internalization by CLEC4M, VWF and FVIII are targeted to early endosomes. We next observed the transport of VWF and FVIII to late endosomes and lysosomes. When CLEC4M-expressing cells were incubated with VWF and FVIII for 30 minutes, we observed colocalization of both proteins with Rab9, a marker for late endosomes. When CLEC4M-expressing cells were incubated with FVIII for 1 hour, we observed colocalization of FVIII with LAMP1, a lysosomal marker, suggesting that the internalization of FVIII by CLEC4M leads to lysosome-mediated degradation of FVIII. In contrast, incubation of CLEC4M-expressing cells with VWF for up to 2 hours did not result in co-localization with LAMP1. These studies confirm that, in the context of the stable cell system used in these experiments, the cell surface lectin receptor CLEC4M mediates endocytosis of FVIII and VWF through a clathrin-coated pit-dependent pathway. Internalization of VWF and FVIII by CLEC4M targets these proteins to early and late endosomes; FVIII is subsequently targeted to lysosomes for degradation. Disclosures: James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.
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Wong, Kok Loon, June Jing-Yi Tai, Wing-Cheong Wong, Hao Han, Xiaohui Sem, Wei-Hseun Yeap, Philippe Kourilsky, and Siew-Cheng Wong. "Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets." Blood 118, no. 5 (August 4, 2011): e16-e31. http://dx.doi.org/10.1182/blood-2010-12-326355.
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Abstract New official nomenclature subdivides human monocytes into 3 subsets: the classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes. This introduces new challenges, as monocyte heterogeneity is mostly understood based on 2 subsets, the CD16− and CD16+ monocytes. Here, we comprehensively defined the 3 circulating human monocyte subsets using microarray, flow cytometry, and cytokine production analysis. We find that intermediate monocytes expressed a large majority (87%) of genes and surface proteins at levels between classical and nonclassical monocytes. This establishes their intermediary nature at the molecular level. We unveil the close relationship between the intermediate and nonclassic monocytes, along with features that separate them. Intermediate monocytes expressed highest levels of major histocompatibility complex class II, GFRα2 and CLEC10A, whereas nonclassic monocytes were distinguished by cytoskeleton rearrangement genes, inflammatory cytokine production, and CD294 and Siglec10 surface expression. In addition, we identify new features for classic monocytes, including AP-1 transcription factor genes, CLEC4D and IL-13Rα1 surface expression. We also find circumstantial evidence supporting the developmental relationship between the 3 subsets, including gradual changes in maturation genes and surface markers. By comprehensively defining the 3 monocyte subsets during healthy conditions, we facilitate target identification and detailed analyses of aberrations that may occur to monocyte subsets during diseases.
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Sang, Yanmei, Wei Zong, Jie Yan, and Min Liu. "The Correlation between the CLEC16A Gene and Genetic Susceptibility to Type 1 Diabetes in Chinese Children." International Journal of Endocrinology 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/245384.
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Objective. The CLEC16A gene is related to the genetic susceptibility to T1DM with racial variability. This study investigated the association between CLEC16A gene polymorphisms and T1DM in Chinese children.Methods. 131 Chinese children with T1DM were selected for study, and 121 healthy adult blood donors were selected as normal controls. PCR and mass spectrometry was used to study the distributions of 17 CLEC16A alleles in patients and controls. The relationship between CLEC16A gene polymorphisms and T1DM was studied.Results. The distributions of two polymorphisms (rs12921922, rs12931878) of CLEC16A in T1DM and healthy controls were significantly different, while the distributions of other CLEC16A polymorphisms show no significant differences. The alleles of rs12921922 are C and T. The frequency of the T allele was significantly increased in patients versus healthy controls. The alleles of rs12931878 are A and C. The frequencies of the A allele are significantly increased in T1DM patients versus healthy controls.Conclusion. Two polymorphisms in the CLEC16A gene correlate with increased susceptibility to T1DM in Chinese children, revealing that it was another new gene that correlates with susceptibility to T1DM in multiple populations.
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Otto, Grant. "The podoplanin–CLEC2 axis in sepsis." Nature Reviews Nephrology 14, no. 3 (January 15, 2018): 143. http://dx.doi.org/10.1038/nrneph.2018.2.
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Haddad, Y., L. Laurans, S. Metghalchi, Z. Zeboudj, A. Giraud, Z. Mallat, and S. Taleb. "The role of CLEC9a in atherosclerosis development." Archives of Cardiovascular Diseases Supplements 9, no. 2 (April 2017): 184. http://dx.doi.org/10.1016/s1878-6480(17)30455-x.
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Katsu, Yoshinao, and Taisen Iguchi. "Tissue-specific expression of Clec2g in mice." European Journal of Cell Biology 85, no. 5 (May 2006): 345–54. http://dx.doi.org/10.1016/j.ejcb.2005.12.004.
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del Fresno, Carlos, and David Sancho. "Clec2d Joins the Cell Death Sensor Ranks." Immunity 52, no. 1 (January 2020): 6–8. http://dx.doi.org/10.1016/j.immuni.2019.12.015.
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Kanemaru, Kazumasa, Emiko Noguchi, Satoko Tahara-Hanaoka, Seiya Mizuno, Hiroaki Tateno, Kaori Denda-Nagai, Tatsuro Irimura, et al. "Clec10a regulates mite-induced dermatitis." Science Immunology 4, no. 42 (December 6, 2019): eaax6908. http://dx.doi.org/10.1126/sciimmunol.aax6908.
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House dust mite (HDM) is a major allergen that causes allergic diseases such as atopic dermatitis. However, the regulatory mechanisms of HDM-induced immune responses are incompletely understood. NC/Nga mice are an inbred strain that is more susceptible to HDM and develops more severe dermatitis than other strains. Using whole-exome sequencing, we found that NC/Nga mice carry a stop-gain mutation in Clec10a, which encodes a C-type lectin receptor, Clec10a (MGL1/CD301a). The repair of this gene mutation using the CRISPR-Cas9 system ameliorated HDM-induced dermatitis, indicating that the Clec10a mutation is responsible for hypersensitivity to HDM in NC/Nga mice. Similarly, Clec10a−/− mice on the C57BL/6J background showed exacerbated HDM-induced dermatitis. Clec10a expressed on skin macrophages inhibits HDM-induced Toll-like receptor 4 (TLR4)–mediated inflammatory cytokine production through the inhibitory immunoreceptor tyrosine activating motif in its cytoplasmic portion. We identified asialoglycoprotein receptor 1 (Asgr1) as a functional homolog of mouse Clec10a in humans. Moreover, we found that a mucin-like molecule in HDM is a ligand for mouse Clec10a and human Asgr1. Skin application of the ligand ameliorated a TLR4 ligand-induced dermatitis in mice. Our findings suggest that Clec10a in mice and Asgr1 in humans play an important role in skin homeostasis against inflammation associated with HDM-induced dermatitis.
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Van Loo, Pieter Fokko, Robert Doornbos, Harry Dolstra, Setareh Shamsili, and Lex Bakker. "Preclinical Evaluation of MCLA117, a CLEC12AxCD3 Bispecific Antibody Efficiently Targeting a Novel Leukemic Stem Cell Associated Antigen in AML." Blood 126, no. 23 (December 3, 2015): 325. http://dx.doi.org/10.1182/blood.v126.23.325.325.
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Abstract Although chemotherapy regimens induce initial remissions in most acute myeloid leukemia (AML) patients, their long term prognosis is very poor. Novel targeted therapies that effectively eradicate both AML blasts and their progenitors are needed.MCLA-117, a potent human CLEC12AxCD3 bispecific IgG antibody redirects patient's cytotoxic T cells to induce AML tumor lysis. CLEC12A is a myeloid differentiation antigen that is expressed on 90-95% of newly diagnosed and relapsed AML. Moreover, CLEC12A is selectively expressed on leukemic stem cells (LSCs) but not normal early hematopoietic progenitors, including hematopoietic stem cells (HSCs). MCLA-117 is a full length human bispecific IgG that incorporates CH2 region amino acid substitutions to abrogate Fcγ receptor and C1q interactions while retaining its binding to FcRn. MCLA-117 specifically binds to CLEC12A expressing myeloid cells and CD3 expressing T cells, as evaluated on healthy donor samples by flow cytometry. In line with the CLEC12A expression profile within the hematopoietic progenitor compartment, MCLA-117 did not bind the HSCs, nor the common myeloid progenitor cells. As determined by Surface Plasmon Resonance, MCLA-117 had an affinity of 3 nM for human CLEC12A and 177 nM for human CD3. The efficacy of MCLA-117 to induce T cell mediated lysis of CLEC12A+ tumor cells was evaluated in cytotoxicity assays. In co-cultures with resting healthy donor T cells and AML tumor cells MCLA-117 efficiently induced CLEC12A antigen dependent T cell activation, T cell proliferation and tumor target cell lysis. MCLA-117 induced tumor target lysis with an EC50 of 66±37 ng/mL. Upregulation of the activation CD69 marker on CD8 T cells was the most sensitive read-out for the activity of MCLA-117 (EC20 of 11±3 ng/mL). Therefore the MABEL (minimum anticipated biological effect level) concentration for MCLA-117 was defined as 10 ng/mL and is used to calculate a safe starting dose in a planned first in human study. MCLA-117 was able to activate and redirect AML patient-derived T cells to CLEC12APOS tumor cells as potently as that of healthy donor-derived T cells. The efficacy of MCLA-117 to induce lysis of AML blasts by autologous T cells in primary AML bone marrow (BM) samples with low T cell to AML blast ratios (E:T ratios of 1:7-1:80) was examined. AML patient BM samples taken at diagnosis were cultured in medium with low amounts of a cytokine cocktail to support in vitro survival of AML blasts. MCLA-117 induced up to 30-fold T cell expansion after 7-10 days. More importantly, MCLA-117 efficiently induced AML blast lysis (up to 88%) in 6/6 tested primary AML patient BM samples, even at very low effector to target ratios (see figure 1). The cytokine release potential was assessed by incubation of human whole blood and PBMC preparations with MCLA-117. Moderate to strong cytokine release in whole blood was observed (IFNγ, IL-6, IL-8, IL-10 and TNFα) after 24 hours at concentrations of 10,000 and 1,000 ng/mL MCLA-117. At concentration of 100 ng/mL and 10 ng/mL, moderate to low cytokine release and no cytokine release was observed, respectively. In PBMC preparations, moderate to strong IFNγ release was observed after 48 hours at MCLA-117 concentrations of 10,000, 1,000 and 100 ng/mL. At these concentrations, only low release of IL-2, IL-10 and TNFα and no release of IL-1β, IL-6 and IL-8 was observed. MCLA-117 may provide a novel treatment option for all subtypes of AML with highly specific targeting of myeloid blasts and progenitors and sparing the normal HSCs, as well as eradicating residual LSCs residing in the BM niche compartments. A first clinical study is planned to evaluate the safety and preliminary efficacy of MCLA-117 in adult AML patients. Disclosures Van Loo: Merus B.V.: Employment. Doornbos:Merus B.V.: Employment. Shamsili:Merus B.V.: Employment. Bakker:Merus B.V.: Employment.
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Claushuis, Theodora A. M., Alex F. de Vos, Bernard Nieswandt, Louis Boon, Joris J. T. H. Roelofs, Onno J. de Boer, Cornelis van ’t Veer, and Tom van der Poll. "Platelet glycoprotein VI aids in local immunity during pneumonia-derived sepsis caused by gram-negative bacteria." Blood 131, no. 8 (February 22, 2018): 864–76. http://dx.doi.org/10.1182/blood-2017-06-788067.
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Rydz, Natalia, Laura L. Swystun, Colleen Notley, Andrew D. Paterson, J. Jacob Riches, Kate Sponagle, Boonchai Boonyawat, Robert R. Montgomery, Paula D. James, and David Lillicrap. "The C-type lectin receptor CLEC4M binds, internalizes, and clears von Willebrand factor and contributes to the variation in plasma von Willebrand factor levels." Blood 121, no. 26 (June 27, 2013): 5228–37. http://dx.doi.org/10.1182/blood-2012-10-457507.
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Fisher, Cynthia E., Tobias M. Hohl, Wenhong Fan, Barry E. Storer, David M. Levine, Lu Ping Zhao, Paul J. Martin, Edus H. Warren, Michael Boeckh, and John A. Hansen. "Validation of single nucleotide polymorphisms in invasive aspergillosis following hematopoietic cell transplantation." Blood 129, no. 19 (May 11, 2017): 2693–701. http://dx.doi.org/10.1182/blood-2016-10-743294.
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Key Points Two SNPs in PTX3 and CLEC7a previously associated with development of proven or probable invasive aspergillosis were validated. Thirteen SNPs in 9 genes were associated at P ≤ .05 with development of IA using a different genetic model than the original study.
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Inoue, Daichi, Jiro Kitaura, Katsuhiro Togami, Koutarou Nishimura, Yutaka Enomoto, Tomoyuki Uchida, Yuki Kagiyama, et al. "C-Terminal-Truncating ASXL1 Mutations Induce MDS Via Inhibition Of PRC2." Blood 122, no. 21 (November 15, 2013): 471. http://dx.doi.org/10.1182/blood.v122.21.471.471.
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Recurrent mutations of ASXL1 (Additional sex combs-like1) are found in various hematological malignancies including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia, and acute myeloid leukemia (AML) with myelodysplasia-related changes. Additionally, ASXL1 mutations are linked with adverse survival in a variety of myeloid malignancies. A previous study demonstrated that loss of ASXL1 in mice promotes myeloid transformation by impairing polycomb repressive complex 2 (PRC2)-mediated gene repression at a number of critical loci and leads to myeloid transformation. However, most ASXL1 mutations are heterozygous and located in the 5’ region of the last exon, indicating a dominant-negative or gain-of-function feature of a truncated ASXL1 protein. Therefore, we investigated if the C-terminal truncated form of ASXL1 (ASXL1-MT) contributes to the development of myeloid malignancies. To this end, we examined the effects of ASXL1-MT using in vitro and in vivo experiments. In in vitro experiment, expression of ASXL1-MT inhibited G-CSF-induced myeloid differentiation of 32Dcl3 cells. In a mouse bone marrow transplantation (BMT) model, ASXL1-MT induced multilineage dysplasia, differentiation block, slowly progressive pancytopenia, BM hyperplasia and splenomegaly. The transduced mice died of severe anemia after a long latency (median survival, 400.5 days), and some of the mice progressed to overt leukemia. Thus, the current model displays all of the features of human MDS. In addition, ASXL1-MT collaborated with N-RAS-G12V, which confers a proliferative advantage, in inducing progression of N-RAS-G12V-induced myeloproliferative neoplasm (MPN) to AML, suggesting that ASXL1-MT contributes to leukemic transformation by inhibiting differentiation of MPN cells. To clarify the molecular mechanism for differentiation block and MDS development induced by ASXL1-MT, we performed expression profiles of 32Dcl3 cells transduced with ASXL1-MT and BM cells of the MDS mice. Of note, gene set enrichment analysis (GSEA) of BM cells of the MDS mice indicated that ASXL1-MT induced an expression profile which inversely correlated with known PRC target genes. In fact, ASXL1-MT remarkably derepressed expression of posterior Hoxa genes, including Hoxa5, Hoxa9 and Hoxa10, which are epigenetically silenced by PRC2 in mature cells. In consistent with this, H3K27me3 was globally reduced in ASXL1-MT transduced cells. We also found ASXL1-MT as well as wild type ASXL1 (ASXL1-WT) can bind to EZH2 and, importantly, co-expression of ASXL1-MT with ASXL1-WT efficiently inhibited the binding between ASXL1-WT and EZH2, suggesting a dominant-negative role of ASXL1-MT against the PRC2 function. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that H3K27me3 and Ezh2-bindig profoundly decreased around the promoter regions of Hoxa5, Hoxa9, and Hoxa10 in the MDS mice, correlating with the upregulation of their mRNA expression. On the other hand, we found that ASXL1-MT reduced the expression of Clec5a, a type 2 transmembrane receptor and that this reduction was associated with differentiation block of the 32Dcl3 cells. Moreover, utilizing an shRNA or a mutant form of Clec5a, we identified that Clec5a plays essential roles in myeloid differentiation of 32Dcl3 cells. Lastly, we searched for microRNAs deregulated by ASXL1-MT since a large subset of microRNAs are found to be transcriptionally regulated by PRC2. Among upregulated microRNAs related to myeloid malignancies, we found that miR-125a targeted 3’UTR of Clec5a gene, repressed Clec5a expression and inhibited granulocytic differentiation in vitro. Intriguingly, H3K27me3 and Ezh2-bindig greatly decreased around the miR-125a gene in the BM cells of the MDS mice, similar to the results of ChIP assays around Hoxa genes. The present results indicate that ASXL1-MT which results in a truncated protein product may (1) inhibit PRC2-function by impairing the interaction of EZH2 with the ASXL1-WT and (2) promote myeloid transformation through impaired PRC2-mediated repression of posterior HOXAs and miR-125a, and subsequent suppression of CLEC5A. HOXA9 and CLEC5A expression were shown to be high and low, respectively, in MDS patients with ASXL1-MT. Our data provide evidence for a novel axis in MDS pathogenesis and implicate both mutant forms of ASXL1 and miR-125a as therapeutic targets in MDS. Disclosures: No relevant conflicts of interest to declare.
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van der Aa, Evelyn, Nadine van Montfoort, and Andrea M. Woltman. "BDCA3+CLEC9A+ human dendritic cell function and development." Seminars in Cell & Developmental Biology 41 (May 2015): 39–48. http://dx.doi.org/10.1016/j.semcdb.2014.05.016.
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Yang, Guo, Huitong Zhou, Jiang Hu, Yuzhu Luo, and Jon G. H. Hickford. "Variation in the Yak Dectin-1 Gene (CLEC7A)." DNA and Cell Biology 30, no. 12 (December 2011): 1069–71. http://dx.doi.org/10.1089/dna.2011.1276.
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Yoshikawa, Fabio Seiti Yamada, Anna Julia Pietrobon, Anna Cláudia Calvielli Castelo Branco, Nátalli Zanete Pereira, Luanda Mara da Silva Oliveira, Clarisse Martins Machado, Alberto José da Silva Duarte, and Maria Notomi Sato. "Zika Virus Infects Newborn Monocytes Without Triggering a Substantial Cytokine Response." Journal of Infectious Diseases 220, no. 1 (February 14, 2019): 32–40. http://dx.doi.org/10.1093/infdis/jiz075.
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Abstract Zika virus (ZIKV) is a clinically important flavivirus that can cause neurological disturbances in newborns. Here, we investigated comparatively the outcome of in vitro infection of newborn monocytes by ZIKV. We observed that neonatal cells show defective production of interleukin 1β, interleukin 10, and monocyte chemoattractant protein 1 in response to ZIKV, although they were as efficient as adult cells in supporting viral infection. Although CLEC5A is a classical flavivirus immune receptor, it is not essential to the cytokine response, but it regulates the viral load only in adult cells. Greater expression of viral entry receptors may create a favorable environment for viral invasion in neonatal monocytes. We are the first to suggest a role for CLEC5A in human monocyte infectivity and to show that newborn monocytes are interesting targets in ZIKV pathogenesis, owing to their ability to carry the virus with only a partial triggering of the immune response, creating a potentially favorable environment for virus-related pathologies in young individuals.
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Tudorache, Madalina, George Ghemes, Andreea Nae, Elena Matei, Ionel Mercioniu, Erhard Kemnitz, Benjamin Ritter, Simona Coman, and Vasile Parvulescu. "Biocatalytic designs for the conversion of renewable glycerol into glycerol carbonate as a value-added product." Open Chemistry 12, no. 12 (December 1, 2014): 1262–70. http://dx.doi.org/10.2478/s11532-014-0547-x.
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AbstractA comparative study of two different biocatalytic models, e.g. enzyme immobilized on magnetic particles (EIMP) and cross-linking enzyme aggregates onto magnetic particles (CLEMPA) was performed. The first model was designed as enzyme-immobilized on the magnetic particles surface (EIMP). The second model was constructed as a network structure with the enzyme aggregates and magnetic particles placed into the nodes and polyglutaraldehyde cross-linker as the network ledges. The design was called cross-linking enzyme aggregates onto magnetic particles (CLEMPA). The biocatalysts were prepared using lipase enzyme from Aspergillus niger for catalyzing the glycerol (Gly) conversion to glycerol carbonate (GlyC). The biocatalyst characteristics for both designs (EIMP and CLEMPA) were evaluated using scanning electron microscopy (SEM), laser light scattering (LLS) and UV-Vis techniques. The EIMP model was strongly influenced by the composition of the polymeric layer covering the particles surface, while the size of the magnetic particles affected mostly the CLEMPA design. Also, the biocatalytic capacity of the tested models was evaluated as maximum 52% Gly conversion with 90% GlyC selectivity for EIMP, and 73% Gly conversion with 77% GlyC selectivity for CLEMPA. Both biocatalytic models were successfully used to prepare GlyC from “crude” glycerol collected directly from the biodiesel process (e.g. 49% Gly conversion with 91% GlyC selectivity for EIMP and 70% Gly conversion with 80% GlyC selectivity for CLEMPA).
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Yang, Jia-Sin, Renn-Chia Lin, Yi-Hsien Hsieh, Heng-Hsiung Wu, Geng-Chung Li, Ya-Chiu Lin, Shun-Fa Yang, and Ko-Hsiu Lu. "CLEFMA Activates the Extrinsic and Intrinsic Apoptotic Processes through JNK1/2 and p38 Pathways in Human Osteosarcoma Cells." Molecules 24, no. 18 (September 9, 2019): 3280. http://dx.doi.org/10.3390/molecules24183280.
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Due to the poor prognosis of metastatic osteosarcoma, chemotherapy is usually employed in the adjuvant situation to improve the prognosis and the chances of long-term survival. 4-[3,5-Bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl]-4-oxo-2-butenoic acid (CLEFMA) is a synthetic analog of curcumin and possesses anti-inflammatory and anticancer properties. To further obtain information regarding the apoptotic pathway induced by CLEFMA in osteosarcoma cells, microculture tetrazolium assay, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. CLEFMA dose-dependently decreased the cell viabilities of human osteosarcoma U2OS and HOS cells and significantly induced apoptosis in human osteosarcoma cells. In addition to the effector caspase 3, CLEFMA significantly activated both extrinsic caspase 8 and intrinsic caspase 9 initiators. Moreover, CLEFMA increased the phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), CLEFMA’s increases of cleaved caspases 3, 8, and 9 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Conclusively, CLEFMA activates both extrinsic and intrinsic apoptotic pathways in human osteosarcoma cells through JNK and p38 signaling. These findings contribute to a better understanding of the mechanisms responsible for CLEFMA’s apoptotic effects on human osteosarcoma cells.
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Kaewkla, Onuma, Wilaiwan Koomsiri, Arinthip Thamchaipenet, and Christopher Milton Mathew Franco. "Microbispora clausenae sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f." International Journal of Systematic and Evolutionary Microbiology 70, no. 12 (December 1, 2020): 6213–19. http://dx.doi.org/10.1099/ijsem.0.004518.
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An endophytic actinobacterium, strain CLES2T, was discovered from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f., collected from the Phujong-Nayoa National Park, Ubon Ratchathani Province, Thailand. The results of a polyphasic taxonomic study identified this strain as a member of the genus Microbispora and a Gram-stain-positive, aerobic actinobacterium. It had well-developed substrate mycelia, which were non-motile and possessed paired spores. A phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this strain in the family Streptosporangiaceae , being most closely related to Microbispora bryophytorum NEAU-TX2-2T (99.4 %), Microbispora camponoti 2C-HV3T (99.2 %), Microbispora catharanthi CR1-09T (99.2 %) and Microbispora amethystogenes JCM 3021T and Microbispora fusca NEAU-HEGS1-5T (both at 99.1 %). The major cellular fatty acid of this strain was iso-C16 : 0 and major menaquinone was MK-9(H4). The polar lipid profile of strain CLES2T contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol and phosphatidylinositol dimannosides. These chemotaxonomic data confirmed the affiliation of strain CLES2T to the genus Microbispora . The DNA G+C content of this strain was 70 mol%. Digital DNA–DNA hybridization and average nucleotide identity blast values between strain CLES2T and M. catharanthi CR1-09T were 62.4 and 94.0 %, respectively. The results of the polyphasic study allowed the genotypic and phenotypic differentiation of strain CLES2T from its closest species with valid names. The name proposed for the new species is Microbispora clausenae sp. nov. The type strain is CLES2T (=DSM 101759T=NRRL B-65340T).
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Riboldi, Elena, Roberta Daniele, Carmen Parola, Antonio Inforzato, Phoebe L. Arnold, Daniela Bosisio, Daved H. Fremont, Antonio Bastone, Marco Colonna, and Silvano Sozzani. "Human C-type Lectin Domain Family 4, Member C (CLEC4C/BDCA-2/CD303) Is a Receptor for Asialo-galactosyl-oligosaccharides." Journal of Biological Chemistry 286, no. 41 (August 31, 2011): 35329–33. http://dx.doi.org/10.1074/jbc.c111.290494.
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Plasmacytoid dendritic cells are specialized in the production of type I interferon (type I IFN), which promotes antiviral and antitumor responses, as well as autoimmune disorders. Activation of type I IFN secretion depends on the pattern recognition receptors TLR7 and TLR9, which sense microbial RNA and DNA, respectively. Type I IFN production is modulated by several receptors, including the type II C-type lectin domain family 4, member C (CLEC4C). The natural ligand of CLEC4C is unknown. To identify it, here we probed a glycan array with a soluble form of the CLEC4C ectodomain. We found that CLEC4C recognizes complex type sugars with terminal galactose. Importantly, soluble CLEC4C bound peripheral blood leukocytes and tumor cells that express glycans with galactose residues at the non-reducing ends. The positive and negative modulation of galactose residues on cell membranes was paralleled by the regulation of type I IFN secretion by plasmacytoid dendritic cells in co-culture experiments in vitro. These results suggest that the modulation in the expression of non-sialylated oligosaccharides by invading pathogens or transformed cells may affect type I IFN response and immune surveillance.
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Pinto, Raimundo Nonato Leite, Nelson Jorge da Silva, and Steven D. Aird. "Human envenomation by the South American opisthoglyph Clelia clelia plumbea (Wied)." Toxicon 29, no. 12 (January 1991): 1512–16. http://dx.doi.org/10.1016/0041-0101(91)90008-f.
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Kauke, Monique, Nikki Ross, Dalia Burzyn, Shelly Martin, Ke Xu, Nuruddeen Lewis, Charan Leng, et al. "703 Engineered exosomes with altered cellular tropism achieve targeted STING agonist delivery and single-agent tumor control in vivo." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A745. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0703.
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BackgroundExosomes are natural, abundant extracellular vesicles capable of transferring complex molecules between neighboring and distant cell types. Translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. Important strategies to maximize the therapeutic potential of exosomes therefore include payload loading, functionalization of the exosome surface with pharmacologically active proteins, and delivery to target cells of interest.MethodsThrough comparative proteomic analysis of purified exosomes, we identified several highly enriched and exosome-specific proteins, including a transmembrane glycoprotein (PTGFRN) belonging to the immunoglobulin superfamily. Leveraging PTGFRN as a scaffold for exosome surface display, we developed our engExTM platform to generate engineered exosomes functionalized with a variety of structurally and biologically diverse proteins.Systemically administered exosomes are primarily taken up by macrophages in the liver and spleen. To redirect exosome uptake to other cell types, we employed our engineering platform to display functional targeting ligands, including single domain antibodies, single chain variable fragments, single chain Fabs (scFabs), and receptor ligands, on the exosome surface at high density. To demonstrate that exosome surface modifications can alter cellular tropism, we generated exosomes displaying anti-Clec9A scFabs to target conventional type 1 dendritic cells (cDC1s), anti-CD3 scFabs to target T cells, and CD40 ligand to target B cells. The engineered exosomes exhibited functional antigen binding that led to greater association with the cell types expressing the cognate receptor both in vitro and in vivo.ResultsIn mice, systemic administration of exosomes engineered to display scFabs targeting Clec9A resulted in a 4-fold increase in the percentage of cDC1 cells in the blood that had taken up exosomes over controls, and a 6-fold increase in the number of exosomes taken up per cell. We further showed that compared to untargeted exosomes, those with altered tropism achieved increased functional payload delivery to the target cell of interest. In primary mouse dendritic cells, anti-Clec9A exosomes loaded with a cyclic dinucleotide STING agonist achieved greater pathway induction, 2.3-fold greater as measured by IFNβ production, 2-fold by IFNα, and 15-fold by IL-12, when compared to an untargeted control. Preliminary in vivo data show that intra-tumorally administered anti-Clec9A exosomes reduce the required STING agonist dose 10-fold to achieve single-agent tumor control and induce immune responses against tumor-associated antigen, compared to controls.ConclusionsThese results demonstrate the potential of our engExTM platform to generate targeted exosome therapeutics capable of immune cell stimulation and tumor growth inhibition in vivo.Ethics ApprovalAll experimental animal studies were performed according to Codiak BioSciences IACUC approved AUP CB2020-001.
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Hamed, GehanM, and MonaF Abdel Fattah. "Value of human CLEC12A expression in acute myeloid leukemia." Egyptian Journal of Haematology 41, no. 4 (2016): 161. http://dx.doi.org/10.4103/1110-1067.198648.
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