Mechanistic insights into the C-type lectin receptor CLEC12A-mediated immune recognition of monosodium urate crystal.

CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals remains unclear. Herein, we reported the crystal structure of the human CLEC12A-C-type lectin-like domain (CTLD) and identified a unique "basic patch" site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at the cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.

CLEC12A Binds to Legionella pneumophila but Has No Impact on the Host's Antibacterial Response.

Legionella pneumophila is an intracellular pathogen that can cause severe pneumonia after the inhalation of contaminated aerosols and replication in alveolar macrophages. Several pattern recognition receptors (PRRs) have been identified that contribute to the recognition of L. pneumophila by the innate immune system. However, the function of the C-type lectin receptors (CLRs), which are mainly expressed by macrophages and other myeloid cells, remains largely unexplored. Here, we used a library of CLR-Fc fusion proteins to search for CLRs that can bind the bacterium and identified the specific binding of CLEC12A to L. pneumophila. Subsequent infection experiments in human and murine macrophages, however, did not provide evidence for a substantial role of CLEC12A in controlling innate immune responses to the bacterium. Consistently, antibacterial and inflammatory responses to Legionella lung infection were not significantly influenced by CLEC12A deficiency. Collectively, CLEC12A is able to bind to L. pneumophila-derived ligands but does not appear to play a major role in the innate defense against L. pneumophila.

Immunophenotypically defined stem cell subsets in paediatric AML are highly heterogeneous and demonstrate differences in BCL-2 expression by cytogenetic subgroups.

In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15-colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL-2 on CD34+ CD38- bone marrow stem cells from 38 paediatric AML patients and seven non-leukaemic, age-matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non-HSC-like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia-associated mutations. Differential BCL-2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non-HSC-like subsets demonstrated BCL-2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia-representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.

The uric acid crystal receptor Clec12A potentiates type I interferon responses.

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.

Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region.

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.

Mapping the CLEC12A expression on myeloid progenitors in normal bone marrow; implications for understanding CLEC12A-related cancer stem cell biology.

The C-type lectin domain family 12, member A (CLEC12A) receptor has emerged as a leukaemia-associated and cancer stem cell marker in myeloid malignancies. However, a detailed delineation of its expression in normal haematopoiesis is lacking. Here, we have characterized the expression pattern of CLEC12A on the earliest stem- and myeloid progenitor subsets in normal bone marrow. We demonstrate distinct CLEC12A expression in the classically defined myeloid progenitors, where on average 39.1% (95% CI [32.5;45.7]) of the common myeloid progenitors (CMPs) expressed CLEC12A, while for granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors (MEPs), the average percentages were 81.0% (95% CI [76.0;85.9]) and 11.9% (95% CI [9.3;14.6]), respectively. In line with the reduced CLEC12A expression on MEPs, functional assessment of purified CLEC12A+/- CMPs and MEPs in the colony-forming unit assay demonstrated CLEC12A+ subsets to favour non-erythroid colony growth. In conclusion, we provide evidence that the earliest CLEC12A+ cell in the haematopoietic tree is the classically defined CMP. Furthermore, we show that CLEC12A-expressing CMPs and MEPs are functionally different than their negative counterparts. Importantly, these data can help determine which cells will be spared during CLEC12A-targeted therapy, and we propose CLEC12A to be included in future studies of myeloid cancer stem cell biology.

Corrigendum: CLEC12A sensitizes differentially responsive breast cancer cells to the anti-cancer effects of artemisinin by repressing autophagy and inflammation.

[This corrects the article DOI: 10.3389/fonc.2023.1242432.].

CLEC12A sensitizes differentially responsive breast cancer cells to the anti-cancer effects of artemisinin by repressing autophagy and inflammation.

Background: Enhanced inflammatory responses promote tumor progression by activating toll-like receptors (TLRs), which in turn are inhibited by C-type lectin like receptors (CTLRs), like CLEC12A. Although the presence of CLEC12A in acute myeloid leukemia is well established, its role in non-hematopoietic tumors is still obscure. In hematopoietic tumors, CLEC12A mostly inhibits TLRs and modulates inflammatory responses via NF-κB signaling. In this study, the fate of tumor progression was determined by modulating CLEC12A using artemisinin (ART), a FDA-approved anti-malarial drug, known for its anti-cancer and immunomodulatory properties with minimal adverse effects on normal cells. Method: Effects of ART were primarily determined on hematological factors and primary metastatic organs, such as lungs, kidney and liver in normal and tumor-bearing BALB/c mice. Tumor-bearing mice were treated with different concentrations of ART and expressions of CLEC12A and associated downstream components were determined. CLEC12A was overexpressed in MDA-MB-231 and 4T1 cells, and the effects of ART were analyzed in the overexpressed cells. Silencing TLR4 using vivo morpholino was performed to elucidate its role in tumor progression in response to ART. Finally, CLEC12A modulation by ART was evaluated in the resident cancer stem cell (CSC) population. Results: ART did not alter physiology of normal mice, in contrast to tumor-bearing mice, where ART led to tumor regression. In addition, ART reduced expression of CLEC12A. Expectedly, TLR4 expression increased, but surprisingly, that of NF-κB (RelA) and JNK/pJNK decreased, along with reduced inflammation, reduced autophagy and increased apoptosis. All the above observations reverted on overexpression of CLEC12A in MDA-MB-231 and 4T1 cells. Inhibition of TLR4, however, indicated no change in the expressions of CLEC12A, NF-κB, or apoptotic markers. The effect of ART showed a similar trend in the CSC population as in cancer cells. Conclusion: This study, for the first time, confirmed a differential role of CLEC12A in non-hematopoietic tumor and cancer stem cells in response to ART. Subsequent interaction and modulation of CLEC12A with ART induced tumor cell death and abrogation of CSCs, confirming a more comprehensive tumor therapy with reduced risk of recurrence. Therefore, ART may be repurposed as an effective drug for cancer treatment in future.

Behçet syndrome: The disturbed balance between anti- (CLEC12A, CLC) and proinflammatory (IFI27) gene expressions.

INTRODUCTION: Behçet syndrome (BS) is a chronic, multisystemic inflammatory condition with unanswered questions regarding its pathogenesis and rational therapeutics. A microarray-based comparative transcriptomic analysis was performed to elucidate the molecular mechanisms of BS and identify any potential therapeutic targets. METHODS: Twenty-nine BS patients (B) and 15 age and sex-matched control subjects (C) were recruited. Patients were grouped as mucocutaneous (M), ocular (O), and vascular (V) according to their clinical phenotypes. GeneChip Human Genome U133 Plus 2.0 arrays were used for expression profiling on peripheral blood samples of the patients and the control subjects. Following documentation of the differentially expressed gene (DEG) sets, the data were further evaluated with bioinformatics analysis, visualization, and enrichment tools. Validation of the microarray data was performed using quantitative reverse transcriptase polymerase chain reaction. RESULTS: When p ≤ 0.05 and fold change ≥2.0 were chosen, the following numbers of DEGs were obtained; B versus C: 28, M versus C: 20, O versus C: 8, V versus C: 555, M versus O: 6, M versus V: 324, O versus V: 142. Venn diagram analysis indicated only two genes, CLEC12A and IFI27, in the intersection of M versus C ∩ O versus C ∩ V versus C. Another noteworthy gene appeared as CLC in the DEG sets. Cluster analyses successfully clustered distinct clinical phenotypes of BS. While innate immunity-related processes were enriched in the M group, adaptive immunity-specific processes were significantly enriched in the O and V groups. CONCLUSIONS: Distinct clinical phenotypes of BS patients displayed distinct expression profiles. In Turkish BS patients, expression differences regarding the genes CLEC12A, IFI27, and CLC seemed to be operative in the disease pathogenesis. Based on these findings, future research should consider the immunogenetic heterogeneity of BS clinical phenotypes. Two anti-inflammatory genes, namely CLEC12A and CLC, may be valuable as therapeutic targets and may also help design an experimental model in BS.

CLEC12A plays an important role in immunomodulatory function and prognostic significance of patients with acute myeloid leukemia.

The physiological function and prognostic significance of C-type lectin domain family 12 member A (CLEC12A) in acute myeloid leukemia (AML) patients are unclear. CLEC12A transcriptional expression in a variety of tumors from several public databases was collected and compared. We found that CLEC12A was highly expressed in AML cell lines and in tissues from AML patients and a higher CLEC12A expression in leukemia stem cells. CLEC12A low expression was associated with poor prognosis in the chemotherapy-only group and high CLEC12A expression may benefit from autologous or allogeneic hematopoietic stem cell transplantation (HSCT). CLEC12A expression was positively correlated with infiltrating levels of type 2 macrophages and monocytes and negatively associated with NK cells and regulatory T cells in AML. CLEC12A high was positively associated with immune checkpoint genes as well as macrophage associated genes. CLEC12A is an ideal chimeric antigen receptor T-cell (CAR-T) therapy target for AML and its expression level was closely linked to treatment response and patients' survival outcome. CLEC12A plays an important immunomodulatory role in AML.

The Inhibitory Receptor CLEC12A Regulates PI3K-Akt Signaling to Inhibit Neutrophil Activation and Cytokine Release.

The myeloid inhibitory C-type lectin receptor CLEC12A limits neutrophil activation, pro-inflammatory pathways and disease in mouse models of inflammatory arthritis by a molecular mechanism that remains poorly understood. We addressed how CLEC12A-mediated inhibitory signaling counteracts activating signaling by cross-linking CLEC12A in human neutrophils. CLEC12A cross-linking induced its translocation to flotillin-rich membrane domains where its ITIM was phosphorylated in a Src-dependent manner. Phosphoproteomic analysis identified candidate signaling molecules regulated by CLEC12A that include MAPKs, phosphoinositol kinases and members of the JAK-STAT pathway. Stimulating neutrophils with uric acid crystals, the etiological agent of gout, drove the hyperphosphorylation of p38 and Akt. Ultimately, one of the pathways through which CLEC12A regulates uric acid crystal-stimulated release of IL-8 by neutrophils is through a p38/PI3K-Akt signaling pathway. In summary this work defines early molecular events that underpin CLEC12A signaling in human neutrophils to modulate cytokine synthesis. Targeting this pathway could be useful therapeutically to dampen inflammation.

Characterization of the Porcine CLEC12A and Analysis of Its Expression on Blood Dendritic Cell Subsets.

CLEC12A has been proposed as a suitable target for delivering antigen to dendritic cells (DCs) to enhance vaccine efficacy both in human and mouse. In this study, we have characterized the porcine homolog of CLEC12A (poCLEC12A). Using new monoclonal antibodies (mAb), raised against its ectodomain, poCLEC12A was found to be expressed on alveolar macrophages, blood conventional type 1 and type 2 DCs and plasmacytoid DCs, but not on monocytes, T cells, B cells or NK cells, in contrast to its human and murine homologs. Western blot analysis showed that in alveolar macrophages this receptor is expressed both as a monomer and a dimer. After binding to DCs, anti- poCLEC12A mAb was efficiently internalized. No significant changes were observed in TNFα or IFNα secretion by plasmacytoid DCs stimulated with either CpGs (ODN2216) or polyinosinic-polycytidylic acid (poly I:C), upon incubation with mAb. These results provide the basis for future investigations aimed to assess the ability of anti-poCLEC12A mAbs to improve vaccine efficacy by targeting antigen to DCs.

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis.

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.

Targeting CLL-1 for acute myeloid leukemia therapy.

Despite major scientific discoveries and novel therapies over the past four decades, the treatment outcomes of acute myeloid leukemia (AML), especially in the adult patient population remain dismal. In the past few years, an increasing number of targets such as CD33, CD123, CLL-1, CD47, CD70, and TIM3, have been developed for immunotherapy of AML. Among them, CLL-1 has attracted the researchers' attention due to its high expression in AML while being absent in normal hematopoietic stem cell. Accumulating evidence have demonstrated CLL-1 is an ideal target for AML. In this paper, we will review the expression of CLL-1 on normal cells and AML, the value of CLL-1 in diagnosis and follow-up, and targeting CLL-1 therapy-based antibody and chimeric antigen receptor T cell therapy as well as providing an overview of CLL-1 as a target for AML.

Prognostic and therapeutic role of CLEC12A in acute myeloid leukemia.

CLEC12A has recently been identified as an antigen, expressed on leukemic stem cells and leukemic blasts. Given the fact that this expression profile seems stable throughout diagnosis, treatment and relapse on leukemic blasts and leukemic stem cells, CLEC12A can be considered a highly potent and reliable marker for the detection of measurable residual disease and therefore applicable for risk stratification and prognostication in AML. Low CLEC12A expression on leukemic blasts seems to be independently associated with lower likelihood of achieving complete remission after 1 cycle of induction chemotherapy, shorter event free survival, as well as overall survival, indicating potential prognostic properties of CLEC12A expression itself. Lack of expression on the normal hematopoietic stem and progenitor cells, in contrast to CD123 and CD33, might result in less toxicity regarding cytopenias, making CLEC12A an interesting target for innovating immunotherapies, including monoclonal and bispecific antibodies, antibody-drug conjugates and CAR-T cells therapy.

The C-type Lectin Receptor CLEC12A Recognizes Plasmodial Hemozoin and Contributes to Cerebral Malaria Development.

Malaria represents a major cause of death from infectious disease. Hemozoin is a Plasmodium-derived product that contributes to progression of cerebral malaria. However, there is a gap of knowledge regarding how hemozoin is recognized by innate immunity. Myeloid C-type lectin receptors (CLRs) encompass a family of carbohydrate-binding receptors that act as pattern recognition receptors in innate immunity. In the present study, we identify the CLR CLEC12A as a receptor for hemozoin. Dendritic cell-T cell co-culture assays indicate that the CLEC12A/hemozoin interaction enhances CD8+ T cell cross-priming. Using the Plasmodium berghei Antwerpen-Kasapa (ANKA) mouse model of experimental cerebral malaria (ECM), we find that CLEC12A deficiency protects mice from ECM, illustrated by reduced ECM incidence and ameliorated clinical symptoms. In conclusion, we identify CLEC12A as an innate sensor of plasmodial hemozoin.

The CLEC12A receptor marks human basophils: Potential implications for minimal residual disease detection in acute myeloid leukemia.

BACKGROUND: The transmembrane receptor C-type lectin domain family 12, member A (CLEC12A) is known to be highly expressed on monocytes and neutrophils and is a reliable leukemia associated marker in acute myeloid leukemia. Consequently, detailed knowledge of the various normal cell types expressing this receptor is essential. We have observed CLEC12A to be expressed on CD45lowSSClowCD14-CD123+ basophils in peripheral blood (PB) and in this study, we aimed at verifying this observation and further delineate the CD45lowSSClowCD14-CD123 + CLEC12A+ subpopulation. METHODS: We analyzed PB from 20 diagnostic chronic myeloid leukemia (CML) samples and eight healthy donors in a six-color multicolor flowcytometry (FCM) based assay. Furthermore, we performed fluorescence activated cell sorting on one CML sample to morphologically confirm the CD45lowSSClowCD14-CD123 + CLEC12A+ subset to be highly enriched for basophils. Finally, to further delineate the CD45lowSSClowCD14-CD123 + CLEC12A+ subpopulation in normal PB, we examined three healthy donors in a 10-color FCM assay enabling further separation of the cell subset into basophils and dendritic cells. RESULTS: The CLEC12A receptor is expressed on basophils. CONCLUSIONS: Identification and enumeration of basophils is of high relevance in diagnostic hematology and immunology. We here show that CLEC12A in a simple FCM assay consistently marks basophils. Importantly, as basophils are characterized by a CD45lowSSClow profile similar to the "blast-gate" used for the evaluation of hematological disorders, awareness of minor normal CLEC12A+ subpopulations is crucial when using CLEC12A as a minimal residual disease marker in myeloid malignancies. © 2017 International Clinical Cytometry Society.