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  1. eLife assessment

    The C-type lectin receptor family recognise pathogens and self-components. Dectin-1 is known to recognize glucan on pathogens. In this fundamental study Dectin-1 and CLEC-2 another - C-type lectin receptor, expressed on platelets - interact through an O-glycosylated ligand presented in the stalk region of Dectin-1. This compelling study demonstrates a potential role for pattern recognition receptors in physiological processes.

  2. Reviewer #1 (Public Review):

    Dectin-1 is a known C-type lectin receptor that has a role in recognizing pathogen glycans, particularly beta-glucan. Haji et al. present evidence that Dectin-1 also has an endogenous ligand, another C-type lectin that is enriched on platelets called CLEC-2. Using a Dectin-1 reporter line, they identify human platelets as a source for the ligand, raising a panel of mAbs to human platelets and identify one (6D11) that prevents signalling and use this to identify CLEC2 as the Dectin-1 receptor by immunopurification. The authors go on to further characterise this interaction by showing that it occurs between the human but not mouse orthologues, showing that it is the stalk region of human Dectin-1 that contains the ligand which consists of sialylated core 1 O-linked glycans displayed on Thr 105 and adjacent amino acids. Finally, the authors over-express human Dectin-1 in mice within the context of a null background for another CLEC-2 ligand (podoplanin) and show that this rescues embryonic lethality. They show that Dectin-1-dependent CLEC-2 signalling is not sufficient to induce platelet aggregation but can rescue perinatal lethality in podoplanin-deficient mice.

    There is a large amount of work in this manuscript and the experiments are well controlled and the results unequivocal and usually verified by several independent techniques. The paper is very well-written making the volume of data accessible. There is novelty here in that this unusually finds that 2 C-type lectin receptors directly interact and the biochemical characterisation of the glycan binding determinants is very well performed. While the authors show that the interaction occurs between the human but not mice orthologues (because mouse Dectin-1 lacks the critical EDxxT motif that is necessary for displaying the o-linked glycan in the correct context) they were able to investigate the functional role of the interaction by generating mice that overexpressed human Dectin-1 in a podoplanin-deficient background. Normally, podoplanin-deficient mice die at birth due to defects in lymphatic vessel development, but this lethality is rescued by overexpression of human Dectin-1. This xeno-overexpression data can be difficult to interpret but suggests that while human Dectin-1 signalling to mouse platelet CLEC-2 is insufficient to drive platelet activation and thrombus formation, it is sufficient to rescue some aspects of platelet function involved in lymph vessel development. They conclude that human Dectin-1 (which is broadly expressed on different tissues) provides a basal level of tonic signalling through platelet-expressed CLEC-2 to establish platelet signalling thresholds.

    I thought the manuscript was comprehensive in its approach, well written, and that the experimental data supported the conclusions.

  3. Reviewer #2 (Public Review):

    C-type lectin receptors are well-known for their pathogen recognition and their immunoregulatory properties. However, most C-type lectins also engage host-derived ligands. While many microbial targets have been identified, the characterization of endogenous ligands has so far lagged behind. In this paper, Haji et al. identified human Dectin-1 as a bonafide self-ligand for the platelet-specific C-type lectin receptor CLEC-2.

    Haji et al. actually identified the first glycan-dependent C-type lectin - C-type lectin interaction, resulting in a 2-way activation cascade downstream of both the Dectin-1 and CLEC-2 receptors. They performed a highly detailed molecular characterization, revealing both the interacting domains with Dectin-1 as well as the interacting glycan sialylated core 1 ligand. Moreover, the authors provide proof of the functional relevance of the Dectin-1 - CLEC-2 interaction in a mouse model deficient for the CLEC-2 ligand podoplanin, demonstrating that human Dectin-1 can rescue the phenotype observed in these podoplanin KO mice.

    The main limitation of this work is the use of Dectin-1 and CLEC-2 transfectants. Glycosylation patterns in transfected 2B4 cells (a T cell line) might not mimic the natural glycosylation pattern on Dectin-1 in vivo. A follow-up study should address which human Dectin-1 positive immune cell subsets are recognized by human CLEC-2 and how human Dectin-1 glycosylation is regulated during immune cell activation and differentiation.
    In addition, Dectin-1 polymorphisms have been identified in the human population, which strongly decreases Dectin-1 expression. Yet, these individuals mainly suffer from fungal infections and so far have not been shown to have lymphatic defects. This leaves the actual in vivo role of the human Dectin-1 - CLEC-2 interaction yet to be resolved.