The large family of C-type lectin (CLEC) receptors comprises carbohydrate-binding proteins that require Ca²⁺ to bind a ligand. The prototypic receptor is the asialoglycoprotein receptor-1 (ASGR1, CLEC4H1) that is expressed primarily by hepatocytes. The early work on ASGR1, which is highly specific for N-acetylgalactosamine (GalNAc), established the foundation for understanding the overall function of CLEC receptors. Cells of the immune system generally express more than one CLEC receptor that serve diverse functions such as pathogen-recognition, initiation of cellular signaling, cellular adhesion, glycoprotein turnover, inflammation and immune responses. The receptor CLEC10A (C-type lectin domain family 10 member A, CD301; also called the macrophage galactose-type lectin, MGL) contains a carbohydrate-recognition domain (CRD) that is homologous to the CRD of ASGR1, and thus, is also specific for GalNAc. CLEC10A is most highly expressed on immature DCs, monocyte-derived DCs, and alternatively activated macrophages (subtype M2a) as well as oocytes and progenitor cells at several stages of embryonic development. This receptor is involved in initiation of T_H1, T_H2, and T_H17 immune responses and induction of tolerance in naïve T cells. Ligand-mediated endocytosis of CLEC receptors initiates a Ca²⁺ signal that interestingly has different outcomes depending on ligand properties, concentration, and frequency of administration. This review summarizes studies that have been carried out on these receptors.

Keratohyalin granules were discovered in the mid-19th century in cells that terminally differentiate to form the outer, cornified layer of the epidermis. The first indications of the composition of these structures emerged in the 1960s from a histochemical stain for histidine, followed by radioautographic evidence of a high incidence of histidine incorporation into newly synthesized proteins in cells containing the granules. Research during the next three decades revealed the structure and function of a major protein in these granules, which was initially called the ‘histidine-rich protein’. Steinert and Dale named the protein ‘filaggrin’ in 1981 because of its ability to aggregate keratin intermediate filaments. The human gene for the precursor, ‘profilaggrin,’ was reported in 1991 to encode 10, 11 or 12 nearly identical repeats. Remarkably, the mouse and rat genes encode up to 20 repeats. The lifetime of filaggrin is the time required for keratinocytes in the granular layer to move into the inner cornified layer. During this transition, filaggrin facilitates the collapse of corneocytes into ‘building blocks’ that become an impermeable surface barrier. The subsequent degradation of filaggrin is as remarkable as its synthesis, and the end-products aid in maintaining moisture in the cornified layer. It was apparent that ichthyosis vulgaris and atopic dermatitis were associated with the absence of this protein. McLean’s team in 2006 identified the cause of these diseases by discovering loss-of-function mutations in the profilaggrin gene, which led to dysfunction of the surface barrier. This story illustrates the complexity in maintaining a healthy, functional epidermis.

Background Receptors specific for the sugar N-acetylgalactosamine (GalNAc) include the human type II, C-type lectin receptor macrophage galactose-type lectin/C-type lectin receptor family member 10A (MGL/CLEC10A/CD301) that is expressed prominently by human peripheral immature dendritic cells, dendritic cells in the skin, alternatively-activated (M2a) macrophages, and to lesser extents by several other types of tissues. CLEC10A is an endocytic receptor on antigen-presenting cells and has been proposed to play an important role in maturation of dendritic cells and initiation of an immune response. In this study, we asked whether a peptide that binds in the GalNAc-binding site of CLEC10A would serve as an effective tool to activate an immune response against ovarian cancer. Methods A 12-mer sequence emerged from a screen of a phage display library with a GalNAc-specific lectin. The peptide, designated svL4, and a shorter peptide consisting of the C-terminal 6 amino acids, designated sv6D, were synthesized as tetravalent structures based on a tri-lysine core. In silico and in vitro binding assays were developed to evaluate binding of the peptides to GalNAc-specific receptors. Endotoxin-negative peptide solutions were administered by subcutaneous injection and biological activity of the peptides was determined by secretion of cytokines and the response of peritoneal immune cells in mice. Anti-cancer activity was studied in a murine model of ovarian cancer. Results The peptides bound to recombinant human CLEC10A with high avidity, with half-maximal binding in the low nanomolar range. Binding to the receptor was Ca²⁺-dependent. Subcutaneous injection of low doses of peptides into mice on alternate days resulted in several-fold expansion of populations of mature immune cells within the peritoneal cavity. Peptide sv6D effectively suppressed development of ascites in a murine ovarian cancer model as a monotherapy and in combination with the chemotherapeutic drug paclitaxel or the immunotherapeutic antibody against the receptor PD-1. Toxicity, including antigenicity and release of cytotoxic levels of cytokines, was not observed. Conclusion sv6D is a functional ligand for CLEC10A and induces maturation of immune cells in the peritoneal cavity. The peptide caused a highly significant extension of survival of mice with implanted ovarian cancer cells with a favorable toxicity and non-antigenic profile.

Phagocytic cells [dendritic cells (DCs), macrophages, monocytes, neutrophils, and mast cells] utilize C-type (Ca²⁺-dependent) lectin-like (CLEC) receptors to identify and internalize pathogens or danger signals. As monitors of environmental imbalances, CLEC receptors are particularly important in the function of DCs. Activation of the immune system requires, in sequence, presentation of antigen to the T cell receptor (TCR) by DCs, interaction of co-stimulatory factors such as CD40/80/86 on DCs with CD40L and CD28 on T cells, and production of IL-12 and/or IFN-α/β to amplify T cell differentiation and expansion. Without this sequence of events within an inflammatory environment, or in a different order, antigen-specific T cells become unresponsive, are deleted or become regulatory T cells. Thus, the mode by which CLEC receptors on DCs are engaged can either elicit activation of T cells to achieve an immune response or induce tolerance. This minireview illustrates these aspects with Dectin-1, DEC205, the mannose receptor and CLEC10A as examples.

Proper immune system function is dependent on precisely evolved sensing and signal transduction events that occur at cellular and subcellular compartment boundaries. Recent immunotherapeutic efforts have generally focused on the roles that two specific protein superfamilies play in such events. This review is directed at a third superfamily, the C-type (Ca²⁺-dependent) lectin-type (CLEC) receptors and the nuanced, less traditionally acknowledged, yet quite important, role of endocytic-based calcium signaling. While extracellular recognition events rely heavily on the sophisticated structural diversity that lectins and glycobiology have to offer, the actual details of CLEC receptor-mediated, endocytic-based, calcium signal transduction have remained less appreciated. Because many CLEC receptor family members are emerging, not only as biomarkers for critical immune cell subpopulations, but also proving to be selective and pivotal modulators of immune function, this review seeks to promote the potential role CLEC receptor-initiated calcium signaling plays in immunotherapy. Given the importance of calcium signaling, these receptors provide a means to initiate a selective physiological response.

We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a K_D in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14⁺ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.