Jin Sung Chung

Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor γ chain to induce innate immune responses

Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DCSIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial) components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that the Fc receptor γ (FcRγ) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcRγ, activation of NF-κB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor α and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcRγ chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcRγ to induce innate immune responses.

Syndecan-4 Mediates the Coinhibitory Function of DC-HIL on T Cell Activation

Receptor-ligand interactions between APCs and T cells determine whether stimulation of the latter leads to activation or inhibition. Previously, we showed that dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) on APC can inhibit T cell activation by binding an unknown ligand expressed on activated T cells. Because DC-HIL binds heparin/heparan sulfate and heparin blocks the inhibitory function of DC-HIL, we hypothesized that a heparin/heparan sulfate proteoglycan on activated T cells is the relevant ligand. Screening assays revealed that syndecan-4 (SD-4) is the sole heparan sulfate proteoglycan immunoprecipitated by DC-HIL from extracts of activated T cells and that blocking SD-4 abrogates binding of DC-HIL to activated T cells. Moreover, cell-bound SD-4 ligated by DC-HIL or cross-linked by anti-SD-4 Ab attenuated anti-CD3 responses, whereas knocked-down SD-4 expression led to enhanced T cell response to APC. Blockade of endogenous SD-4 using specific Ab or soluble SD-4 receptor led to augmented T cell reactions to syngeneic and allogeneic stimulation in vitro and exacerbated contact hypersensitivity responses in vivo. We conclude that SD-4 is the T cell ligand through which DC-HIL mediates its negative coregulatory function.

Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion.

Abstract:  Gpnmb is a glycosylated transmembrane protein implicated in the development of glaucoma in mice and melanoma in humans. It shares significant amino acid sequence homology with the melanosome protein Pmel‐17. Its extracellular domain contains a RGD motif for binding to integrin and its intracellular domain has a putative endosomal and/or melanosomal‐sorting motif. These features led us to posit that Gpnmb is associated with melanosomes and involved in cell adhesion. We showed that human Gpnmb is expressed constitutively by melanoma cell lines, primary‐cultured melanocytes and epidermal melanocytes in situ, with most of it found intracellularly within melanosomes and to a lesser degree in lysosomes. Our newly developed monoclonal antibody revealed surface expression of Gpnmb on these pigment cells, albeit to a lesser degree than the intracellular fraction. Gpnmb expression was upregulated by UVA (but not UVB) irradiation and by α‐melanocyte‐stimulating hormone (MSH) (but not β‐MSH); its cell surface expression on melanocytes (but not on melanoma cells) was increased markedly by IFN‐γ and TNF‐α. PAM212 keratinocytes adhered to immobilized Gpnmb in a RGD‐dependent manner. These results indicate that Gpnmb is a melanosome‐associated glycoprotein that contributes to the adhesion of melanocytes with keratinocytes.

The DC-HIL/syndecan-4 pathway inhibits human allogeneic T cell responses

T‐cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC‐HIL on APC to syndecan‐4 (SD‐4) on activated T cells potently inhibits T‐cell activation. In humans, we now show that DC‐HIL also binds to SD‐4 on activated T cells through recognition of its heparinase‐sensitive saccharide moiety. DC‐HIL blocks anti‐CD3‐induced T‐cell responses, reducing secretion of pro‐inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC‐HIL phosphorylates SD‐4s intracellular tyrosine and serine residues. Anti‐SD‐4 Ab mimics the ability of DC‐HIL to attenuate anti‐CD3 response more potently than Ab directed against other inhibitory receptors (CTLA‐4 or programmed cell death‐1). Among leukocytes, DC‐HIL is expressed highest by CD14+ monocytes and this expression can be upregulated markedly by TGF‐β. Among APC, DC‐HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC‐HIL expression on CD14+ monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF‐β reduced this capacity, whereas knocking down the DC‐HIL gene augmented it. Our findings indicate that the DC‐HIL/SD‐4 pathway can be manipulated to treat T‐cell‐driven disorders in humans.

DC-HIL/glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells.

DC-HIL/glycoprotein nmb (Gpnmb) expressed on antigen-presenting cells attenuates T-cell activation by binding to syndecan-4 (SD-4) on activated T cells. Because DC-HIL/Gpnmb is expressed abundantly by mouse and human melanoma lines, we posited that melanoma-associated DC-HIL/Gpnmb exerts similar inhibitory function on melanoma-reactive T cells. We generated small interfering RNA-transfected B16F10 melanoma cells to completely knock down DC-HIL/Gpnmb expression, with no alteration in cell morphology, melanin synthesis, or MHC class I expression. This knockdown had no effect on B16F10 proliferation in vitro or entry into the cell cycle following growth stimulation, but it markedly reduced the growth of these cells in vivo following their s.c. injection into syngeneic immunocompetent (but not immunodeficient) mice. This reduction in tumor growth was due most likely to an augmented capacity of DC-HIL-knocked down B16F10 cells (compared with controls) to activate melanoma-reactive T cells as documented in vitro and in mice. Whereas DC-HIL knockdown had no effect on susceptibility of melanoma to killing by cytotoxic T cells, blocking SD-4 function enhanced the reactivity of CD8(+) T cells to melanoma-associated antigens on parental B16F10 cells. Using an assay examining the spread to the lung following i.v. injection, DC-HIL-knocked down cells produced lung foci at similar numbers compared with that produced by control cells, but the size of the former foci was significantly smaller than the latter. We conclude that DC-HIL/Gpnmb confers upon melanoma the ability to downregulate the activation of melanoma-reactive T cells, thereby allowing melanoma to evade immunologic recognition and destruction. As such, the DC-HIL/SD-4 pathway is a potentially useful target for antimelanoma immunotherapy.

Sézary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-β on the cell surface

Because syndecan-4 (SD-4) on effector and memory T cells inhibits T-cell activation by binding dendritic cell-associated heparan sulfate proteoglycan-integrin ligand (DC-HIL) on antigen presenting cells and because malignant cells of the cutaneous T-cell lymphoma (CTCL) subset, Sézary syndrome (SS), exhibit memory T-cell phenotype, we posited SS cells to express SD-4. Indeed, malignant T cells from patients with SS and from CTCL cell lines constitutively expressed SD-4 at high levels, in contrast to T cells from healthy volunteers and patients with other inflammatory skin diseases and to non-CTCL cell lines that did not. SS cells also bound to DC-HIL at a level higher than normal T cells activated in vitro, resulting in their inhibited proliferation to anti-CD3 antibody. SD-4 on SS cells also trapped transforming growth factor-β1 to their cell surface, enhancing their ability to inhibit activation of syngeneic and allogeneic normal T cells. All of these inhibitory properties were dependent on overexpression of distinct heparan sulfate (HS) moieties by SD-4 on SS cells. Finally, we showed toxin-conjugated DC-HIL to abrogate the ability of SS cells to proliferate in vitro. These findings indicate that SD-4 bearing distinct HS moieties plays a pathogenic role in SS and may be targeted for treatment.

Binding of DC-HIL to dermatophytic fungi induces tyrosine phosphorylation and potentiates antigen presenting cell function.

APCs express receptors recognizing microbes and regulating immune responses by binding to corresponding ligands on immune cells. Having discovered a novel inhibitory pathway triggered by ligation of DC-HIL on APC to a heparin/heparan sulfate-like saccharide of syndecan-4 on activated T cells, we posited DC-HIL can recognize microbial pathogens in a similar manner. We showed soluble recombinant DC-HIL to bind the dermatophytes Trichophyton rubrum and Microsporum audouinii, but not several bacteria nor Candida albicans. Dermatophyte binding was inhibited completely by the addition of heparin. Because DC-HIL contains an ITAM-like intracellular sequence, we questioned whether its binding to dermatophytes can induce tyrosine phosphorylation in dendritic cells (DC). Culturing DC with T. rubrum (but not with C. albicans pseudohyphae) induced phosphorylation of DC-HIL, but not when the tyrosine residue of the ITAM-like sequence was mutated to phenylalanine. To examine the functional significance of such signaling on DC, we cross-linked DC-HIL with mAb (surrogate ligand), which not only induced tyrosine phosphorylation but also up-regulated expression of 23 genes among 662 genes analyzed by gene-array, including genes for profilin-1, myristoylated alanine rich protein kinase C substrate like-1, C/EBP, LOX-1, IL-1β, and TNF-α. This cross-linking also up-regulated expression of the activation markers CD80/CD86 and heightened APC capacity of DC to activate syngeneic T cells. Our findings support a dual role for DC-HIL: inhibition of adaptive immunity following ligation of syndecan-4 on activated T cells and induction of innate immunity against dermatophytic fungi.

The DC‐HIL ligand syndecan‐4 is a negative regulator of T‐cell allo‐reactivity responsible for graft‐versus‐host disease

Acute graft‐versus‐host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. Allo‐reactive T cells are the major mediators of GVHD and the process is regulated by positive and negative regulators on antigen‐presenting cells (APC). Because the significance of negative regulators in GVHD pathogenesis is not fully understood, and having discovered that syndecan‐4 (SD‐4) on effector T cells mediates the inhibitory function of DC‐HIL on APC, we proposed that SD‐4 negatively regulates the T‐cell response to allo‐stimulation in acute GVHD, using SD‐4 knockout mice. Although not different from their wild‐type counterparts in responsiveness to anti‐CD3 stimulation, SD‐4−/− T cells lost the capacity to mediate the inhibitory function of DC‐HIL and were hyper‐reactive to allogeneic APC. Moreover, infusion of SD‐4−/− T cells into sub‐lethally γ‐irradiated allogeneic mice worsened mortality, with hyper‐proliferation of infused T cells in recipients. Although there my be little or no involvement of regulatory T cells in this model because SD‐4 deletion had no deleterious effect on T‐cell‐suppressive activity compared with SD‐4+/+ regulatory T cells. We conclude that SD‐4, as the T‐cell ligand of DC‐HIL, is a potent inhibitor of allo‐reactive T cells responsible for GVHD and a potentially useful target for treating this disease.

The DC-HIL/Syndecan-4 Pathway Regulates Autoimmune Responses through Myeloid-Derived Suppressor Cells

Having discovered that the dendritic cell (DC)–associated heparan sulfate proteoglycan–dependent integrin ligand (DC-HIL) receptor on APCs inhibits T cell activation by binding to syndecan-4 (SD-4) on T cells, we hypothesized that the DC-HIL/SD-4 pathway may regulate autoimmune responses. Using experimental autoimmune encephalomyelitis (EAE) as a disease model, we noted an increase in SD-4+ T cells in lymphoid organs of wild-type (WT) mice immunized for EAE. The autoimmune disease was also more severely induced (clinically, histologically, and immunophenotypically) in mice knocked out for SD-4 compared with WT cohorts. Moreover, infusion of SD-4−/− naive T cells during EAE induction into Rag2−/− mice also led to increased severity of EAE in these animals. Similar to SD-4 on T cells, DC-HIL expression was upregulated on myeloid cells during EAE induction, with CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) as the most expanded population and most potent T cell suppressor among the myeloid cells examined. The critical role of DC-HIL was supported by DC-HIL gene deletion or anti–DC-HIL treatment, which abrogated T cell suppressor activity of MDSCs, and also by DC-HIL activation inducing MDSC expression of IFN-γ, NO, and reactive oxygen species. Akin to SD-4−/− mice, DC-HIL−/− mice manifested exacerbated EAE. Adoptive transfer of MDSCs from EAE-affected WT mice into DC-HIL−/− mice reduced EAE severity to the level of EAE-immunized WT mice, an outcome that was precluded by depleting DC-HIL+ cells from the infused MDSC preparation. Our findings indicate that the DC-HIL/SD-4 pathway regulates autoimmune responses by mediating the T cell suppressor function of MDSCs.

Inhibition of T-cell activation by syndecan-4 is mediated by CD148 through protein tyrosine phosphatase activity.

Most coinhibitory receptors regulate T‐cell responses through an ITIM that recruits protein tyrosine phosphatases (PTPs) to mediate inhibitory function. Because syndecan‐4 (SD‐4), the coinhibitor for DC‐associated heparan sulfate proteoglycan integrin ligand (DC‐HIL), lacks such an ITIM, we posited that SD‐4 links with a PTP in an ITIM‐independent manner. We show that SD‐4 associates constitutively with the intracellular protein syntenin but not with the receptor‐like PTP CD148 on human CD4+ T cells. Binding to DC‐HIL allowed SD‐4 to assemble with CD148 through the help of syntenin as a bridge, and this process upregulated the PTP activity of CD148, which is required for SD‐4 to mediate DC‐HILs inhibitory function. Using a mouse model, we found SD‐4 to be located away from the immunological synapse formed between T cells and APCs during activation of T cells. These findings indicate that SD‐4 is unique among known T‐cell coinhibitors, in employing CD148 to inhibit T‐cell activation at a site distal from the synapse.