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 DC-SIGN) 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 ␥ (FcR␥) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcR␥, activation of NF-B, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor ␣ and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcR␥ chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcR␥ to induce innate immune responses.

IntroductionT-cell activation is dependent on signals delivered by antigenpresenting cells (APCs) to the antigen (Ag)-specific T-cell receptor (TCR) and accessory receptors on T cells. 1 The principal stimulatory accessory signal is transmitted by B7-1 (CD80) or B7-2 (CD86) on APCs to the CD28 receptor on T cells. 2 Interestingly, engagement of the same B7-1 or B7-2 ligand to CTLA-4 (CD152) on T cells markedly attenuates T-cell responses. 3,4 The importance of CTLA-4 as an inhibitory regulator of T-cell activation is illustrated by death of CTLA-4-deficient mice within 4 weeks of birth because of massive lymphocytic infiltration destroying critical organs. 5 More recently, other inhibitory regulators of T-cell activation were identified, including PD-L1 (B7-H1) and PD-L2 (B7-DC) on APCs and PD-1 on T cells, 6 BTLA on B cells and T helper 1 (Th1) effector cells and its ligand (herpes virus entry mediator) on T cells, 7,8 and Tim-3 on APCs and Th1 effector cells and Tim-3 ligand on CD4 ϩ T cells. [9][10][11] The T-cell ligands possess a single immunoglobulin (Ig)-like variable (IgV) domain, and the APC receptors contain both IgV and Ig constant (IgC) domains. 12 Interactions between ligand-receptor pairs are mediated predominantly by residues of Ig-like domains. 12 Because of their structural and functional similarities to B7 molecules, these ligands/receptors are considered members of the B7 receptor superfamily. 12 Ligation of PD-1 on T cells leads to inhibited T-cell responses that can be rescued by exogenous IL-2 or CD28 costimulation, [13][14][15] although one report showed that binding of PD-L1 (B7-H1) to PD-1 stimulated T-cell proliferation and IL-10 secretion. [16][17][18] PD-1 deficiency leads to exaggerated autoimmunity since PD-1 knockout mice develop splenomegaly, increased numbers of B and myeloid cells, increased serum IgG and IgA, and a lupus erythematosuslike disease with age. 19,20 These mice are also markedly susceptible to Ag-induced experimental autoimmune encephalomyelitis (EAE). 19,20 BTLA knockout mice do not exhibit developmental Tor B-cell defects, but their lymphocytes have heightened responses to anti-CD3 antibody (Ab) and to anti-IgM Ab; 8 these mice are also prone to developing EAE. 8 In the case of the Tim-3 pathway, its blockade by monoclonal Ab (mAb), Fc-fused soluble receptor, or gene disruption leads to exacerbated Th1-mediated autoimmune diabetes mellitus in nonobese diabetic (NOD) mice. 10,11 T-cell expression of PD-1, BTLA, or Tim-3 resembles CTLA-4 in that it is not constitutive, but is induced by activation. 21 Moreover, the costimulation delivered by each appears to be mediated through the TCR. 12 By contrast, expression of PD-1, BTLA, Tim-3, or their ligands differ from CTLA-4 in that it is not restricted to T cells, but is expressed more widely to include B cells and APCs. Indeed, some of these ligands (PD-L1 and PD-L2) are also expressed in nonlymphoid tissues. 12 Such broad expression profiles suggest that these molecules can modulate immune responses in secondary lympho...

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 glycosylated transmembrane protein implicated in 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-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 adhesion of melanocytes with keratinocytes.

No abstract

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-4's 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 1 monocytes and this expression can be upregulated markedly by TGF-b. 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 1 monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF-b 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 (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. Cancer Res; 70(14); 5778-87. ©2010 AACR.

Two kinds of bacteria having different-structured angular dioxygenases-a dibenzofuran (DF)-utilizing bacterium, Terrabacter sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp. strain CA10-were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all monoto triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.