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.

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.

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2 ؉ cells after intravenous injection and in CD11c ؉ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8 ؉ T-cell response in HLA-A2 transgenic mice and stimulated a CD4 ؉ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A b . As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.

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.

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.

Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1

ABSTRACT. Age-associated changes of visual evoked potentials by flash stimulation (flash VEP) were evaluated in 53 beagle dogs aged from 1-to 15-year-old. Among the components of flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) peaks by 150 msec, the latency of P2 and the later peaks (N2 and P3) were significantly delayed with aging. Both amplitudes of the P2-N2 and N2-P3 also showed a significant correlation with aging. The flash VEP is considered to be an available and useful technique to evaluate not only for visual pathway, but also some disturbance of neurological functions, like as those reported in demented human. KEY WORDS: age-associated change, canine, flash VEP.J. Vet. Med. Sci. 68(1): 79-82, 2006 Visual evoked potentials (VEPs) indicated electrophysiological responses from retina to visual cortex is reported to be an useful technique for evaluating optic pathway [8] and also to be a sensitive indicator for dementia in human [4,18,22]. Visser et al. [21] demonstrated that the latency of the flash VEP components was delayed in the patients with dementia, especially Alzheimer's disease. The latency of the positive peak 2 (P2 :mean latency was 100 msec) and the later components were significantly delayed, although the similar results were sometimes detected in aged human. The flash VEP recorded in dogs showed 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) components by 150 msec after the flash stimuli, like as those reported in human [11,17,19]. In addition, various age-associated pathological and/or physiological changes were commonly observed in aged dogs as well as aged human [5,10]. Since there are few diagnostic techniques for dementia in dogs, age-associated changes of the flash VEP were evaluated using various aged beagle dogs.Total 53 beagle dogs (25 male and 28 female) aged from 1-to 15-year-old kept in Medical Safety Research Laboratories, Sankyo Co., Ltd., were used.The flash VEP was recorded using needle electrodes inserted in the midline of the scalp according to the method reported by Strain et al. [18]. Briefly, electrodes of recording, reference, and ground were placed over the midline of the nuchal crest, forehead, and vertex, respectively. Prior to recording, the dogs were instilled the mydriatic drug, including tropicamide and phenylephrine hydrochloride, and subcutaneously given atropine sulfate (0.05 mg/kg) and intramuscularly given xylazine (2-3 mg/kg) for the sedation. Then the dogs were dark-adapted at least 1 hr and the flash VEP were recorded in the dark shielded room. The white flash stimulus was given 1/sec with the intensity of 0.6 joule using a photostimulator (SLS-3100 Photostimulator, Nihon Kohden Co., Tokyo, Japan). The xenon lamp was located about 15 cm in front of the examined eye and another eye was covered with the black cloth. The responses were recorded with signal averager (Neuropack Four Mini, Nihon Kohden Co, Tokyo, Japan). The bandpass filter was set at 1 Hz low and 1,000 Hz high. In this study, consecutive 30 responses wer...

SummaryAcute graft-versus-host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. Alloreactive 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 À/À 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 À/À T cells into sub-lethally c-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.