RESUMEN
The epidermis is located in the outermost layer of the living body and is the place where external stimuli such as ultraviolet rays and microorganisms first come into contact. Melanocytes and melanin play a wide range of roles such as adsorption of metals, thermoregulation, and protection from foreign enemies by camouflage. Pigmentary disorders are observed in diseases associated with immunodeficiency such as Griscelli syndrome, indicating molecular sharing between immune systems and the machineries of pigment formation. Melanocytes express functional toll-like receptors (TLRs), and innate immune stimulation via TLRs affects melanin synthesis and melanosome transport to modulate skin pigmentation. TLR2 enhances melanogenetic gene expression to augment melanogenesis. In contrast, TLR3 increases melanosome transport to transfer to keratinocytes through Rab27A, the responsible molecule of Griscelli syndrome. TLR4 and TLR9 enhance tyrosinase expression and melanogenesis through p38 MAPK (mitogen-activated protein kinase) and NFκB signaling pathway, respectively. TLR7 suppresses microphthalmia-associated transcription factor (MITF), and MITF reduction leads to melanocyte apoptosis. Accumulating knowledge of the TLRs function of melanocytes has enlightened the link between melanogenesis and innate immune system.
Asunto(s)
Inmunidad Innata/inmunología , Melaninas/metabolismo , Melanocitos/citología , Melanosomas/metabolismo , Pigmentación de la Piel , Receptores Toll-Like/metabolismo , Animales , Humanos , Melanocitos/inmunología , Melanocitos/metabolismo , Melanosomas/inmunologíaRESUMEN
Hofmeister ions are thought to play fundamentally important roles in protein solubility, folding, stability, and function. Salt ions profoundly influence the course of protein misfolding, aggregation, and amyloid formation associated with devastating human diseases. However, the molecular origin of the salt-effect in protein aggregation remains elusive. Here, we report an unusual biphasic amyloidogenesis of a pH-responsive, intrinsically disordered, oligopeptide repeat domain of a melanosomal protein, Pmel17, that regulates the amyloid-assisted melanin synthesis in mammals via functional amyloid formation. We demonstrate that a symphony of molecular events involving charge-peptide interactions and hydration, in conjunction with secondary phenomena, critically governs the course of this biphasic amyloid assembly. We show that at mildly acidic pH, typical of melanosomes, highly amyloidogenic oligomeric units assemble into metastable, dendritic, fractal networks following the forward Hofmeister series. However, the subsequent condensation of fractal networks via conformational maturation into amyloid fibrils follows an inverse Hofmeister series due to fragmentation events coupled with secondary nucleation processes. Our results indicate that ions exert a strong influence on the aggregation kinetics as well as on the nanoscale morphology and also modulate the autocatalytic amplification processes during amyloid assembly via an intriguing dual Hofmeister effect. This unique interplay of molecular drivers will be of prime importance in delineating the aggregation pathways of a multitude of intrinsically disordered proteins involved in physiology and disease.
Asunto(s)
Amiloide/genética , Proteínas Amiloidogénicas/genética , Amiloidosis/genética , Antígeno gp100 del Melanoma/genética , Amiloide/química , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Amiloidosis/patología , Humanos , Concentración de Iones de Hidrógeno , Proteínas Intrínsecamente Desordenadas , Iones , Cinética , Melaninas/biosíntesis , Melanosomas/genética , Melanosomas/inmunología , Agregado de Proteínas/genética , Antígeno gp100 del Melanoma/metabolismoRESUMEN
Skin depigmentation diseases, such as vitiligo, are pigmentation disorders that often destroy melanocytes. However, their pathological mechanisms remain unclear, and therefore, promising treatments or prevention has been lacking. Here, we demonstrate that a zebrafish insertional mutant showing a significant reduction of nicastrin transcript possesses melanosome maturation defect, Tyrosinase-dependent mitochondrial swelling, and melanophore cell death. The depigmentation phenotypes are proven to be a result of γ-secretase inactivation. Furthermore, live imaging demonstrates that macrophages are recruited to and can phagocytose melanophore debris. Thus, we characterize a potential zebrafish depigmentation disease model, a nicastrinhi1384 mutant, which can be used for further treatment or drug development of diseases related to skin depigmentation and/or inflammation.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/genética , Hipopigmentación/genética , Glicoproteínas de Membrana/genética , Monofenol Monooxigenasa/metabolismo , Piel/inmunología , Proteínas de Pez Cebra/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Embrión no Mamífero , Humanos , Hipopigmentación/inmunología , Hipopigmentación/patología , Melanosomas/inmunología , Melanosomas/metabolismo , Melanosomas/ultraestructura , Glicoproteínas de Membrana/metabolismo , Microscopía Electrónica de Transmisión , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/genética , Mutación , Piel/patología , Pigmentación de la Piel/efectos de los fármacos , Pigmentación de la Piel/genética , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Vitiligo is a common depigmentation disorder characterized by a loss of functional melanocytes and melanin from epidermis, in which the autoantigens and subsequent autoimmunity caused by oxidative stress play significant roles according to hypotheses. Various factors lead to reactive oxygen species (ROS) overproduction in the melanocytes of vitiligo: the exogenous and endogenous stimuli that cause ROS production, low levels of enzymatic and non-enzymatic antioxidants, disturbed antioxidant pathways and polymorphisms of ROS-associated genes. These factors synergistically contribute to the accumulation of ROS in melanocytes, finally leading to melanocyte damage and the production of autoantigens through the following ways: apoptosis, accumulation of misfolded peptides and cytokines induced by endoplasmic reticulum stress as well as the sustained unfolded protein response, and an 'eat me' signal for phagocytic cells triggered by calreticulin. Subsequently, autoantigens presentation and dendritic cells maturation occurred mediated by the release of antigen-containing exosomes, adenosine triphosphate and melanosomal autophagy. With the involvement of inducible heat shock protein 70, cellular immunity targeting autoantigens takes the essential place in the destruction of melanocytes, which eventually results in vitiligo. Several treatments, such as narrow band ultraviolet, quercetin and α-melanophore-stimulating hormone, are reported to be able to lower ROS thereby achieving repigmentation in vitiligo. In therapies targeting autoimmunity, restore of regulatory T cells is absorbing attention, in which narrow band ultraviolet also plays a role.
Asunto(s)
Autoantígenos/biosíntesis , Estrés Oxidativo , Vitíligo/inmunología , Vitíligo/metabolismo , Animales , Autoinmunidad , Daño del ADN/inmunología , Humanos , Melanocitos/inmunología , Melanocitos/metabolismo , Melanosomas/inmunología , Melanosomas/metabolismo , Modelos Inmunológicos , Especies Reactivas de Oxígeno/metabolismo , Vitíligo/terapiaRESUMEN
BACKGROUND: Patients with oculocutaneous albinism (OCA) have severely decreased pigmentation of their skin, hair and eyes. OCA2 and OCA4 result from mutations of the OCA2 and SLC45A2 genes, respectively, both of which disrupt the trafficking of the critical melanogenic enzyme tyrosinase to melanosomes. Both proteins encoded by those loci (termed P and MATP, respectively) have 12 putative transmembrane regions and are thought to function as transporters, although their functions and subcellular localizations remain to be characterized. OBJECTIVE: To generate specific antibodies against unique synthetic peptides encoded by P and MATP that could be used to characterize their functions and subcellular localizations. METHODS: Western blotting and immunohistochemistry were used to assess the specificity of antibodies and to colocalize P and MATP proteins with various subcellular markers. RESULTS: Specific antibodies to the P and MATP proteins were generated that work well for Western blotting and immunohistochemistry. The localizations of P and MATP with various subcellular organelles were characterized using confocal microscopy, which revealed that they colocalize to some extent with LAMP2, but do not significantly colocalize with markers of the ER, Golgi or melanosomes. Interestingly, both P and MATP colocalize significantly with BLOC-1, a sorting component involved in the intracellular trafficking of melanosomal/lysosomal constituents. CONCLUSION: These results provide a basis to understand how disrupted functions of P or MATP result in the misrouting of tyrosinase and cause the hypopigmentation seen in OCA2 and OCA4.
Asunto(s)
Albinismo Oculocutáneo/inmunología , Anticuerpos/química , Hipopigmentación/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/metabolismo , Transporte Biológico , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Inmunohistoquímica , Masculino , Melanocitos/citología , Melanosomas/inmunología , Melanosomas/metabolismo , Datos de Secuencia Molecular , Monofenol Monooxigenasa/inmunología , Monofenol Monooxigenasa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Péptidos/química , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
PMEL, also known as Pmel17 or gp100, is a melanocyte-specific glycoprotein that is essential for the formation of stage II melanosomes. As it has a highly restricted expression pattern in normal tissues and a transient presence on the cell surface, PMEL is believed to be a potential target for antibody drug conjugate therapy in some pigmentary diseases. The production of a high specificity and high affinity monoclonal antibody against human PMEL was helpful for the antibody drug conjugate therapy study. In the present study, monoclonal antibodies (MAbs) against PMEL were obtained by immunizing BALB/c mice with the recombinant PMEL-GST fusion protein. Three mAbs (A3F, G11B, and J7E) with a titer of 1:6000, 1:10,000, and 1:3000, respectively, were obtained. Immunoglobulin subclass assay revealed that A3F was IgG2b, G11B was IgG1, and J7E was IgG2a. Specificity analysis by Western blotting demonstrated that A3F and J7E cross-reacted with GPNMB or LAMP; however, G11B reacted with PMEL only. Immunohistochemistry experiments showed that G11B could bind human PMEL antigen in normal skin. Flow cytometry assay demonstrated that G11B could bind to the surface of PMEL positive melanoma cells but not PMEL negative cells. Taken together, these results show that this G11B provides a useful tool for the antibody drug conjugate therapy study in some pigmentary diseases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígeno gp100 del Melanoma/inmunología , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Reacciones Cruzadas/inmunología , Humanos , Inmunoglobulinas/inmunología , Células Jurkat , Melanocitos/inmunología , Melanoma/inmunología , Melanosomas/inmunología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicans is an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicans supernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection.
Asunto(s)
Candida albicans/patogenicidad , Candidiasis/inmunología , Melaninas/metabolismo , Melanocitos/inmunología , Receptor Toll-Like 4/inmunología , Anticuerpos Antifúngicos/inmunología , Antifúngicos/inmunología , Antifúngicos/metabolismo , Antígenos Fúngicos/inmunología , Antígenos Fúngicos/metabolismo , Candida albicans/inmunología , Candida albicans/metabolismo , Candidiasis/microbiología , Interacciones Huésped-Patógeno , Humanos , Hifa/fisiología , Inmunidad Innata , Melaninas/inmunología , Melanocitos/metabolismo , Melanocitos/microbiología , Melanosomas/inmunología , Esporas Fúngicas/fisiología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Short-lived protein translation products are proposed to be a major source of substrates for major histocompatibility complex (MHC) class I antigen processing and presentation; however, a direct link between protein stability and the presentation level of MHC class I-peptide complexes has not been made. We have recently discovered that the peptide Tyr((369-377)) , derived from the tyrosinase protein is highly presented by HLA-A2 on the surface of melanoma cells. To examine the molecular mechanisms responsible for this presentation, we compared characteristics of tyrosinase in melanoma cells lines that present high or low levels of HLA-A2-Tyr((369-377)) complexes. We found no correlation between mRNA levels and the levels of HLA-A2-Tyr((369-377)) presentation. Co-localization experiments revealed that, in cell lines presenting low levels of HLA-A2-Tyr((369-377)) complexes, tyrosinase co-localizes with LAMP-1, a melanosome marker, whereas in cell lines presenting high HLA-A2-Tyr((369-377)) levels, tyrosinase localizes to the endoplasmic reticulum. We also observed differences in tyrosinase molecular weight and glycosylation composition as well as major differences in protein stability (t(1/2) ). By stabilizing the tyrosinase protein, we observed a dramatic decrease in HLA-A2-tyrosinase presentation. Our findings suggest that aberrant processing and instability of tyrosinase are responsible for the high presentation of HLA-A2-Tyr((369-377)) complexes and thus shed new light on the relationship between intracellular processing, stability of proteins, and MHC-restricted peptide presentation.
Asunto(s)
Presentación de Antígeno , Antígeno HLA-A2/inmunología , Melanoma/inmunología , Monofenol Monooxigenasa/inmunología , Proteínas de Neoplasias/inmunología , Línea Celular Tumoral , Glicosilación , Antígeno HLA-A2/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas/inmunología , Proteínas de Membrana de los Lisosomas/metabolismo , Melanoma/metabolismo , Melanosomas/inmunología , Melanosomas/metabolismo , Monofenol Monooxigenasa/metabolismo , Proteínas de Neoplasias/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Estabilidad Proteica , ARN Mensajero/inmunología , ARN Mensajero/metabolismoRESUMEN
Melanocyte destruction in the skin of vitiligo patients has been considered to be a consequence of an autoimmune response against melanosomal proteins. However, little is known about the molecular mechanisms by which the immune system recognizes these sequestered intracellular self-proteins, which are confined in specialized organelles termed melanosomes, and is provoked into an autoimmune response to melanocytes. Here, we utilize a sucrose density-gradient ultracentrifugation protocol to enrich melanosomal components from dopachrome tautomerase (Dct)-mutant or wild-type melanocytes exposed to a pulse of hydrogen peroxide at a noncytotoxic concentration to evaluate their immunogenicity in mice challenged with the corresponding melanosomal proteins. The results demonstrate that enhanced humoral and cellular immune responses to a challenge with late-stage melanosomal proteins, especially with those derived from Dct-mutant melanocytes, are found in the immunized mice. To elucidate whether a reduced 5,6-dihydroxyindole-2-carboxylic acid (DHICA) content in melanin might cause a loss in antioxidative protection to the proteins, we incubated these melanosomal proteins in vitro with synthetic 5,6-dihydroindole (DHI)-melanin or DHI/DHICA (1:1)-melanin and then used them to immunize mice. T cell proliferation and IgG antibody responsiveness to the challenges were significantly induced by melanosomal proteins treated with DHI-melanin, but not by those treated with DHI/DHICA (1:1)-melanin. Moreover, we observed that melanosomal proteins derived from Dct-mutant melanocytes are subject to oxidative modifications that alter their antigenic configurations to attain an enhanced immunogenicity compared with those derived from wild-type melanocytes. From these results, we conclude that DHICA-mediated antioxidation plays a critical role in the maintenance of immune hyporesponsiveness to melanosomal proteins.
Asunto(s)
Tolerancia Inmunológica , Indoles/metabolismo , Oxidorreductasas Intramoleculares/inmunología , Melaninas , Vitíligo/inmunología , Animales , Formación de Anticuerpos/inmunología , Antioxidantes/metabolismo , Autoinmunidad , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Peróxido de Hidrógeno/metabolismo , Inmunización Secundaria , Indoles/química , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Melaninas/inmunología , Melaninas/metabolismo , Melanocitos/inmunología , Melanocitos/metabolismo , Melanosomas/inmunología , Melanosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Mutación/inmunología , Oxidación-Reducción , Piel/inmunología , Piel/metabolismo , Piel/patología , Linfocitos T/inmunología , Vitíligo/metabolismo , Vitíligo/patologíaRESUMEN
Many human solid tumors express MHC class II (MHC-II) molecules, and proteins normally localized to melanosomes give rise to MHC-II-restricted epitopes in melanoma. However, the pathways by which this response occurs have not been defined. We analyzed the processing of one such epitope, gp100(44-59), derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1(high)/MHC-II(+) late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp100(44-59) presentation. By depletion of the AP-2 adaptor protein using small interfering RNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp100(44-59) epitope production. The gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC-II molecules. The gp100(44-59) presentation is dramatically reduced, and processing occurs entirely in early endosomes or stage I melanosomes. This occurrence suggests that melanosomes are inefficient Ag-processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC-II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Endosomas/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/inmunología , Melanoma/inmunología , Melanosomas/inmunología , Glicoproteínas de Membrana/inmunología , Complejo 2 de Proteína Adaptadora/inmunología , Moléculas de Adhesión Celular Neuronal/inmunología , Desdiferenciación Celular/inmunología , Línea Celular Tumoral , Proteínas Ligadas a GPI , Humanos , Transporte de Proteínas/inmunología , ARN Interferente Pequeño/inmunología , Antígeno gp100 del MelanomaRESUMEN
Vitiligo is characterized by progressive skin depigmentation resulting from an autoimmune response targeting epidermal melanocytes. Melanocytes are particularly immunogenic by virtue of the contents of their melanosomes, generating the complex radical scavenging molecule melanin in a process that involves melanogenic enzymes and structural components, including tyrosinase, MART-1, gp100, TRP-2 and TRP-1. These molecules are also prime targets of the immune response in both vitiligo and melanoma. The immunogenicity of melanosomal proteins can partly be explained by the dual role of melanosomes, involved both in melanin synthesis and processing of exogenous antigens. Melanocytes are capable of presenting antigens in the context of MHC class II, providing HLA-DR+ melanocytes in perilesional vitiligo skin the option of presenting melanosomal antigens in response to trauma and local inflammation. Type I cytokine-mediated immunity to melanocytes in vitiligo involves T cells reactive with melanosomal antigens, similar to T cells observed in melanoma. In vitiligo, however, T cell tuning allows T cells with higher affinity for melanocyte differentiation antigens to enter the circulation after escaping clonal deletion in primary lymphoid organs. The resulting efficacious and progressive autoimmune response to melanocytes provides a roadmap for melanoma therapy.
Asunto(s)
Autoantígenos/inmunología , Autoinmunidad , Melanosomas/inmunología , Pigmentación/inmunología , Linfocitos T/inmunología , Vitíligo/inmunología , Animales , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Supresión Clonal , Antígenos HLA-DR/inmunología , Humanos , Oxidorreductasas Intramoleculares/inmunología , Antígeno MART-1 , Melaninas/inmunología , Melanosomas/enzimología , Melanosomas/patología , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Oxidorreductasas/inmunología , Linfocitos T/enzimología , Linfocitos T/patología , Vitíligo/enzimología , Vitíligo/patología , Antígeno gp100 del MelanomaRESUMEN
Dendritic cells (DCs) can capture apoptotic cells and present them to immune competent cells as self-antigens (Ags). Langerhans cells (LCs), DCs in the epidermis, are capable of presenting tissue-associated Ags in the steady state, suggesting that LCs may also capture apoptotic cells and transport them to skin regional lymph nodes (LNs). However, to what extent LCs utilize apoptotic cells as self-Ags in vivo is still unclear. To clarify this point, we examined the contribution of milk fat globule EGF factor 8 (MFG-E8), a secreted glycoprotein, to capturing skin Ags. MFG-E8 is expressed in several subsets of macrophages (M phi s) and DCs, including LCs, and crucial for recognizing and engulfing apoptotic cells. Using a skin-hyperpigmented KRT14-Kitl-Tg (Kitl-Tg) mouse system, we measured the accumulation of melanin granules (MGs), a marker of skin Ags, transported from the skin to regional LNs in Mfge8-deficient mice. Unexpectedly, their accumulation in Mfge8-deficient Kitl-Tg mice was comparable to that in Mfge8-heterozygous littermates. Mfge8-deficient DCs engulfed skin-derived MGs efficiently in vitro. The results indicate that MFG-E8 does not contribute critically or functions redundantly to capturing and trafficking of skin Ags in the steady state, and suggest a possibility that LCs may capture skin Ags in forms other than apoptotic cells in vivo.
Asunto(s)
Antígenos de Superficie/metabolismo , Apoptosis , Células de Langerhans/inmunología , Proteínas de la Leche/metabolismo , Piel/inmunología , Animales , Antígenos de Superficie/genética , Gránulos Citoplasmáticos/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células de Langerhans/citología , Células de Langerhans/metabolismo , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Melaninas/inmunología , Melanosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Proteínas de la Leche/genética , Pigmentación de la PielRESUMEN
CD4+ T lymphocytes play an important role in CD8+ T cell-mediated responses against tumors. Considering that approximately 20% of melanomas express MHC class II, it is plausible that concomitant presentation by MHC class I and class II shapes positive (helper T cells) or negative (regulatory T cells) antitumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we showed that gp100 putative NH2-terminal signal sequence and the last 70 residues in COOH terminus are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1+/HLA-DR+ endosomal/MIIC. Gp100 targeting sequences were characterized by deleting different sections in the COOH terminus (last 70 residues). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in COOH terminus resulted in decreased MHC class II presentation, without effects on class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100 targeting sequences to mobilize green fluorescent protein to endosomal compartments and to allow MHC class II and class I presentation of minimal endogenous epitopes. We conclude that these specific sequences are MIIC-targeting motifs, which could be included in expression cassettes for endogenously expressed tumor or viral antigens for MHC class II and class I presentation and optimize in vivo T-cell responses or as an in vitro tool for characterization of new MHC class II epitopes.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos HLA-DR/inmunología , Melanosomas/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Antígenos CD40/inmunología , Endosomas/inmunología , Endosomas/metabolismo , Epítopos/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Antígenos HLA-DR/genética , Humanos , Proteínas de Membrana de los Lisosomas/inmunología , Melanosomas/genética , Melanosomas/metabolismo , Glicoproteínas de Membrana/farmacología , Proteínas de Neoplasias/farmacología , Linfocitos T/inmunología , Transfección , Antígeno gp100 del MelanomaRESUMEN
Subclasses of immunoglobulin G (IgG) display substantial differences in their ability to mediate effector responses, contributing to variable activity of antibodies against microbes and tumors. We demonstrate that the mechanism underlying this long-standing observation of subclass dominance in function is provided by the differential affinities of IgG subclasses for specific activating IgG Fc receptors compared with their affinities for the inhibitory IgG Fc receptor. The significant differences in the ratios of activating-to-inhibitory receptor binding predicted the in vivo activity. We suggest that these highly predictable functions assigned by Fc binding will be an important consideration in the design of therapeutic antibodies and vaccines.
Asunto(s)
Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Animales , Plaquetas/inmunología , Línea Celular , Femenino , Melanosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Receptores de IgG/clasificaciónRESUMEN
Melanosomes are specialized intracellular compartments within melanocytes and retinal pigment epithelial cells that function in the synthesis, storage, and secretion of melanins, which are the major pigments made by mammals. The mechanisms that regulate the formation of melanosomes, and the pathways by which constituent proteins are targeted to them, are related to those involved in the biogenesis of major histocompatibility complex (MHC) class II antigen-processing compartments. Consequently, diseases that affect pigmentation may also affect antigen presentation to T cells. Moreover, many of the tissue-specific proteins that localize to melanosomes and participate in melanin formation double as tumor-associated antigens that are targets for T cells in patients with melanoma. Our studies on melanosome biogenesis are providing new ways of thinking about antigen-processing compartments and the mechanisms regulating presentation of tumor-associated antigens.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Melanosomas/inmunología , Animales , Humanos , Transporte de Proteínas/inmunologíaRESUMEN
To identify novel, tumor-specific target antigens for vaccine development, we studied immune responses to P.polypeptide, an M(r) 110,000 integral melanosomal membrane protein associated with the Prader-Willi syndrome. Together with expressed sequence tag (EST) and serial analyses of gene expression (SAGE) library analyses, reverse transcription-PCR and Northern blotting verified that P.polypeptide expression was limited to melanoma and melanocytes. A single dominant epitope corresponding to positions 427-435 (IMLCLIAAV) was identified using allele-specific epitope forecasting combined with work in HLA-A*0201/K(b) transgenic mice. This epitope was then used to generate de novo human P.polypeptide-specific CD8+ T cells capable of recognizing P.polypeptide expressing human tumor cell lines in an HLA-A*0201-restricted fashion. Thus, P.polypeptide may be valuable in the creation of novel therapeutic anticancer vaccines.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Melanoma/inmunología , Melanosomas/inmunología , Péptidos/inmunología , Animales , Presentación de Antígeno , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Epítopos de Linfocito T/inmunología , Antígenos H-2/inmunología , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide-specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross-reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte-specific markers, tyrosinase, tyrosinase-related protein (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.
Asunto(s)
Especificidad de Anticuerpos , Lentigo/patología , Melanocitos/química , Melanocitos/inmunología , Glicoproteínas de Membrana , Oxidorreductasas , Piel/química , Adulto , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secciones por Congelación , Humanos , Inmunohistoquímica , Recién Nacido , Oxidorreductasas Intramoleculares/análisis , Oxidorreductasas Intramoleculares/inmunología , Queratinocitos/química , Queratinocitos/enzimología , Queratinocitos/inmunología , Melanocitos/enzimología , Melanoma/patología , Melanosomas/química , Melanosomas/enzimología , Melanosomas/inmunología , Datos de Secuencia Molecular , Monofenol Monooxigenasa/análisis , Monofenol Monooxigenasa/inmunología , Nevo Intradérmico/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas/análisis , Proteínas/inmunología , Conejos , Piel/citología , Piel/enzimología , Neoplasias Cutáneas/patología , Pigmentación de la Piel , Antígeno gp100 del MelanomaRESUMEN
Melanogenesis cascade may be directly or indirectly linked to the dynamics of endosome-lysosome biogenesis. This study aims to identify how and to what extent the endosome-lysosome system is involved in melanosome biogenesis, by utilizing a novel melanogenesis marker, J1, which we identified in the process of developing monoclonal antibodies (MoAbs) against human melanosomes. The antigenic epitope of MoAb J1 was expressed by all of the melanotic and nonmelanotic cells examined. It was expressed primarily by granular structures located in regions proximal to the Golgi complex. Most of MoAb J1 positive granules were co-stained with melanogenic markers, tyrosinase or tyrosinase-related protein (TRP-1). The epitope of MoAb J1 was also coexpressed by most, but not all, of LGP85 (a lysosomal marker) positive granules in both melanoma and non-melanoma cells, indicating that MoAb J1 recognizes a subset of lysosomal vesicles. MoAb J1 did not, however, react with vesicles with late/early (syntaxin 8/ EEA1) endosomal markers. Further examination using fluorophore-labeled pepstatin, a marker of lysosomal luminal content, confirmed that MoAb J1 specifically recognizes the luminal surface of lysosomes. These results indicate that MoAb J1 possesses an antigen epitope that is expressed in the luminal component of prelysosomal granules which are involved in the biogenesis cascade common to both melanosomes and lysosomes. We suggest that tyrosinase family protein, tyrosinase and TRP-1 are transported to melanosomes from TGN via these prelysosomal granules after being transiently transported to late endosomes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Lisosomas/inmunología , Melanosomas/inmunología , Animales , Ratones , Conejos , Células Tumorales CultivadasRESUMEN
Melanosomal membrane proteins are frequently recognized by the immune system of patients with melanoma and vitiligo. Melanosomal glycoproteins are transported to melanosomes by a dileucine-based melanosomal transport signal (MTS). To investigate whether this sorting signal could be involved in presentation of melanosome membrane proteins to the immune system, we devised a fusion construct containing the MTS from the mouse brown locus product gp75/tyrosinase-related protein-1 and full-length OVA as a reporter Ag. The fusion protein was expressed as an intracellular membrane protein, sorted to the endocytic pathway, processed, and presented by class II MHC molecules. DNA immunization with this construct elicited CD4+ T cell proliferative responses in vivo. Ag presentation and T cell responses in vitro and in vivo required a functional MTS. Mutations of either the upstream leucine in MTS or elimination of the entire MTS negated in vitro Ag presentation and in vivo T cell responses. In a mouse melanoma model, DNA immunization with MTS constructs protected mice from tumor challenge in a CD4+ T cell-dependent manner, but complete deletion of MTS decreased tumor rejection. Therefore, MTS can target epitopes to the endocytic pathway leading to presentation by class II MHC molecules to helper T cells.
Asunto(s)
Secuencia de Aminoácidos , Presentación de Antígeno , Linfocitos T CD4-Positivos/inmunología , Antígenos de Histocompatibilidad Clase II , Melanoma Experimental/inmunología , Melanosomas/inmunología , Animales , Transporte Biológico , Vacunas contra el Cáncer/inmunología , Endocitosis , Femenino , Glicoproteínas/genética , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Melanoma Experimental/prevención & control , Melanosomas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Vacunación , Vacunas de ADN/inmunologíaRESUMEN
The adoptive transfer of tumor-infiltrating lymphocytes (TIL) along with interleukin (IL)-2 into autologous patients with cancer resulted in the objective regression of tumor, indicating that T cells play an important role in tumor regression. In the last few years, efforts have been made towards understanding the molecular basis of T-cell-mediated antitumor immunity and elucidating the molecular nature of tumor antigens recognized by T cells. Tumor antigens identified thus far could be classified into several categories: tissue-specific differentiation antigens, tumor-specific shared antigens and tumor-specific unique antigens. CD4+ T cells play a central role in orchestrating the host immune response against cancer, infectious diseases and autoimmune diseases, and we thus have attempted to identify major histocompatibility complex (MHC) class II-restricted tumor antigens as well. The identification of tumor rejection antigens provides new opportunities for the development of therapeutic strategies against cancer. This review will summarize the current status of MHC class I- and class II-restricted human tumor antigens, and their potential application to cancer treatment.