RESUMO
Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags.
Assuntos
Apresentação de Antígeno/imunologia , Autofagia/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Linfócitos B/imunologia , Separação Celular , Citoplasma/imunologia , Eletroporação , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Isoformas de Proteínas/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND: Recurrent genomic changes in B-lymphoblastic leukemia (B-ALL) identified by genome-wide single-nucleotide polymorphism (SNP) microarray analysis provide important prognostic information, but gene copy number analysis of its rare lymphoma counterpart, B-lymphoblastic lymphoma (B-LBL), is limited by the low incidence and lack of fresh tissue for genomic testing. PROCEDURE: We used molecular inversion probe (MIP) technology to analyze and compare copy number alterations (CNAs) in archival formalin-fixed paraffin-embedded pediatric B-LBL (n = 23) and B-ALL (n = 55). RESULTS: Similar to B-ALL, CDKN2A/B deletions were the most common alteration identified in 6/23 (26%) B-LBL cases. Eleven of 23 (48%) B-LBL patients were hyperdiploid, but none showed triple trisomies (chromosomes 4, 10, and 17) characteristic of B-ALL. IKZF1 and PAX5 deletions were observed in 13 and 17% of B-LBL, respectively, which was similar to the reported frequency in B-ALL. Immunoglobulin light chain lambda (IGL) locus deletions consistent with normal light chain rearrangement were observed in 5/23 (22%) B-LBL cases, compared with only 1% in B-ALL samples. None of the B-LBL cases showed abnormal, isolated VPREB1 deletion adjacent to IGL locus, which we identified in 25% of B-ALL. CONCLUSIONS: Our study demonstrates that the copy number profile of B-LBL is distinct from B-ALL, suggesting possible differences in pathogenesis between these closely related diseases.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Feminino , Formaldeído , Humanos , Leucemia de Células B/genética , Linfoma de Células B/genética , Masculino , Inclusão em Parafina , Fixação de TecidosRESUMO
Development of novel strategies to treat noninfectious posterior uveitis is an ongoing challenge, in part because of limited availability of animal models that mimic the naturally occurring disease in humans. Mice deficient in the autoimmune regulatory gene Aire develop a spontaneous T-cell and macrophage-mediated autoimmune uveitis that closely recapitulates human endogenous uveitis and thus provide a useful model for mechanistic and therapeutic investigations. Lymphocytic and mononuclear infiltration of the retina in Aire knockout (KO) mice triggers the onset of uveitis from initial retinal inflammation to eventual destruction of the neuroretina with loss of photoreceptors. The C-C chemokine receptor type 2 protein (CCR2) functions in directing monocyte and macrophage migration to inflamed tissues via interaction with monocyte chemotactic proteins. Using the Aire KO mouse model, we demonstrated an essential role for CCR2 in the pathogenesis of autoimmune-mediated uveitis. Loss of functional CCR2 effectively reduced immune cell infiltration and rescued the retina from destruction. CCR2-dependent migration of bone marrow-derived cells provided the driving force for retinal inflammation, with CCR2-expressing mononuclear cells contributing to retinal damage via recruitment of CD4(+) T cells. These studies identify the CCR2 pathway as a promising therapeutic target that may prove an effective approach to treat uveitis associated with autoimmunity.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Movimento Celular/imunologia , Receptores CCR2/imunologia , Retina/imunologia , Uveíte/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/patologia , Movimento Celular/genética , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/imunologia , Monócitos/patologia , Receptores CCR2/genética , Retina/patologia , Uveíte/genética , Uveíte/patologiaRESUMO
Lymphocytic infiltration of the lacrimal gland and ocular surface in autoimmune diseases such as Sjögren's syndrome (SS) causes an aqueous-deficient dry eye that is associated with significant morbidity. Previous studies from our laboratory and others have established autoimmune regulator (Aire)-deficient mice as a useful model to examine exocrinopathy and ocular surface disease associated with SS. Consistent with human SS, autoreactive CD4(+) T cells play an indispensible role in the development of exocrine and ocular surface disease in Aire knockout mice. We report that in addition to CD4(+) T cells, a large number of macrophages infiltrate the corneal stroma, limbus, and lacrimal glands of diseased mice. Adoptive transfer of autoreactive CD4(+) T cells from Aire knockout mice led to local infiltration of macrophages and ocular surface damage in immunodeficient recipients. Depletion of local macrophages, through subconjunctival injection of clodronate liposome, attenuated lissamine green staining and improved ocular phenotype. Alternatively, systemic depletion of macrophages had no effect on ocular phenotype but led to significant improvements in lacrimal gland exocrinopathy and tear secretion. Our results suggested that autoreactive CD4(+) T cells provoked macrophage infiltration to the eye and lacrimal gland, where they played a functional role in directing the development of autoimmune dry eye.
Assuntos
Movimento Celular/imunologia , Síndromes do Olho Seco/complicações , Olho/patologia , Macrófagos/imunologia , Macrófagos/patologia , Síndrome de Sjogren/complicações , Síndrome de Sjogren/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Ácido Clodrônico , Substância Própria/patologia , Síndromes do Olho Seco/imunologia , Olho/imunologia , Humanos , Limbo da Córnea/patologia , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Fenótipo , Síndrome de Sjogren/patologia , Lágrimas/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Proteína AIRERESUMO
Keratinizing squamous metaplasia (SQM) of the ocular mucosal epithelium is a blinding corneal disease characterized by the loss of conjunctival goblet cells (GCs), pathological ocular surface keratinization and tissue recruitment of immune cells. Using the autoimmune regulator (Aire)-deficient mouse as a model for Sjögren's syndrome (SS)-associated SQM, we identified CD4(+) T lymphocytes as the main immune effectors driving SQM and uncovered a pathogenic role for interleukin-1 (IL-1). IL-1, a pleiotropic cytokine family enriched in ocular epithelia, governs tissue homeostasis and mucosal immunity. Here, we used adoptive transfer of autoreactive CD4(+) T cells to dissect the mechanism whereby IL-1 promotes SQM. CD4(+) T cells adoptively transferred from both Aire knockout (KO) and Aire/IL-1 receptor type 1 (IL-1R1) double KO donors conferred SQM to severe-combined immunodeficiency (scid) recipients with functional IL-1R1, but not scid recipients lacking IL-1R1. In the lacrimal gland, IL-1R1 was primarily immunolocalized to ductal epithelium surrounded by CD4(+) T cells. In the eye, IL-1R1 was expressed on local mucosal epithelial and stromal cells, but not on resident antigen-presenting cells or infiltrating immune cells. In both tissues, autoreactive CD4(+) T-cell infiltration was only observed in the presence of IL-1R1-postive resident cells. Moreover, persistent activation of IL-1R1 signaling led to chronic immune-mediated inflammation by retaining CD4(+) T cells in the local microenvironment. Following IL-1R1-dependent infiltration of CD4(+) T cells, we observed SQM hallmarks in local tissues-corneal keratinization, conjunctival GC mucin acidification and epithelial cell hyperplasia throughout the ocular surface mucosa. Proinflammatory IL-1 expression in ocular epithelial cells significantly correlated with reduced tear secretion, while CD4(+) T-cell infiltration of the lacrimal gland predicted the development of ocular SQM. Collectively, data in this study indicated a central role for IL-1 in orchestrating a functional interplay between immune cells and resident cells of SS-targeted tissues in the pathogenesis of SQM.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Ceratoconjuntivite Seca/etiologia , Receptores de Interleucina-1/metabolismo , Síndrome de Sjogren/complicações , Transferência Adotiva , Animais , Células Caliciformes/patologia , Interleucina-1/metabolismo , Ceratoconjuntivite Seca/metabolismo , Ceratoconjuntivite Seca/patologia , Aparelho Lacrimal/imunologia , Aparelho Lacrimal/metabolismo , Metaplasia/imunologia , Metaplasia/metabolismo , Camundongos , Camundongos Knockout , Mucosa/imunologia , Mucosa/metabolismo , Mucosa/patologia , Transdução de Sinais , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologiaRESUMO
Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4(+) T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide-binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II-mediated antigen presentation. Lysosome-associated membrane proteins such as LAMP-2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP-2 were examined for changes in MHC class II-restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4(+) T cells was impaired in the LAMP-2-deficient B cells. Peptide-binding to MHC class II on LAMP-2-deficient B cells was reduced at physiological pH compared with wild-type cells. However, peptide-binding and class II-restricted antigen presentation were restored by incubation of LAMP-2-negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP-2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP-2-deficient B cells. Consequently, LAMP-2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.
Assuntos
Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Doença de Depósito de Glicogênio Tipo IIb/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Apresentação de Antígeno/genética , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linhagem Celular Tumoral , Doença de Depósito de Glicogênio Tipo IIb/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Ativação Linfocitária , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Camundongos , Mutação/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genéticaAssuntos
Linfoma de Burkitt/complicações , Linfoma de Burkitt/diagnóstico , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/etiologia , Adulto , Anticorpos Monoclonais Murinos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma de Burkitt/tratamento farmacológico , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Endoscopia Gastrointestinal , Endossonografia , Humanos , Masculino , Prednisona/uso terapêutico , Rituximab , Neoplasias Gástricas/tratamento farmacológico , Vincristina/uso terapêuticoRESUMO
OBJECTIVES: We describe a rare case of a male child with X-linked lymphoproliferative disease type 1 (XLP1) who presented with Burkitt lymphoma (BL) when he was 6 years old, achieved a complete response to therapy, and developed a second BL after seven years. METHODS: Diagnostic H&E stained slides and ancillary studies were reviewed for both lymphomas. B-cell clonality by PCR and SNP array studies were performed on both specimens. RESULTS: Both lymphomas were Epstein-Barr virus (EBV) negative. Flow cytometry showed λ light chain restriction in the initial BL and κ light chain restriction in the subsequent BL. B-cell clonality testing indicated that the two lymphomas are not clonally related. SNP array analysis of the second BL showed genomic changes that were not present in the first BL. CONCLUSIONS: These results confirm that these two tumors represent unrelated BLs. Pathologists and clinicians should be aware that second lymphomas in XLP1 patients may represent new neoplasms rather than late relapses.
Assuntos
Linfoma de Burkitt/patologia , Transtornos Linfoproliferativos/complicações , Segunda Neoplasia Primária/patologia , Adolescente , Biomarcadores Tumorais/análise , Linfoma de Burkitt/genética , Criança , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Masculino , Segunda Neoplasia Primária/genéticaRESUMO
Major histocompatibility (MHC) class II molecules function to present antigenic peptides to CD4 T lymphocytes. The pathways by which these molecules present exogenous antigens have been extensively studied. However by contrast, far less is known about the processing and trafficking of cytosolic antigens, which can also serve as an alternative source of ligands for MHC class II molecules. Self-proteins, tumor antigens, as well as viral proteins found within the cytosol of cells, can be presented via MHC class II molecules, resulting in the activation of specific CD4 T cells. Studies have begun to reveal unique steps as well as some similarities in the pathways for cytosolic and exogenous antigen presentation. Recent developments in this area are summarized here.
Assuntos
Apresentação de Antígeno , Citosol/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologiaRESUMO
Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder characterized by immune-mediated destruction of the salivary and lacrimal glands with unknown etiology. Due to recent research utilizing human subjects as well as laboratory animal models, our understanding of the pathophysiological and immunological mechanisms of pSS has made great strides. As a consequence, targeted, immune-based therapies are gaining increased attention as the ideal way to conquer autoimmune diseases like pSS. Currently, however, there is no effective treatment to target specific immunological events or effector immune cells in the pathogenesis of pSS (discussed in other reviews of the current issue). Here, we summarize our current understanding and knowledge of the roles of monocytes/macrophages in the pathogenesis of pSS. Human studies, especially utilizing salivary gland biopsies, demonstrate the infiltration of macrophages and its correlation with disease severity. Moreover, animal model studies have shown the functional involvement of macrophages in promoting the ocular component of pSS.
Assuntos
Macrófagos/imunologia , Síndrome de Sjogren/imunologia , Animais , HumanosRESUMO
Nemaline myopathy (NM) is a genetically and clinically heterogeneous disorder resulting from a disruption of the thin filament proteins of the striated muscle sarcomere. The disorder is typically characterized by muscle weakness including the face, neck, respiratory, and limb muscles and is clinically classified based on the age of onset and severity. Mutations in the ACTA1 gene contribute to a significant proportion of NM cases. The majority of ACTA1 gene mutations are missense mutations causing autosomal dominant NM by producing an abnormal protein. However, approximately 10% of ACTA1 gene mutations are associated with autosomal recessive NM; these mutations are associated with loss of protein function. We report the first case of a large deletion in the ACTA1 gene contributing to autosomal recessive NM. This case illustrates the importance of understanding disease mechanisms at the molecular level to accurately infer the inheritance pattern and potentially aid with clinical management.
Assuntos
Actinas/genética , Miopatias da Nemalina/genética , Deleção de Sequência , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Músculo Esquelético/patologia , Miopatias da Nemalina/patologiaRESUMO
SHP-1 is a cytoplasm protein tyrosine phosphatase expressed primarily in hematopoietic cells. In the immune system, SHP-1 plays critical roles in regulation of many receptor-mediated signaling cascades, and SHP-1 deficiency in mice causes spontaneous inflammation and autoimmunity. Here, we report a unique requirement for SHP-1 in interleukin-12/23 p40 (IL-12p40) production in response to Toll-like receptor (TLR) stimulation in macrophages. Bone marrow-derived macrophages (BMDMs) lacking significant SHP-1 activity display a profound defect in IL-12p40 synthesis in response to lipopolysaccharide, peptidoglycan, and synthetic TLR ligands, while producing normal amounts of other proinflammatory cytokines, such as TNFalpha and IL-6. Inhibition of SHP-1 function in wild-type BMDMs decreases IL-12p40, and expression of functional SHP-1 protein in mutant cells restores IL-12p40 production following TLR ligation. SHP-1 regulation of IL-12p40 transcription requires both its catalytic activity and phosphotyrosine binding by its N-terminal SH2 domain and is mediated via repression of, and interaction with, phosphatidylinositol 3-kinase, without affecting c-Rel activation. In contrast to normal NF-kappaB activation, SHP-1-defective me(v)/me(v) macrophages display a defect in nucleosome remodeling at the IL-12p40 promoter, and phosphatidylinositol 3-kinase inhibition significantly restores normal nucleosome remodeling in me(v)/me(v) macrophages. Thus, there is a critical role for the tyrosine phosphatase activity of SHP-1 for induction of IL-12p40 production in macrophages in response to TLR ligands, a novel mechanism for host regulation of a specific proinflammatory cytokine important in both innate and adaptive immunity.
Assuntos
Subunidade p40 da Interleucina-12/biossíntese , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Toll-Like/metabolismo , Animais , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunoprecipitação , Subunidade p40 da Interleucina-12/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologiaRESUMO
During antigen processing, peptides are generated and displayed in the context of major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells (APCs) to modulate immune responses to foreign antigens and guide self-tolerance. Exogenous and cytoplasmic antigens are processed by distinct routes within APCs to yield class II ligands. Exogenous antigens are internalized, processed, and bound to class II molecules within endosomal and lysosomal compartments of APCs. Studies reviewed here demonstrate the importance of reduction in regulating exogenous antigen presentation. The differential expression of a gamma-interferon-inducible lysosomal thiol reductase in professional APCs and melanomas is discussed in the context of tumor immune evasion. Cytoplasmic autoantigens, by contrast, are degraded by the proteasome and other enzymes in the cytosol, with the resulting peptides translocating to endosomal and lysosomal compartments for intersection with class II molecules. Processing and editing of these antigenic peptides within endosomes and lysosomes may be critical in regulating their display via class II proteins. Multiple pathways may regulate the transit of cytosolic peptides to class II molecules. The role of lysosome-associated membrane protein-2a and heat-shock cognate protein 70 in promoting cytoplasmic peptide presentation by MHC class II molecules is discussed.
Assuntos
Apresentação de Antígeno/imunologia , Citosol/metabolismo , Endossomos/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Citosol/imunologia , Endossomos/imunologia , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Proteínas de Membrana Lisossomal , Melanoma/imunologiaRESUMO
Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe II/metabolismo , Vaccinia virus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Linfócitos B/imunologia , Linfócitos B/virologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Chlorocebus aethiops , DNA Complementar/genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/imunologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transdução de Sinais/imunologia , Vaccinia virus/genéticaRESUMO
Extracellular antigens are internalized and processed before binding MHC class II molecules within endosomal and lysosomal compartments of professional antigen presenting cells (APC) for subsequent presentation to T cells. Yet select cytoplasmic peptides derived from autoantigens also intersect and bind class II molecules via an unknown mechanism. In human B lymphoblasts, inhibition of the peptide transporter associated with antigen processing (TAP) failed to alter class II-restricted cytoplasmic epitope presentation. By contrast, decreased display of cytoplasmic epitopes via class II molecules was observed in cells with diminished expression of the lysosome-associated membrane protein-2 (Lamp-2). Overexpression of Lamp-2 isoform A (Lamp-2a), an established component of chaperone-mediated autophagy, enhanced cytoplasmic autoantigen presentation. Manipulating APC expression of heat shock cognate protein 70 (hsc70), a cofactor for Lamp-2a, also altered cytoplasmic class II peptide presentation. These results demonstrate a novel role for the lysosomal Lamp-2a-hsc70 complex in promoting immunological recognition and antigen presentation.
Assuntos
Apresentação de Antígeno , Antígenos CD/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Transportadores de Cassetes de Ligação de ATP , Antígenos CD/genética , Linhagem Celular , Citoplasma/imunologia , Eletroporação , Endossomos/imunologia , Glutamato Descarboxilase/imunologia , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Membrana Lisossomal , Lisossomos/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
HLA-DM stabilizes peptide-receptive class II alphabeta dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273-285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.