RESUMO
AIMS/HYPOTHESIS: Heterogeneity in individuals with type 1 diabetes has become more generally appreciated, but has not yet been extensively and systematically characterised. Here, we aimed to characterise type 1 diabetes heterogeneity by creating immunological, genetic and clinical profiles for individuals with juvenile-onset type 1 diabetes in a cross-sectional study. METHODS: Participants were HLA-genotyped to determine HLA-DR-DQ risk, and SNP-genotyped to generate a non-HLA genetic risk score (GRS) based on 93 type 1 diabetes-associated SNP variants outside the MHC region. Islet autoimmunity was assessed as T cell proliferation upon stimulation with the beta cell antigens GAD65, islet antigen-2 (IA-2), preproinsulin (PPI) and defective ribosomal product of the insulin gene (INS-DRIP). Clinical parameters were collected retrospectively. RESULTS: Of 80 individuals, 67 had proliferation responses to one or more islet antigens, with vast differences in the extent of proliferation. Based on the multitude and amplitude of the proliferation responses, individuals were clustered into non-, intermediate and high responders. High responders could not be characterised entirely by enrichment for the highest risk HLA-DR3-DQ2/DR4-DQ8 genotype. However, high responders did have a significantly higher non-HLA GRS. Clinically, high T cell responses to beta cell antigens did not reflect in worsened glycaemic control, increased complications, development of associated autoimmunity or younger age at disease onset. The number of beta cell antigens that an individual responded to increased with disease duration, pointing to chronic islet autoimmunity and epitope spreading. CONCLUSIONS/INTERPRETATION: Collectively, these data provide new insights into type 1 diabetes disease heterogeneity and highlight the importance of stratifying patients on the basis of their genetic and autoimmune signatures for immunotherapy and personalised disease management.
Assuntos
Autoimunidade/fisiologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Adolescente , Adulto , Autoimunidade/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Criança , Pré-Escolar , Estudos Transversais , Diabetes Mellitus Tipo 1/genética , Feminino , Genótipo , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Insulina/metabolismo , Masculino , Análise de Componente Principal , Precursores de Proteínas/metabolismo , Estudos Retrospectivos , Linfócitos T/metabolismo , Adulto JovemRESUMO
Human CD4+ T lymphocytes play an important role in inducing potent immune responses. T cells are activated and stimulated by peptides presented in human leucocyte antigen (HLA)-class II molecules. These HLA-class II molecules typically present peptides of between 12 and 20 amino acids in length. The region that interacts with the HLA molecule, designated as the peptide-binding core, is highly conserved in the residues which anchor the peptide to the molecule. In addition, as these peptides are the product of proteolytic cleavages, certain conserved residues may be expected at the N- and C-termini outside the binding core. To study whether similar conserved residues are present in different cell types, potentially harbouring different proteolytic enzymes, the ligandomes of HLA-DRB1*03:01/HLA-DRB > 1 derived from two different cell types (dendritic cells and EBV-transformed B cells) were identified with mass spectrometry and the binding core and N- and C-terminal residues of a total of 16,568 peptides were analysed using the frequencies of the amino acids in the human proteome. Similar binding motifs were found as well as comparable conservations in the N- and C-terminal residues. Furthermore, the terminal conservations of these ligandomes were compared to the N- and C-terminal conservations of the ligandome acquired from dendritic cells homozygous for HLA-DRB1*04:01. Again, comparable conservations were evident with only minor differences. Taken together, these data show that there are conservations in the terminal residues of peptides, presumably the result of the activity of proteases involved in antigen processing.
Assuntos
Linfócitos B/metabolismo , Células Dendríticas/metabolismo , Antígenos HLA-DR/classificação , Antígenos HLA-DR/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteoma/metabolismo , Motivos de Aminoácidos , Linfócitos B/citologia , Células Cultivadas , Células Dendríticas/citologia , Humanos , Ligantes , Ligação ProteicaRESUMO
Identifying T cell epitopes of islet autoantigens is important for understanding type 1 diabetes (T1D) immunopathogenesis and to design immune monitoring and intervention strategies in relationship to disease progression. Naturally processed T cell epitopes have been discovered by elution from HLA-DR4 of pulsed B lymphocytes. The designated professional APC directing immune responses is the dendritic cell (DC). To identify naturally processed epitopes, monocyte-derived DC were pulsed with preproinsulin (PPI), glutamic acid decarboxylase (65-kDa isoform; GAD65), and insulinoma-associated Ag-2 (IA-2), and peptides were eluted of HLA-DR3 and -DR4, which are associated with highest risk for T1D development. Proteome analysis confirmed uptake and processing of islet Ags by DC. PPI peptides generated by DC differed from those processed by B lymphocytes; PPI signal-sequence peptides were eluted from HLA-DR4 and -DR3/4 that proved completely identical to a primary target epitope of diabetogenic HLA-A2-restricted CD8 T cells. HLA-DR4 binding was confirmed. GAD65 peptides, eluted from HLA-DR3 and -DR4, encompassed two core regions overlapping the two most immunodominant and frequently studied CD4 T cell targets. GAD65 peptides bound to HLA-DR3. Strikingly, the IA-2 ligandome of HLA-DR was exclusively generated from the extracellular part of IA-2, whereas most previous immune studies have focused on intracellular IA-2 epitopes. The newly identified IA-2 peptides bound to HLA-DR3 and -DR4. Differential T cell responses were detected against the newly identified IA-2 epitopes in blood from T1D patients. The core regions to which DC may draw attention from autoreactive T cells are largely distinct and more restricted than are those of B cells. GAD65 peptides presented by DC focus on highly immunogenic T cell targets, whereas HLA-DR-binding peptides derived from IA-2 are distinct from the target regions of IA-2 autoantibodies.
Assuntos
Autoimunidade/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígeno HLA-DR3/imunologia , Antígeno HLA-DR4/imunologia , Ilhotas Pancreáticas/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Epitopos de Linfócito T/imunologia , Glutamato Descarboxilase/metabolismo , Humanos , Insulina/metabolismo , Ativação Linfocitária/imunologia , Ligação Proteica/imunologia , Precursores de Proteínas/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismoRESUMO
Autoreactive T cells specific for islet autoantigens develop in type 1 diabetes (T1D) by escaping central as well as peripheral tolerance. The current paradigm for development of islet autoimmunity is just beginning to include the contribution of posttranslationally modified (PTM) islet autoantigens, for which the immune system may be ignorant rather than tolerant. As a result, PTM is the latest promising lead in the quest to understand how the break in peripheral tolerance occurs in T1D. However, it is not completely clear how, where, or when these modifications take place. Currently, only a few PTM antigens have been well-thought-out or identified in T1D, and methods for identifying and characterizing new PTM antigens are rapidly improving. This review will address both reported and potential new sources of modified islet autoantigens and discuss how islet neo-autoantigen generation may contribute to the development and progression of T1D.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Autoimunidade , Diabetes Mellitus Tipo 1/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Fatores de RiscoRESUMO
HLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared with HLA-DQ2 or HLA-DQ8 homozygote subjects that may result from the presence of a transdimer formed between the α-chain of HLA-DQ2 (DQA1*05:01) and the ß-chain of HLA-DQ8 (DQB1*03:02). We generated cells exclusively expressing this transdimer (HLA-DQ8trans), characterized its peptide binding repertoire, and defined a unique transdimer-specific peptide binding motif that was found to be distinct from those of HLA-DQ2 and HLA-DQ8. This motif predicts an array of peptides of islet autoantigens as candidate T cell epitopes, many of which selectively bind to the HLA transdimer, whereas others bind to both HLA-DQ8 and transdimer with similar affinity. Our findings provide a molecular basis for the association between HLA-DQ transdimers and T1D and set the stage for rational testing of potential diabetogenic peptide epitopes.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Antígenos HLA-DQ/química , Antígenos HLA-DQ/metabolismo , Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Diabetes Mellitus Tipo 1/genética , Dimerização , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Humanos , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Because susceptibility to celiac disease is associated strongly with HLA-DQ2 (DQA1*05/DQB1*02) and weakly with HLA-DQ8 (DQA1*03/DQB1*03), a subset of patients carries both HLA-DQ2 and HLA-DQ8. As a result, these patients may express two types of mixed HLA-DQ2/8 transdimers (encoded by DQA1*05/DQB1*03 and DQA1*03/DQB1*02) in addition to HLA-DQ2 and HLA-DQ8. Using T cells from a celiac disease patient expressing HLA-DQ8trans (encoded by DQA*0501/DQB*0302), but neither HLA-DQ2 nor HLA-DQ8, we demonstrate that this transdimer is expressed on the cell surface and can present multiple gluten peptides to T cell clones isolated from the duodenum of this patient. Furthermore, T cell clones derived from this patient and HLA-DQ2/8 heterozygous celiac disease patients respond to gluten peptides presented by HLA-DQ8trans, as well as HLA-DQ8, in a similar fashion. Finally, one gluten peptide is recognized better when presented by HLA-DQ8trans, which correlates with preferential binding of this peptide to HLA-DQ8trans. These results implicate HLA-DQ8trans in celiac disease pathogenesis and demonstrate extensive T cell cross-reactivity between HLA-DQ8 and HLA-DQ8trans. Because type 1 diabetes is strongly associated with the presence of HLA-DQ8trans, our findings may bear relevance to this disease as well.
Assuntos
Epitopos de Linfócito T/imunologia , Gliadina/imunologia , Gliadina/metabolismo , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Cadeias beta de HLA-DQ/metabolismo , Subpopulações de Linfócitos T/imunologia , Apresentação de Antígeno/imunologia , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Células Clonais , Reações Cruzadas/imunologia , Epitopos de Linfócito T/metabolismo , Células HEK293 , Antígenos HLA-DQ/química , Cadeias beta de HLA-DQ/química , Humanos , Multimerização Proteica , Subpopulações de Linfócitos T/metabolismoRESUMO
For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.
Assuntos
Doxorrubicina/farmacologia , Glucosilceramidas/química , Nanoestruturas/química , Neoplasias/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Sistemas de Liberação de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Neoplasias/patologia , Resultado do Tratamento , Lipossomas Unilamelares/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.
Assuntos
Éteres Fosfolipídicos/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Ligante Fas/metabolismo , Inositol Polifosfato 5-Fosfatases , Linfoma/patologia , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Lisofosfolipídeos/farmacologia , Esfingomielinas/metabolismo , Receptor fas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Proteína Ligante Fas/farmacologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Leupeptinas/farmacologia , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Microscopia Confocal , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingomielinas/deficiência , Transfecção , Receptor fas/genéticaRESUMO
The heterozygous DQ2/8 (DQA1*05:01-DQB1*02:01/DQA1*03:01-DQB1*03:02) genotype confers the highest risk in type 1 diabetes (T1D), whereas the DQ6/8 (DQA1*02:01-DQB1*06:02/DQA1*03:01-DQB1*03:02) genotype is protective. The mechanism of dominant protection by DQ6 (DQB1*06:02) is unknown. We tested the hypothesis that DQ6 interferes with peptide binding to DQ8 by competition for islet epitope ("epitope stealing") by analysis of the islet ligandome presented by HLA-DQ6/8 and -DQ8/8 on dendritic cells pulsed with islet autoantigens preproinsulin (PPI), GAD65, and IA-2, followed by competition assays using a newly established "epitope-stealing" HLA/peptide-binding assay. HLA-DQ ligandome analysis revealed a distinct DQ6 peptide-binding motif compared with the susceptible DQ2/8 molecules. PPI and IA-2 peptides were identified from DQ6, of DQ6/8 heterozygous dendritic cells, but no DQ8 islet peptides were retrieved. Insulin B6-23, a highly immunogenic CD4 T-cell epitope in patients with T1D, bound to both DQ6 and DQ8. Yet, binding of InsB6-23 to DQ8 was prevented by DQ6. We obtained first functional evidence of a mechanism of dominant protection from disease, in which HLA molecules associated with protection bind islet epitopes in a different, competing, HLA-binding register, leading to "epitope stealing" and conceivably diverting the immune response from islet epitopes presented by disease-susceptible HLA molecules in the absence of protective HLA.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-DQ/imunologia , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 1/genética , Antígenos HLA-DQ/genética , Haplótipos , HumanosRESUMO
Identification of epitopes that are recognized by diabetogenic T cells and cause selective beta cell destruction in type 1 diabetes (T1D) has focused on peptides originating from native beta cell proteins. Translational errors represent a major potential source of antigenic peptides to which central immune tolerance is lacking. Here, we describe an alternative open reading frame within human insulin mRNA encoding a highly immunogenic polypeptide that is targeted by T cells in T1D patients. We show that cytotoxic T cells directed against the N-terminal peptide of this nonconventional product are present in the circulation of individuals diagnosed with T1D, and we provide direct evidence that such CD8+ T cells are capable of killing human beta cells and thereby may be diabetogenic. This study reveals a new source of nonconventional polypeptides that act as self-epitopes in clinical autoimmune disease.
Assuntos
Autoantígenos/imunologia , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Insulina/genética , Peptídeos/imunologia , RNA Mensageiro/genética , Linfócitos T Citotóxicos/imunologia , Adolescente , Adulto , Autoantígenos/genética , Autoimunidade/genética , Linfócitos T CD8-Positivos/imunologia , Criança , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/genética , Feminino , Antígenos HLA-DQ/imunologia , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/imunologia , Masculino , Fases de Leitura Aberta , Peptídeos/genética , Biossíntese de Proteínas , Adulto JovemRESUMO
Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of the insulin-producing beta cells. Beta cell dysfunction caused by an inflammatory microenvironment is believed to trigger the peripheral activation of CD4 and CD8 autoreactive T cells. This review will compile post-transcriptional and post-translational modifications (PTM) involved in the generation of beta cell neoantigens and proposes a reconstruction of the sequence of events connecting environmental changes and autoimmunity.
Assuntos
Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Sistema Imunitário , Células Secretoras de Insulina/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Autoimunidade , Interação Gene-Ambiente , Humanos , Inflamação , Processamento de Proteína Pós-TraducionalRESUMO
HLA-DQ2/8 heterozygous individuals are at far greater risk for type 1 diabetes (T1D) development by expressing HLA-DQ8trans on antigen-presenting cells compared with HLA-DQ2 or -DQ8 homozygous individuals. Dendritic cells (DC) initiate and shape adaptive immune responses by presenting HLA-epitope complexes to naïve T cells. To dissect the role of HLA-DQ8trans in presenting natural islet epitopes, we analyzed the islet peptidome of HLA-DQ2, -DQ8, and -DQ2/8 by pulsing DC with preproinsulin (PPI), IA-2, and GAD65. Quality and quantity of islet epitopes presented by HLA-DQ2/8 differed from -DQ2 or -DQ8. We identified two PPI epitopes solely processed and presented by HLA-DQ2/8 DC: an HLA-DQ8trans-binding signal-sequence epitope previously identified as CD8 T-cell epitope and a second epitope that we previously identified as CD4 T-cell epitope with increased binding to HLA-DQ8trans upon posttranslational modification. IA-2 epitopes retrieved from HLA-DQ2/8 and -DQ8 DC bound to HLA-DQ8cis/trans. No GAD65 epitopes were eluted from HLA-DQ. T-cell responses were detected against the novel islet epitopes in blood from patients with T1D but scantly detected in healthy donor subjects. We report the first PPI and IA-2 natural epitopes presented by highest-risk HLA-DQ8trans. The selective processing and presentation of HLA-DQ8trans-binding islet epitopes provides insight in the mechanism of excessive genetic risk imposed by HLA-DQ2/8 heterozygosity and may assist immune monitoring of disease progression and therapeutic intervention as well as provide therapeutic targets for immunotherapy in subjects at risk for T1D.
Assuntos
Autoantígenos/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Antígenos HLA-DQ/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/genética , Feminino , Glutamato Descarboxilase/imunologia , Antígenos HLA-DQ/genética , Heterozigoto , Homozigoto , Humanos , Insulina/imunologia , Masculino , Peptídeos , Precursores de Proteínas/imunologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Adulto JovemRESUMO
OBJECTIVE: We previously showed that mycobacterial Hsp70-derived peptide B29 induced B29-specific Treg cells that suppressed experimental arthritis in mice via cross-recognition of their mammalian Hsp70 homologs. The aim of the current study was to characterize B29 binding and specific CD4+ T cell responses in the context of human major histocompatibility complex (MHC) molecules. METHODS: Competitive binding assays were performed to examine binding of peptide B29 and its mammalian homologs to HLA molecules. The effect of B29 immunization in HLA-DQ8-transgenic mice with proteoglycan-induced arthritis was assessed, followed by ex vivo restimulation with B29 to examine the T cell response. Human peripheral blood mononuclear cells were used to investigate the presence of B29-specific T cells with immunoregulatory potential. RESULTS: The binding affinity of the B29 peptide was high to moderate for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was considered to be functional, because B29 immunization resulted in the suppression of arthritis and T cell responses in HLA-DQ8-transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4+ T cells, which were cross-reactive with the mammalian homologs. Using HLA-DR4+ tetramers specific for B29 or the mammalian homolog mB29b, we showed expansion of cross-reactive T cells, especially the human FoxP3+ CD4+CD25+ T cell population, after in vitro stimulation with B29. CONCLUSION: These results demonstrated a conserved fine specificity and functionality of B29-induced Treg cell responses in the context of the human MHC. Based on these findings, a path for translation of the experimental findings for B29 into a clinical immunomodulatory therapeutic approach is within reach.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-DQ/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Animais , Ligação Competitiva , Separação Celular , Células Cultivadas , Reações Cruzadas , Encefalinas/imunologia , Feminino , Fatores de Transcrição Forkhead/imunologia , Humanos , Técnicas In Vitro , Integrina beta1/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Precursores de Proteínas/imunologiaRESUMO
The presence of antiphospholipid antibodies in plasma is a risk factor for thrombo-embolic complications. In vitro, however, the same antibodies can prolong clotting times in coagulation assays, a classic marker for a bleeding tendency. For years this contradiction puzzles many scientists.We now know that the term antiphospholipid antibodies comprises a heterogeneous population of antibodies and there is growing evidence that only subpopulations of antiphospholipid antibodies are relevant for the clinical complication. In combination with new information on the complex interaction between antiphospholipid antibodies, the protein beta2-Glycoprotein I, and cellular surfaces have opened new avenues for the understanding of the pathology of this syndrome.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Glicoproteínas/fisiologia , Receptores de LDL/fisiologia , Animais , Anticorpos Antifosfolipídeos/sangue , Anticorpos Antifosfolipídeos/química , Humanos , Modelos Biológicos , Ativação Plaquetária , Receptores de LDL/imunologia , Tromboembolia/patologia , beta 2-Glicoproteína IRESUMO
Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified 31 candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis-restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-γ, whereas most healthy donors produced interleukin-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.
Assuntos
Autoantígenos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Antígenos HLA-DQ/metabolismo , Processamento de Proteína Pós-Traducional , Linfócitos T/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Criança , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Epitopos , Feminino , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Linfócitos T/metabolismoRESUMO
Celiac disease is a T cell-mediated disease induced by dietary gluten, a component of which is gliadin. 95% of individuals with celiac disease carry the HLA (human leukocyte antigen)-DQ2 locus. Here we determined the T-cell receptor (TCR) usage and fine specificity of patient-derived T-cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and DQ2.5-glia-α2. We determined the ternary structures of four distinct biased TCRs specific for those epitopes. All three TCRs specific for DQ2.5-glia-α2 docked centrally above HLA-DQ2, which together with mutagenesis and affinity measurements provided a basis for the biased TCR usage. A non-germline encoded arginine residue within the CDR3ß loop acted as the lynchpin within this common docking footprint. Although the TCRs specific for DQ2.5-glia-α1a and DQ2.5-glia-α2 docked similarly, their interactions with the respective gliadin determinants differed markedly, thereby providing a basis for epitope specificity.
Assuntos
Doença Celíaca/imunologia , Epitopos de Linfócito T/química , Gliadina/química , Antígenos HLA-DQ/química , Receptores de Antígenos de Linfócitos T/química , Gliadina/imunologia , Humanos , Fenômenos Imunogenéticos , Modelos Moleculares , Conformação Molecular , TriticumRESUMO
PURPOSE OF REVIEW: Description on post-translational modification of islet-autoantigens in type 1 diabetes (T1D). RECENT FINDINGS: T1D is an autoimmune disease characterized by progressive destruction of the insulin-producing beta-cells. It is a complex disease process that results from the loss of tolerance to beta-cell autoantigens. This loss of tolerance can be caused by modification of beta-cell autoantigens, generating 'neo-autoantigens', and inducing T-cell responses. Post-translational modifications (PTMs) within the endoplasmic reticulum of stressed beta-cells might impact on the autoantigen T-cell epitope repertoire and on T1D pathogenesis progression. This review summarizes the processes involved in beta-cell stress and PTM of beta-cell autoantigens in T1D. SUMMARY: PTMs of beta-cell autoantigens provide a novel hypothesis to understand how autoreactive T-cells can escape immune tolerance and cause destruction of beta-cells ('beta-cell homicide'). Additionally, aberrant proteins produced by stressed beta-cells can cause their own destruction ('beta-cell suicide'). Upon endoplasmic reticulum-stress, proteins are misfolded or modified changing the protein structure. In T1D, this may generate new beta-cell (neo)autoantigens. PTM of islet-autoantigens provides a mechanism by which pathogenic T-cells can escape thymic deletion. This amplifies the immune response when encountering a modified beta-cell neo-autoantigen bound to T1D predisposing human leucocyte antigen molecules in the periphery.
Assuntos
Antígenos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Antígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Retículo Endoplasmático/metabolismo , HumanosRESUMO
The paradoxical correlation between thrombosis and the lupus anticoagulant (LAC) effect is an enigmatic feature of the antiphospholipid (aPL) syndrome. The Dutch authors previously reported that thrombosis-related anti-beta2-glycoprotein I (beta2GPI) antibodies recognize domain I and cause LAC. The American authors reported that aPLs disrupt an anticoagulant annexin A5 (AnxA5) crystal shield. We investigated whether antidomain I antibodies correlate with disruption of AnxA5-anticoagulant activity. We studied a well-characterized group of 33 patients including subgroups with beta2GPI-dependent LAC that recognize domain I (n=11), with beta2GPI-independent LAC (n=12), and lacking LAC (n=10). The effects on AnxA5-anticoagulant activity were determined with an AnxA5 resistance assay that measures coagulation times with and without AnxA5. Patients with beta2GPI-dependent LAC (group A, all with thrombosis) had significantly lower AnxA5-anticoagulant ratios than those with beta2GPI-independent LAC (group B, thrombosis n=4; 157.8% versus 235.6%, P<.001) and those without LAC (group C, thrombosis n=2; 157.8% versus 232.5%, P<.001). There was no difference in the ratios between groups B and C (P=.92). Plasmas with beta2GPI-dependent LAC that recognize domain I displayed significantly increased AnxA5 resistance, suggesting that specifically anti-beta2GPI antibodies compete with AnxA5 for anionic phospholipids. These results are consistent with a model in which aPL antibodies may promote thrombosis by interfering with the anticoagulant activity of AnxA5.
Assuntos
Anexina A5/fisiologia , Anticorpos Antifosfolipídeos/imunologia , Trombofilia/etiologia , beta 2-Glicoproteína I/imunologia , Adulto , Sítios de Ligação/imunologia , Coagulação Sanguínea , Estudos de Casos e Controles , Epitopos , Feminino , Humanos , Inibidor de Coagulação do Lúpus/sangue , Masculino , Trombose/etiologiaRESUMO
Recently, we published the existence of 2 populations of anti-beta2-glycoprotein I (beta2-GPI) IgG antibodies. Type A antibodies recognize epitope G40-R43 in domain I of beta2-GPI and are strongly associated with thrombosis. Type B antibodies recognize other parts of beta2-GPI and are not associated with thrombosis. In this study we demonstrate that type A antibodies only recognize plasma-purified beta2-GPI when coated onto a negatively charged surface and not when coated onto a neutrally charged surface. The affinity of type B antibodies toward plasma-purified beta2-GPI was independent of the charge of the surface to which beta2-GPI was coated. Type A antibodies did not recognize plasma-purified beta2-GPI in solution, whereas they did recognize recombinant beta2-GPI both in solution and coated onto a neutrally charged plate. When the carbohydrate chains were removed from plasma-purified beta2-GPI, we found that type A antibodies did recognize the protein in solution. This supports the hypothesis that the difference in recognition of plasma-purified and recombinant beta2-GPI is caused by the difference in glycosylation and that epitope G40-R43 of plasma-purified beta2-GPI is covered by a carbohydrate chain. Type A anti-beta2-GPI antibodies can only recognize this epitope when this carbohydrate chain is displaced as a result of a conformational change. This finding has major implications both for the detection of pathogenic anti-beta2-GPI antibodies and the comprehension of the pathophysiology of the antiphospholipid syndrome.