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1.
Diabetes ; 68(9): 1830-1840, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31175101

RESUMO

We recently established that hybrid insulin peptides (HIPs) are present in human islets and that T cells reactive to HIPs are found in the residual islets of organ donors with type 1 diabetes (T1D). Here, we investigate whether HIP-reactive T cells are indicative of ongoing autoimmunity in patients with T1D. We used interferon-γ enzyme-linked immune absorbent spot analyses on peripheral blood mononuclear cells (PBMCs) to determine whether patients with new-onset T1D or control subjects displayed T-cell reactivity to a panel of 16 HIPs. We observed that nearly one-half of the patients responded to one or more HIPs. Responses to four HIPs were significantly elevated in patients with T1D but not in control subjects. To characterize the T cells reactive to HIPs, we used a carboxyfluorescein succinimidyl ester-based assay to clone T cells from PBMCs. We isolated six nonredundant, antigen-specific T-cell clones, most of which reacting to their target HIPs in the low nanomolar range. One T-cell clone was isolated from the same patient on two different blood draws, indicating persistence of this T-cell clone in the peripheral blood. This work suggests that HIPs are important target antigens in human subjects with T1D and may play a critical role in disease.

2.
J Proteome Res ; 18(3): 814-825, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30585061

RESUMO

We recently discovered hybrid insulin peptides (HIPs) as a novel class of post-translationally modified peptides in murine-derived beta cell tumors, and we demonstrated that these molecules are autoantigens in type 1 diabetes (T1D). A HIP consists of an insulin fragment linked to another secretory granule peptide via a peptide bond. We verified that autoreactive CD4 T cells in both mouse and human autoimmune diabetes recognize these modified peptides. Here, we use mass spectrometric analyses to confirm the presence of HIPs in both mouse and human pancreatic islets. We also present criteria for the confident identification of these peptides. This work supports the hypothesis that HIPs are autoantigens in human T1D and provides a foundation for future efforts to interrogate this previously unknown component of the beta cell proteome.

3.
Diabetes ; 67(9): 1836-1846, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29976617

RESUMO

We recently established that hybrid insulin peptides (HIPs), formed in islet ß-cells by fusion of insulin C-peptide fragments to peptides of chromogranin A or islet amyloid polypeptide, are ligands for diabetogenic CD4 T-cell clones. The goal of this study was to investigate whether HIP-reactive T cells were indicative of ongoing autoimmunity. MHC class II tetramers were used to investigate the presence, phenotype, and function of HIP-reactive and insulin-reactive T cells in NOD mice. Insulin-reactive T cells encounter their antigen early in disease, but they express FoxP3 and therefore may contribute to immune regulation. In contrast, HIP-reactive T cells are proinflammatory and highly diabetogenic in an adoptive transfer model. Because the frequency of antigen-experienced HIP-reactive T cells increases over progression of disease, they may serve as biomarkers of autoimmune diabetes.


Assuntos
Autoantígenos/metabolismo , Peptídeo C/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Cromogranina A/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Recombinação Genética , Animais , Autoantígenos/química , Autoantígenos/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Autoimunidade , Biomarcadores/sangue , Peptídeo C/química , Peptídeo C/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Cromogranina A/química , Cromogranina A/genética , Células Clonais , Cruzamentos Genéticos , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Progressão da Doença , Feminino , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Ativação Linfocitária , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Organismos Livres de Patógenos Específicos
6.
J Autoimmun ; 78: 11-18, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27802879

RESUMO

BDC-6.9, a diabetogenic CD4 T cell clone isolated from a non-obese diabetic (NOD) mouse, responds to pancreatic islet cells from NOD but not BALB/c mice. We recently reported that a hybrid insulin peptide (HIP), 6.9HIP, formed by linkage of an insulin C-peptide fragment and a fragment of islet amyloid polypeptide (IAPP), is the antigen for BDC-6.9. We report here that the core 12-mer peptide from 6.9HIP, centered on the hybrid peptide junction, is also highly antigenic for BDC-6.9. In agreement with the observation that BALB/c islet cells fail to stimulate the T cell clone, a single amino acid difference in the BALB/c IAPP sequence renders the BALB/c version of the HIP only weakly antigenic. Mutant peptide analysis indicates that each parent molecule-insulin C-peptide and IAPP-donates residues critical for antigenicity. Through mass spectrometric analysis, we determine the distribution of naturally occurring 6.9HIP across chromatographic fractions of proteins from pancreatic beta cells. This distribution closely matches the profile of the T cell response to the fractions, confirming that 6.9HIP is the endogenous islet antigen for the clone. Using a new MHC II tetramer reagent, 6.9HIP-tet, we show that T cells specific for the 6.9HIP peptide are prevalent in the pancreas of diabetic NOD mice. Further study of HIPs and HIP-reactive T cells could yield valuable insight into key factors driving progression to diabetes and thereby inform efforts to prevent or reverse this disease.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Insulina/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Sequência de Aminoácidos , Animais , Autoantígenos/química , Peptídeo C/química , Peptídeo C/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Epitopos de Linfócito T/química , Insulina/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout
7.
Nat Med ; 22(12): 1482-1487, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27798614

RESUMO

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.


Assuntos
Autoantígenos/imunologia , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígeno HLA-A2/imunologia , Antígenos HLA-DQ/imunologia , Ilhotas Pancreáticas/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Criança , Feminino , Humanos , Masculino , Adulto Jovem
8.
Science ; 351(6274): 711-4, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26912858

RESUMO

T cell-mediated destruction of insulin-producing ß cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in ß cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in ß cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in ß cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.


Assuntos
Peptídeo C/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Células Secretoras de Insulina/imunologia , Sequência de Aminoácidos , Animais , Peptídeo C/química , Células Clonais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Tolerância Imunológica , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia
9.
J Immunol ; 196(1): 39-43, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26608914

RESUMO

T cells reactive to ß cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the ß cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD.ChgA(+/+) mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Cromogranina A/genética , Diabetes Mellitus Tipo 1/genética , Transferência Adotiva , Animais , Autoimunidade/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Cromogranina A/imunologia , Diabetes Mellitus Tipo 1/imunologia , Citometria de Fluxo , Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Repetições de Microssatélites/genética
10.
J Autoimmun ; 50: 38-41, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24239002

RESUMO

Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n = 27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n = 31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n = 11) and control subjects (n = 11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.


Assuntos
Antígenos CD4/imunologia , Cromogranina A/imunologia , Diabetes Mellitus Tipo 1/genética , Peptídeos/imunologia , Adolescente , Adulto , Autoantígenos/genética , Autoantígenos/imunologia , Antígenos CD4/genética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Criança , Cromogranina A/genética , Cromogranina A/farmacologia , Diabetes Mellitus Tipo 1/imunologia , Feminino , Expressão Gênica , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Teste de Histocompatibilidade , Humanos , Masculino , Peptídeos/genética , Peptídeos/farmacologia , Cultura Primária de Células , Transglutaminases/metabolismo , Transglutaminases/farmacologia
11.
J Immunol ; 191(8): 3990-4, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24043895

RESUMO

We previously reported a peptide KS20 from islet amyloid polypeptide (IAPP) to be the target Ag for a highly diabetogenic CD4 T cell clone BDC-5.2.9. To track IAPP-reactive T cells in NOD mice and determine how they contribute to the pathogenesis of type 1 diabetes, we designed a new I-Ag7 tetramer with high affinity for BDC-5.2.9 that contains the peptide KS20. We found that significant numbers of KS20 tetramer(+) CD4 T cells can be detected in the pancreas of prediabetic and diabetic NOD mice. To verify pathogenicity of IAPP-reactive cells, we sorted KS20 tetramer(+) cells and cloned them from uncloned T cell lines isolated from spleen and lymph nodes of diabetic mice. We isolated a new KS20-reactive Th1 CD4 T cell clone that rapidly transfers diabetes. Our results suggest that IAPP triggers a broad autoimmune response by CD4 T cells in NOD mice.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Estado Pré-Diabético/imunologia , Transferência Adotiva , Animais , Autoantígenos/imunologia , Autoimunidade/imunologia , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/imunologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Pâncreas/imunologia , Baço/citologia
12.
Immunol Res ; 55(1-3): 167-72, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22971988

RESUMO

Autoreactive CD4 T cells play a central role in the development of type 1 diabetes. The BDC panel of diabetogenic T cell clones was originally isolated from non-obese diabetic mice and has been used to study the role of autoreactive CD4 T cells and T cell autoantigens in the development of diabetes. Recent studies by our group have led to the identification of two new target antigens for clones of this panel. This review describes the proteomic strategy used for antigen identification, the antigens identified, and the potential contribution of post-translational modification to autoantigen generation. In addition, we compare peptide epitopes for the T cell clones and discuss their potential applications in investigating the role of T cell autoantigens in the pathogenesis and regulation of disease.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Animais , Humanos , Proteômica
13.
Diabetes ; 61(12): 3239-46, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22912420

RESUMO

Chromogranin A (ChgA) has been identified as the antigen target for three NOD-derived, diabetogenic CD4 T-cell clones, including the well-known BDC-2.5. These T-cell clones respond weakly to the peptide WE14, a naturally occurring proteolytic cleavage product from ChgA. We show here that WE14 can be converted into a highly antigenic T-cell epitope through treatment with the enzyme transglutaminase (TGase). The WE14 responses of three NOD-derived CD4 T-cell clones, each with different T-cell receptors (TCRs), and of T cells from BDC-2.5 TCR transgenic mice are increased after TGase conversion of the peptide. Primary CD4 T cells isolated from NOD mice also respond to high concentrations of WE14 and significantly lower concentrations of TGase-treated WE14. We hypothesize that posttranslational modification plays a critical role in the generation of T-cell epitopes in type 1 diabetes.


Assuntos
Cromogranina A/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Cromatografia em Gel , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Transglutaminases/metabolismo
14.
Diabetes ; 60(9): 2325-30, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21734016

RESUMO

OBJECTIVE: To investigate autoantigens in ß-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9. RESEARCH DESIGN AND METHODS: To purify ß-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of ß-cell tumors. The presence of antigen was monitored by measuring the interferon-γ production of BDC-5.2.9 in response to chromatographic fractions in the presence of NOD antigen-presenting cells. Peak antigenic fractions were analyzed by ion-trap mass spectrometry, and candidate proteins were further investigated through peptide analysis and, where possible, testing of islet tissue from gene knockout mice. RESULTS: Mass-spectrometric analysis revealed the presence of islet amyloid polypeptide (IAPP) in antigen-containing fractions. Confirmation of IAPP as the antigen target was demonstrated by the inability of islets from IAPP-deficient mice to stimulate BDC-5.2.9 in vitro and in vivo and by the existence of an IAPP-derived peptide that strongly stimulates BCD-5.2.9. CONCLUSIONS: IAPP is the target antigen for the diabetogenic CD4 T-cell clone BDC-5.2.9.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos NOD
15.
Nat Immunol ; 11(3): 225-31, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20139986

RESUMO

Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.


Assuntos
Autoantígenos/imunologia , Cromogranina A/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Fragmentos de Peptídeos/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Epitopos/imunologia , Antígenos HLA-A , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Dados de Sequência Molecular
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