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1.
JCI Insight ; 4(23)2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31671072

RESUMO

At diagnosis, most people with type 1 diabetes (T1D) produce measurable levels of endogenous insulin, but the rate at which insulin secretion declines is heterogeneous. To explain this heterogeneity, we sought to identify a composite signature predictive of insulin secretion, using a collaborative assay evaluation and analysis pipeline that incorporated multiple cellular and serum measures reflecting ß cell health and immune system activity. The ability to predict decline in insulin secretion would be useful for patient stratification for clinical trial enrollment or therapeutic selection. Analytes from 12 qualified assays were measured in shared samples from subjects newly diagnosed with T1D. We developed a computational tool (DIFAcTO, Data Integration Flexible to Account for different Types of data and Outcomes) to identify a composite panel associated with decline in insulin secretion over 2 years following diagnosis. DIFAcTO uses multiple filtering steps to reduce data dimensionality, incorporates error estimation techniques including cross-validation and sensitivity analysis, and is flexible to assay type, clinical outcome, and disease setting. Using this novel analytical tool, we identified a panel of immune markers that, in combination, are highly associated with loss of insulin secretion. The methods used here represent a potentially novel process for identifying combined immune signatures that predict outcomes relevant for complex and heterogeneous diseases like T1D.

2.
Cancer Immunol Res ; 7(10): 1727-1739, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31405946

RESUMO

Although CD4+ T cells likely play key roles in antitumor immune responses, most immuno-oncology studies have been limited to CD8+ T-cell responses due to multiple technical barriers and a lack of shared antigens across patients. Merkel cell carcinoma (MCC) is an aggressive skin cancer caused by Merkel cell polyomavirus (MCPyV) oncoproteins in 80% of cases. Because MCPyV oncoproteins are shared across most patients with MCC, it is unusually feasible to identify, characterize, and potentially augment tumor-specific CD4+ T cells. Here, we report the identification of CD4+ T-cell responses against six MCPyV epitopes, one of which included a conserved, essential viral oncogenic domain that binds/disables the cellular retinoblastoma (Rb) tumor suppressor. We found that this epitope (WEDLT209-228) could be presented by three population-prevalent HLA class II alleles, making it a relevant target in 64% of virus-positive MCC patients. Cellular staining with a WEDLT209-228-HLA-DRB1*0401 tetramer indicated that specific CD4+ T cells were detectable in 78% (14 of 18) of evaluable MCC patients, were 250-fold enriched within MCC tumors relative to peripheral blood, and had diverse T-cell receptor sequences. We also identified a modification of this domain that still allowed recognition by these CD4+ T cells but disabled binding to the Rb tumor suppressor, a key step in the detoxification of a possible therapeutic vaccine. The use of these new tools for deeper study of MCPyV-specific CD4+ T cells may provide broader insight into cancer-specific CD4+ T-cell responses.

3.
Arthritis Rheumatol ; 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31403245

RESUMO

OBJECTIVE: Autoantibodies targeting histidyl-transfer RNA synthetase (HisRS; anti-Jo-1) are common in the idiopathic inflammatory myopathies (IIMs) and antisynthetase syndrome. This study was undertaken to investigate immunity against HisRS in the blood and lungs of patients with IIM/antisynthetase syndrome. METHODS: Bronchoalveolar lavage (BAL) fluid, BAL fluid cells, and peripheral blood mononuclear cells (PBMCs) from patients with IIM/antisynthetase syndrome (n = 24) were stimulated with full-length HisRS protein or a HisRS-derived peptide (HisRS11-23 ). BAL fluid and PBMCs from patients with sarcoidosis (n = 7) and healthy subjects (n = 12) were included as controls. The CD4+ T cell response was determined according to levels of CD40L up-regulation and cytokine expression using flow cytometry. Anti-Jo-1 autoantibody responses in the serum and BAL fluid were assessed by enzyme-linked immunosorbent assay. Lung biopsy samples from patients with IIM/antisynthetase syndrome (n = 14) were investigated by immunohistochemistry. RESULTS: In BAL fluid, CD4+ T cells from 3 of 4 patients with IIM/antisynthetase syndrome responded to stimulation with HisRS protein, as measured by the median fold change in CD40L expresssion in stimulated cells compared to unstimulated cells (median fold change 3.6, interquartile range [IQR] 2.7-14.7), and 2 of 3 patients with IIM/antisynthetase syndrome had the highest responses to HisRS11-23 (median fold change 88, IQR 27-149). In PBMCs, CD4+ T cells from 14 of 18 patients with IIM/antisynthetase syndrome responded to HisRS protein (median fold change 7.38, IQR 2.69-31.86; P < 0.001), whereas a HisRS11-23 response was present in 11 of 14 patients with IIM/antisynthetase syndrome (median fold change 3.4, IQR 1.87-10.9; P < 0.001). In the control group, there was a HisRS11-23 response in 3 of 7 patients with sarcoidosis (median fold change 2.09, IQR 1.45-3.29) and in 5 of 12 healthy controls (median fold change 2, IQR 1.89-2.42). CD4+ T cells from patients with IIM/antisynthetase syndrome displayed a pronounced Th1 phenotype in the BAL fluid when compared to the PBMCs (P < 0.001), producing high amounts of interferon-γ and interleukin-2 following stimulation. Anti-Jo-1 autoantibodies were detected in BAL fluid and germinal center (GC)-like structures were seen in the lung biopsy samples from patients with IIM/antisynthetase syndrome. CONCLUSION: The results of this study demonstrate a pronounced presence of HisRS-reactive CD4+ T cells in PBMCs and BAL fluid cells from patients with IIM/antisynthetase syndrome as compared to patients with sarcoidosis and healthy controls. These findings, combined with the presence of anti-Jo-1 autoantibodies in BAL fluid and GC-like structures in the lungs, suggest that immune activation against HisRS might take place within the lungs of patients with IIM/antisynthetase syndrome.

4.
Sci Adv ; 5(8): eaaw9336, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31457096

RESUMO

Human leukocyte antigen (HLA)-DQ8 transdimer (HLA-DQA1*0501/DQB1*0302) confers exceptionally high risk in autoimmune diabetes. However, little is known about HLA-DQ8 transdimer-restricted CD4 T cell recognition, an event crucial for triggering HLA-DQ8 transdimer-specific anti-islet immunity. Here, we report a high degree of epitope overlap and T cell promiscuity between susceptible HLA-DQ8 and HLA-DQ8 transdimer. Despite preservation of putative residues for T cell receptor (TCR) contact, stronger disease-associated responses to cross-reactive, immunodominant islet epitopes are elicited by HLA-DQ8 transdimer. Mutagenesis at the α chain of HLA-DQ8 transdimer in complex with the disease-relevant GAD65250-266 peptide and in silico analysis reveal the DQ α52 residue located within the N-terminal edge of the peptide-binding cleft for the enhanced T cell reactivity, altering avidity and biophysical affinity between TCR and HLA-peptide complexes. Accordingly, a structurally promiscuous but nondegenerate TCR-HLA-peptide interface is pivotal for HLA-DQ8 transdimer-mediated autoimmune diabetes.

5.
J Immunol ; 203(5): 1276-1287, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31308093

RESUMO

CD4+ T cells are essential for immune protection against viruses, yet their multiple roles remain ill-defined at the single-cell level in humans. Using HLA class II tetramers, we studied the functional properties and clonotypic architecture of EBV-specific CD4+ T cells in patients with infectious mononucleosis, a symptomatic manifestation of primary EBV infection, and in long-term healthy carriers of EBV. We found that primary infection elicited oligoclonal expansions of TH1-like EBV-specific CD4+ T cells armed with cytotoxic proteins that responded immediately ex vivo to challenge with EBV-infected B cells. Importantly, these acutely generated cytotoxic CD4+ T cells were highly activated and transcriptionally distinct from classically described cytotoxic CD4+ memory T cells that accumulate during other persistent viral infections, including CMV and HIV. In contrast, EBV-specific memory CD4+ T cells displayed increased cytokine polyfunctionality but lacked cytotoxic activity. These findings suggested an important effector role for acutely generated cytotoxic CD4+ T cells that could potentially be harnessed to improve the efficacy of vaccines against EBV.

6.
Arthritis Rheumatol ; 2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30390384

RESUMO

OBJECTIVE: Recognition of citrullinated antigens such as vimentin, fibrinogen, and alpha-enolase is associated with rheumatoid arthritis (RA). Emerging data suggests that the matrix protein aggrecan is also recognized as a citrullinated antigen. The goal of this study was to directly visualize cit-aggrecan specific T cells and characterize them in subjects with RA. METHODS: Citrullinated aggrecan peptides with likely DRB1*04:01 binding motifs were predicted using a previously published scanning algorithm. Peptides with detectable binding were assessed for immunogenicity by HLA tetramer staining, followed by single cell cloning. Selectivity for citrullinated peptide was assessed through tetramer staining and proliferation assays. Ex vivo tetramer staining was then employed to assess frequencies of aggrecan specific T cells in peripheral blood. Finally, disease association was assessed by comparing T cell frequencies in RA patients and controls and correlating aggrecan specific T cells with levels of aggrecan specific antibodies. RESULTS: We identified six immunogenic peptides, two of which were the predominant T cell targets in peripheral blood. These two epitopes were citrullinated at HLA binding residues and shared homologous sequences. RA patients had significantly higher frequencies of cit-aggrecan-specific T cells than healthy subjects. Furthermore, T cell frequencies were significantly correlated with antibodies against citrullinated aggrecan. CONCLUSION: Our findings indicate that T cells that recognize citrullinated aggrecan are present in subjects with RA and correlate with antibodies that target this same antigen. Consequently, aggrecan-specific T cells and antibodies are potentially relevant markers that could be utilized to monitor patients with RA or at risk subjects. This article is protected by copyright. All rights reserved.

7.
Cell Immunol ; 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30428974

RESUMO

CD4+ T cell responses are thought to play a role in type 1 diabetes (T1D). However, detection and characterization of T cells that respond to beta cell epitopes in subjects with T1D has been limited by technical obstacles, including the inherently low frequencies in peripheral blood and variable responsiveness of individual subjects to single epitopes. We implemented a multicolor staining approach that allows direct ex vivo characterization of multiple CD4+ T cell specificities in a single sample. Here we demonstrate and apply that multicolor approach to directly measure the frequency and phenotype of beta cell specific CD4+ T cells in T1D patients and HLA matched controls. For this work we utilized five DR0401 restricted peptides from proinsulin, GAD65, IA-2, and IGRP, which were previously reported as disease relevant epitopes. Surprisingly, although responses to each of these peptides can be readily detected after in vitro expansion, our results indicated that only proinsulin specific T cells were consistently detectable at moderate frequencies in subjects with T1D. Characterization of beta cell specific CD4+ T cells revealed only modest differences between subjects with T1D and healthy controls. Subjects with T1D did have higher proportions of CD45RA negative epitope specific T cells than controls. In patients epitope specific T cells were often CXCR3 positive and a substantial proportion were CCR7 negative, suggesting a Th1-like effector phenotype. Finally, we demonstrated that our multicolor staining approach is compatible with class I multimer analysis, facilitating the characterization of self-reactive CD4+ and CD8+ T cells using a single sample.

8.
J Immunol ; 2018 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-30455401

RESUMO

DRB4*01:01 (DRB4) is a secondary HLA-DR product that is part of the high-risk DR4/DQ8 haplotype that is associated with type 1 diabetes (T1D). DRB4 shares considerable homology with HLA-DR4 alleles that predispose to autoimmunity, including DRB1*04:01 and DRB1*04:04. However, the DRB4 protein sequence includes distinct residues that would be expected to alter the characteristics of its binding pockets. To identify high-affinity peptides that are recognized in the context of DRB4, we used an HLA class II tetramer-based approach to identify epitopes within multiple viral Ags. We applied a similar approach to identify antigenic sequences within glutamic acid decarboxylase 65 and pre-proinsulin that are recognized in the context of DRB4. Seven sequences were immunogenic, eliciting high-affinity T cell responses in DRB4+ subjects. DRB1*04:01-restricted responses toward many of these peptides have been previously described, but responses to a novel pre-proinsulin 9-28 peptide were commonly observed in subjects with T1D. Furthermore, T cells that recognized this peptide in the context of DRB4 were present at significantly higher frequencies in patients with T1D than in healthy controls, implicating this as a disease-relevant specificity that may contribute to the breakdown of ß cell tolerance in genetically susceptible individuals. We then deduced a DRB4 motif and confirmed its key features through structural modeling. This modeling suggested that the core epitope within the pre-proinsulin 9-28 peptide has a somewhat unusual binding motif, with tryptophan in the fourth binding pocket of DRB4, perhaps influencing the availability of this complex for T cell selection.

10.
Diabetes ; 67(11): 2337-2348, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30348823

RESUMO

The ß-cell has become recognized as a central player in the pathogenesis of type 1 diabetes with the generation of neoantigens as potential triggers for breaking immune tolerance. We report that posttranslationally modified glucose-regulated protein 78 (GRP78) is a novel autoantigen in human type 1 diabetes. When human islets were exposed to inflammatory stress induced by interleukin-1ß, tumor necrosis factor-α, and interferon-γ, arginine residue R510 within GRP78 was converted into citrulline, as evidenced by liquid chromatography-tandem mass spectrometry. This conversion, known as citrullination, led to the generation of neoepitopes, which effectively could be presented by HLA-DRB1*04:01 molecules. With the use of HLA-DRB1*04:01 tetramers and ELISA techniques, we demonstrate enhanced antigenicity of citrullinated GRP78 with significantly increased CD4+ T-cell responses and autoantibody titers in patients with type 1 diabetes compared with healthy control subjects. Of note, patients with type 1 diabetes had a predominantly higher percentage of central memory cells and a lower percentage of effector memory cells directed against citrullinated GRP78 compared with the native epitope. These results strongly suggest that citrullination of ß-cell proteins, exemplified here by the citrullination of GRP78, contributes to loss of self-tolerance toward ß-cells in human type 1 diabetes, indicating that ß-cells actively participate in their own demise.

11.
PLoS One ; 13(10): e0204620, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30304054

RESUMO

Whole extract or allergen-specific IgE testing has become increasingly popular in the diagnosis of peanut allergy. However, much less is known about T cell responses in peanut allergy and how it relates to different clinical phenotypes. CD4+ T cells play a major role in the pathophysiology of peanut allergy as well as tolerance induction during oral desensitization regimens. We set out to characterize and phenotype the T cell responses and their targets in peanut sensitized patients. Using PBMC from peanut-allergic and non-allergic patients, we mapped T cell epitopes for three major peanut allergens, Ara h 1, 2 and 3 (27 from Ara h 1, 4 from Ara h 2 and 43 from Ara h 3) associated with release of IFNγ (representative Th1 cytokine) and IL5 (representative Th2 cytokine). A pool containing 19 immunodominant peptides, selected to account for 60% of the total Ara h 1-3-specific T cell response in allergics, but only 20% in non-allergics, was shown to discriminate T cell responses in peanut-sensitized, symptomatic vs non-symptomatic individuals more effectively than peanut extract. This pool elicited positive T cell responses above a defined threshold in 12/15 sensitized, symptomatic patients, whereas in the sensitized but non-symptomatic cohort only, 4/14 reacted. The reactivity against this peptide pool in symptomatic patients was dominated by IL-10, IL-17 and to a lesser extend IL-5. For four distinct epitopes, HLA class II restrictions were determined, enabling production of tetrameric reagents. Tetramer staining in four donors (2 symptomatic, 2 non-symptomatic) revealed a trend for increased numbers of peanut epitope-specific T cells in symptomatic patients compared to non-symptomatic patients, which was associated with elevated CRTh2 expression whereas cells from non-symptomatic patients exhibited higher levels of Integrin ß7 expression. Our results demonstrate differences in T cell response magnitude, epitope specificity and phenotype between symptomatic and non-symptomatic peanut-sensitized patients. In addition to IgE reactivity, analysis of peanut-specific T cells may be useful to improve our understanding of different clinical manifestations in peanut allergy.

12.
J Autoimmun ; 92: 47-56, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29853344

RESUMO

ACPA-positive rheumatoid arthritis (RA) is associated with distinct HLA-DR alleles and immune responses to many citrullinated self-antigens. Herein we investigated the T cell epitope confined within α-enolase326-340 in the context of HLA-DRB1*04:01 and assessed the corresponding CD4+ T cells in both the circulation and in the rheumatic joint. Comparative crystallographic analyses were performed for the native and citrullinated α-enolase326-340 peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for ex vivo and in vitro assessment of α-enolase-specific T cells in peripheral blood, synovial fluid and synovial tissue by flow cytometry. The native and modified peptides take a completely conserved structural conformation within the peptide-binding cleft of HLA-DRB1*04:01. The citrulline residue-327 was located N-terminally, protruding towards TCRs. The frequencies of T cells recognizing native eno326-340 were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno326-340 was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, p = 0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, p = 0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within α-enolase326-340 does not appear to lead to complete negative selection of cognate CD4+ T cells. In RA patient samples, only T cells recognizing the citrullinated version of α-enolase326-340 were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance.

13.
Diabetes ; 67(6): 1035-1042, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29784651

RESUMO

Prior to the onset of type 1 diabetes, there is progressive loss of immune self-tolerance, evidenced by the accumulation of islet autoantibodies and emergence of autoreactive T cells. Continued autoimmune activity leads to the destruction of pancreatic ß-cells and loss of insulin secretion. Studies of samples from patients with type 1 diabetes and of murine disease models have generated important insights about genetic and environmental factors that contribute to susceptibility and immune pathways that are important for pathogenesis. However, important unanswered questions remain regarding the events that surround the initial loss of tolerance and subsequent failure of regulatory mechanisms to arrest autoimmunity and preserve functional ß-cells. In this Perspective, we discuss various processes that lead to the generation of neoepitopes in pancreatic ß-cells, their recognition by autoreactive T cells and antibodies, and potential roles for such responses in the pathology of disease. Emerging evidence supports the relevance of neoepitopes generated through processes that are mechanistically linked with ß-cell stress. Together, these observations support a paradigm in which neoepitope generation leads to the activation of pathogenic immune cells that initiate a feed-forward loop that can amplify the antigenic repertoire toward pancreatic ß-cell proteins.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/metabolismo , Células Secretoras de Insulina/imunologia , Modelos Imunológicos , Linfócitos T/imunologia , Animais , Apresentação do Antígeno , Autoanticorpos/biossíntese , Doenças Autoimunes/sangue , Doenças Autoimunes/metabolismo , Doenças Autoimunes/fisiopatologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Progressão da Doença , Humanos , Tolerância Imunológica , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ativação Linfocitária , Processamento de Proteína Pós-Traducional , Linfócitos T/metabolismo
14.
Diabetes ; 67(7): 1356-1368, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29654212

RESUMO

In spite of tolerance mechanisms, some individuals develop T-cell-mediated autoimmunity. Posttranslational modifications that increase the affinity of epitope presentation and/or recognition represent one means through which self-tolerance mechanisms can be circumvented. We investigated T-cell recognition of peptides that correspond to modified ß-cell antigens in subjects with type 1 diabetes. Modified peptides elicited enhanced proliferation by autoreactive T-cell clones. Endoplasmic reticulum (ER) stress in insulinoma cells increased cytosolic calcium and the activity of tissue transglutaminase 2 (tTG2). Furthermore, stressed human islets and insulinomas elicited effector responses from T cells specific for modified peptides, suggesting that ER stress-derived tTG2 activity generated deamidated neoepitopes that autoreactive T cells recognized. Patients with type 1 diabetes had large numbers of T cells specific for these epitopes in their peripheral blood. T cells with these specificities were also isolated from the pancreatic draining lymph nodes of cadaveric donors with established diabetes. Together, these results suggest that self-antigens are enzymatically modified in ß-cells during ER stress, giving rise to modified epitopes that could serve to initiate autoimmunity or to further broaden the antigenic repertoire, activating potentially pathogenic CD4+ T cells that may not be effectively eliminated by negative selection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Estresse do Retículo Endoplasmático/fisiologia , Epitopos de Linfócito T/metabolismo , Células Secretoras de Insulina/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apresentação do Antígeno , Autoantígenos/imunologia , Autoimunidade/imunologia , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Ativação Enzimática , Epitopos de Linfócito T/imunologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Insetos , Células Secretoras de Insulina/imunologia , Processamento de Proteína Pós-Traducional/fisiologia , Desiminases de Arginina em Proteínas/metabolismo , Transglutaminases/metabolismo
15.
Circulation ; 138(11): 1130-1143, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-29588316

RESUMO

BACKGROUND: CD4+ T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules. METHODS: We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe-/-) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined. RESULTS: In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4+ T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3+ regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe-/- mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4+ T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-Ab-p18 tetramer identified the expansion of p18-specific CD4+ T cells on vaccination, which were enriched for interleukin-10-producing Tregs. CONCLUSIONS: These findings show that APOB p18-specific CD4+ T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.

16.
Blood Adv ; 2(4): 309-322, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29444872

RESUMO

Factor VIII (FVIII)-neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 contains an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells from HA subjects. The aim of this study was to identify amino acid substitutions to deimmunize this epitope while retaining procoagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The shortest DRB1*01:01-binding peptide was FVIII2194-2205, and residues important for affinity were identified as F2196, M2199, A2201, and S2204. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation of clones specific for the WT sequence. T-cell clones that were stimulated by recombinant WT-FVIII-C2 (rWT-FVIII-C2) protein did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic peptide includes a naturally processed T-cell epitope. Additional amino acid substitutions at F2196 and M2199 were evaluated by peptide-MHC class II (MHCII)-binding assays, T-cell proliferation assays, epitope prediction algorithms, and sequence homologies. Six B-domain-deleted (BDD)-FVIII proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W, or M2199R were produced. Proliferation of T-cell clones and polyclonal lines in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A was reduced compared with responses to WT-BDD-FVIII. The BDD-FVIII-F2196K sequence modification appears to be the most promising sequence variant tested here, due to its effectiveness at eliminating DRB1*01:01-restricted immunogenicity, low potential immunogenicity in the context of other MHCII alleles, expression level comparable to WT-BDD-FVIII, and retained procoagulant activity. These results provide proof of principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients.

17.
Diabetologia ; 61(3): 658-670, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29196783

RESUMO

AIMS/HYPOTHESIS: Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8+ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting. METHODS: The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers. RESULTS: The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8+ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8+ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors. CONCLUSIONS/INTERPRETATION: Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Antígeno HLA-A2/metabolismo , Adulto , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Adulto Jovem
18.
Front Immunol ; 9: 2811, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619245

RESUMO

Infection and vaccination can lead to activation of autoreactive T cells, including the activation of cross-reactive T cells. However, detecting these cross-reactive T cells and identifying the non-self and self-antigen epitopes is difficult. The current study demonstrates the utility of a novel approach that effectively accomplishes both. We utilized surface expression of CD38 on newly activated CD4 memory T cells as a strategy to identify type 1 diabetes associated autoreactive T cells activated by influenza vaccination in healthy subjects. We identified an influenza A matrix protein (MP) specific CD4+ T cell clone that cross-recognizes an immunodominant epitope from Glutamic Acid Decarboxylase 65 (GAD65) protein. The sequences of the MP and GAD65 peptides are rather distinct, with only 2 identical amino acids within the HLA-DR binding region. This result suggests that activation of autoreactive T cells by microbial infection under certain physiological conditions can occur amongst peptides with minimum amino acid sequence homology. This novel strategy also provides a new research pathway in which to examine activation of autoreactive CD4+ T cells after vaccination or natural infection.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Epitopos Imunodominantes/imunologia , ADP-Ribosil Ciclase 1/imunologia , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/virologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/virologia , Glutamato Descarboxilase/imunologia , Antígenos HLA-DR/imunologia , Humanos , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Peptídeos/imunologia
19.
Diabetes ; 66(12): 2940-2941, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29162735
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