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Studies of individual T cell antigen receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, the general rules that can be used to predict whether TCRs are self-reactive have not been fully elucidated. Here we found that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the complementarity-determining region CDR3ß robustly promoted the development of self-reactive TCRs. This property was found irrespective of the member of the ß-chain variable region (Vß) family present in the TCR or the length of the CDR3ß. An index based on these findings distinguished Vß2(+), Vß6(+) and Vß8.2(+) regulatory T cells from conventional T cells and also distinguished CD4(+) T cells selected by the major histocompatibility complex (MHC) class II molecule I-A(g7) (associated with the development of type 1 diabetes in NOD mice) from those selected by a non-autoimmunity-promoting MHC class II molecule I-A(b). Our results provide a means for distinguishing normal T cell repertoires versus autoimmunity-prone T cell repertoires.
Assuntos
Autoimunidade , Regiões Determinantes de Complementaridade/genética , Diabetes Mellitus Tipo 1/imunologia , Subpopulações de Linfócitos T/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Autoantígenos/imunologia , Autoantígenos/metabolismo , Diferenciação Celular , Tolerância Central , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos KnockoutRESUMO
Adiponectin is a circulating hormone secreted by adipose tissue that exerts, unlike other adipokines such as leptin, anti-inflammatory, anti-atherosclerotic and other protective effects on health. Adiponectin receptor agonists are being tested in clinical trials and are expected to show benefits in many diseases. In a recent article, LW Chen's group used monocyte chemoattractant protein-1 (MCP-1/CCL2) to improve plasma levels of adiponectin, suggesting the involvement of dipeptidyl peptidase 4 (DPP4/CD26) in the mechanism. Here, we discuss the significance of the role of DPP4, favoring the increase in DPP4-positive interstitial progenitor cells, a finding that fits with the greater stemness and persistence of other DPP4/CD26-positive cells.
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Adipogenia , Tecido Adiposo , Dipeptidil Peptidase 4 , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidase 4/genética , Adipogenia/genética , Adipogenia/efeitos dos fármacos , Humanos , Tecido Adiposo/metabolismo , Animais , Adiponectina/metabolismo , Adiponectina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Células Estromais/metabolismo , Adipócitos/metabolismo , Adipócitos/efeitos dos fármacosRESUMO
AIMS/HYPOTHESIS: Antigen-specific therapy aims to modify inflammatory T cell responses in type 1 diabetes and restore immune tolerance. One strategy employs GAD65 conjugated to aluminium hydroxide (GAD-alum) to take advantage of the T helper (Th)2-biasing adjuvant properties of alum and thereby regulate pathological Th1 autoimmunity. We explored the cellular and molecular mechanism of GAD-alum action in the setting of a previously reported randomised placebo-controlled clinical trial conducted by Type 1 Diabetes TrialNet. METHODS: In the clinical trial conducted by Type 1 Diabetes TrialNet, participants were immunised with 20 µg GAD-alum (twice or three times) or alum alone and peripheral blood mononuclear cell samples were banked at baseline and post treatment. In the present study, GAD-specific T cell responses were measured in these samples and GAD-specific T cell lines and clones were generated, which were then further characterised. RESULTS: At day 91 post immunisation, we detected GAD-specific IL-13+ CD4 T cell responses significantly more frequently in participants immunised with GAD-alum (71% and 94% treated twice or three times, respectively) compared with those immunised with alum alone (38%; p = 0.003 and p = 0.0002, respectively) accompanied by high secreted levels of IL-13, IL-4 and IL-5, confirming a GAD-specific, GAD-alum-induced Th2 response. Of note, GAD-specific, IL-13+ CD4 T cells observed after immunisation co-secreted IFN-γ, displaying a bifunctional Th1/Th2 phenotype. Single-cell transcriptome analysis identified IL13 and IFNG expression in concert with the canonical Th2 and Th1 transcription factor genes GATA3 and TBX21, respectively. T cell receptor ß-chain (TCRB) CDR3 regions of GAD-specific bifunctional T cells were identified in circulating naive and central memory CD4 T cell pools of non-immunised participants with new-onset type 1 diabetes and healthy individuals, suggesting the potential for bifunctional responses to be generated de novo by GAD-alum immunisation or via expansion from an existing public repertoire. CONCLUSIONS/INTERPRETATION: GAD-alum immunisation activates and propagates GAD-specific CD4 T cells with a distinctive bifunctional phenotype, the functional analysis of which might be important in understanding therapeutic responses.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Imunoterapia/métodos , Células Th1/imunologia , Células Th2/imunologia , Linhagem Celular , Criopreservação , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores de Antígenos de Linfócitos T/metabolismo , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Type 1 diabetes (T1D) results from the destruction of pancreatic ß-cells by the immune system, and CD8+ T lymphocytes are critical actors in this autoimmune response. Pancreatic islets are surrounded by a mesh of nervous cells, the peri-insular Schwann cells, which are also targeted by autoreactive T lymphocytes and express specific antigens, such as the neurotrophic factor S100-ß. Previous work has shown increased proliferative responses to whole S100-ß in both human T1D patients and the nonobese diabetic (NOD) mouse model. We describe for the first time naturally processed and presented epitopes (NPPEs) presented by class I human leukocyte antigen-A*02:01 (A2.1) molecules derived from S100-ß. These NPPEs triggered IFN-γ responses more frequently in both newly diagnosed and long-term T1D patients compared with healthy donors. Furthermore, the same NPPEs are recognized during the autoimmune response leading to diabetes in A2.1-transgenic NOD mice as early as 4 wk of age. Interestingly, when these NPPEs are used to prevent diabetes in this animal model, an acceleration of the disease is observed together with an exacerbation in insulitis and an increase in S100-ß-specific cytotoxicity in vaccinated animals. Whether these can be used in diabetes prevention needs to be carefully evaluated in animal models before use in future clinical assays.-Calviño-Sampedro, C., Gomez-Tourino, I., Cordero, O. J., Reche, P. A., Gómez-Perosanz, M., Sánchez-Trincado, J. L., Rodríguez, M. Á., Sueiro, A. M., Viñuela, J. E., Calviño, R. V. Naturally presented HLA class I-restricted epitopes from the neurotrophic factor S100-ß are targets of the autoimmune response in type 1 diabetes.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Epitopos/farmacologia , Antígeno HLA-A2/imunologia , Subunidade beta da Proteína Ligante de Cálcio S100/farmacologia , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Antígeno HLA-A2/genética , Humanos , Interferon gama/genética , Interferon gama/imunologia , Células K562 , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos TransgênicosRESUMO
We recently demonstrated that the major effector function of neonatal CD4+ T cells is to produce CXCL8, a prototypic cytokine of innate immune cells. In this article, we show that CXCL8 expression, prior to proliferation, is common in newly arising T cells (so-called "recent thymic emigrants") in adults, as well as in babies. This effector potential is acquired in the human thymus, prior to TCR signaling, but rather than describing end-stage differentiation, such cells, whether isolated from neonates or adults, can further differentiate into IFN-γ-producing CD4+ T cells. Thus, the temporal transition of host defense from innate to adaptive immunity is unexpectedly mirrored at the cellular level by the capacity of human innate-like CXCL8-producing CD4+ T cells to transition directly into Th1 cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Neuroblastoma/imunologia , Timócitos/imunologia , Tumor de Wilms/imunologia , Imunidade Adaptativa , Adulto , Animais , Células Cultivadas , Humanos , Imunidade Inata , Recém-Nascido , Interferon gama/metabolismo , Interleucina-8/metabolismo , Camundongos , Camundongos SCID , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Type 1 diabetes was one of the earliest disorders to be associated with the phenomenon of autoimmunity and is one of the most studied organ-specific autoimmune diseases at the epidemiologic, immunologic and genetic level. Despite this, and the emergence of a plethora of strategies for trying to intervene in, or prevent the disease, it remains at some distance from being reliably and safely tractable by immunotherapy, a source of great frustration in this research field. In this article we review some of the key concepts that might impact upon this lack of success in the clinic going forward. These include new insights into autoreactive CD4 and CD8 T cell biology and a discussion of the concept of disease heterogeneity as it applies to type 1 diabetes. The onset of disease is characterised by a delicate equilibrium of proinflammatory and regulatory T cells, which we have termed "balanced autoreactive set-point", and which may be amenable to antigen-specific immunotherapies that alter the rate of disease progression. Advances in the characterization of T cells, especially at the single cell level, could be rewarding, notably from the vantage point of biomarker and surrogate discovery. A better understanding of T cell targeting, autoantigen processing and the ß-cell:immune interface is also needed, although access to diseased tissues is a major limitation in this effort. Finally, the existence of disease heterogeneity is an emerging theme in this and other complex immunopathologies, and could be both a blessing (finding the right drug for the right person) and a hindrance (compromising the power of early-stage trials of emerging therapeutics).
Assuntos
Autoantígenos/metabolismo , Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Linfócitos T Reguladores/imunologia , Fatores Etários , Anticorpos Monoclonais/uso terapêutico , Apresentação de Antígeno , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/terapia , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Inflamação/imunologia , Células Secretoras de Insulina/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/metabolismoAssuntos
Infecções por HIV , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Intervenção Médica Precoce , Feminino , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Homofobia , Humanos , Lactente , Prêmio Nobel , Vacinas contra a SAIDS/imunologia , Sexo Seguro , Direitos da MulherRESUMO
In this editorial we comment on the article by Agatsuma et al published in the World Journal of Gastroenterology. They suggest policies for more effective colorectal screening. Screening is the main policy that has led to lower mortality rates in later years among the population that was eligible for screening. Colonoscopy is the gold standard tool for screening and has preventive effects by removing precancerous or early malignant polyps. However, colonoscopy is an invasive process, and fecal tests such as the current hemoglobin immunodetection were developed, followed by endoscopy, as the general tool for population screening, avoiding logistical and economic problems. Even so, participation and adherence rates are low. Different screening options are being developed with the idea that if people could choose between the ones that best suit them, participation in population-based screening programs would increase. Blood tests, such as a recent one that detects cell-free DNA shed by tumors called circulating tumor DNA, showed a similar accuracy rate to stool tests for cancer, but were less sensitive for advanced precancerous lesions. At the time when the crosstalk between the immune system and cancer was being established as a new hallmark of cancer, novel immune system-related biomarkers and information on patients' immune parameters, such as cell counts of different immune populations, were studied for the early detection of colorectal cancer, since they could be effective in asymptomatic people, appearing earlier in the adenoma-carcinoma development compared to the presence of fecal blood. sCD26, for example, detected 80.37% of advanced adenomas. To reach as many eligible people as possible, starting at an earlier age than current programs, the direction could be to apply tests based on blood, urine or salivary fluid to samples taken during routine visits to the primary health system.
Assuntos
Colonoscopia , Neoplasias Colorretais , Detecção Precoce de Câncer , Humanos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/prevenção & controle , Detecção Precoce de Câncer/métodos , Detecção Precoce de Câncer/normas , Programas de Rastreamento/métodos , Programas de Rastreamento/estatística & dados numéricos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Sangue Oculto , Fezes/química , Adenoma/diagnóstico , Adenoma/prevenção & controleRESUMO
Soluble CD26 (sCD26), a glycoprotein with dipeptidyl peptidase (DPP4) enzymatic activity, can contribute to early diagnosis of colorectal cancer and advanced adenomas and has been studied, including for prognostic purposes, across various other types of cancer and disease. The latest research in this field has confirmed that most, though not all, serum/plasma sCD26 is related to inflammation. The shedding and/or secretion of sCD26 from different immune cells are being investigated, and blood DPP4 activity levels do not correlate very strongly with protein titers. Some of the main substrates of this enzyme are key chemokines involved in immune cell migration, and both soluble and cell-surface CD26 can bind adenosine deaminase (ADA), an enzyme involved in the metabolism of immunosuppressor extracellular adenosine. Of note, there are T cells enriched in CD26 expression and, in mice tumor models, tumor infiltrating lymphocytes exhibited heightened percentages of CD26+ correlating with tumor regression. We employed sCD26 as a biomarker in the follow-up after curative resection of colorectal cancer for the early detection of tumor recurrence. Changes after treatment with different biological disease-modifying antirheumatic drugs, including Ig-CTLA4, were also observed in rheumatoid arthritis. Serum soluble CD26/DPP4 titer variation has recently been proposed as a potential prognostic biomarker after a phase I trial in cancer immunotherapy with a humanized anti-CD26 antibody. We propose that dynamic monitoring of sCD26/DPP4 changes, in addition to well-known inflammatory biomarkers such as CRP already in use as informative for immune checkpoint immunotherapy, may indicate resistance or response during the successive steps of the treatment. As tumor cells expressing CD26 can also produce sCD26, the possibility of sorting immune- from non-immune-system-originated sCD26 is discussed.
RESUMO
Melanoma is the main cause of death among skin cancers and its incidence worldwide has been experiencing an appalling increase. However, traditional treatments lack effectiveness in advanced or metastatic patients. Immunotherapy, meanwhile, has been shown to be an effective treatment option, but the rate of cancers responding remains far from ideal. Here we have developed a personalized neoantigen peptide-based cancer vaccine by encapsulating patient derived melanoma neoantigens in polyethylenimine (PEI)-functionalised poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and coating them with polyinosinic:polycytidylic acid (poly(I:C)). We found that PLGA NPs can be effectively modified to be coated with the immunoadjuvant poly(I:C), as well as to encapsulate neoantigens. In addition, we found that both dendritic cells (DCs) and lymphocytes were effectively stimulated. Moreover, the developed NP was found to have a better immune activation profile than NP without poly(I:C) or without antigen. Our results demonstrate that the developed vaccine has a high capacity to activate the immune system, efficiently maturing DCs to present the antigen of choice and promoting the activity of lymphocytes to exert their cytotoxic function. Therefore, the immune response generated is optimal and specific for the elimination of melanoma tumour cells.
RESUMO
In the advanced renal cell carcinoma (RCC) scenario, there are no consistent biomarkers to predict the clinical benefit patients derived from immune checkpoint blockade (ICB). Taking this into consideration, herein, we conducted a retrospective study in order to develop and validate a gene expression score for predicting clinical benefit to the anti-PD-1 antibody nivolumab in the context of patients diagnosed with advanced clear cell RCC enrolled in the CheckMate-009, CheckMate-010, and CheckMate-025 clinical trials. First, a three-gene expression score (3GES) with prognostic value for overall survival integrating HMGA1, NUP62, and ARHGAP42 transcripts was developed in a cohort of patients treated with nivolumab. Its prognostic value was then validated in the TCGA-KIRC cohort. Second, the predictive value for nivolumab was confirmed in a set of patients from the CheckMate-025 phase 3 clinical trial. Lastly, we explored the correlation of our 3GES with different clinical, molecular, and immune tumor characteristics. If the results of this study are definitively validated in other retrospective and large-scale, prospective studies, the 3GES will represent a valuable tool for guiding the design of ICB-based clinical trials in the aRCC scenario in the near future.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Renais , Inibidores de Checkpoint Imunológico , Neoplasias Renais , Nivolumabe , Feminino , Humanos , Masculino , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/imunologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/imunologia , Nivolumabe/uso terapêutico , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The modulation of the A2B adenosine receptor is a promising strategy in cancer (immuno) therapy, with A2BAR antagonists emerging as immune checkpoint inhibitors. Herein, we report a systematic assessment of the impact of (di- and mono-)halogenation at positions 7 and/or 8 on both A2BAR affinity and pharmacokinetic properties of a collection of A2BAR antagonists and its study with structure-based free energy perturbation simulations. Monohalogenation at position 8 produced potent A2BAR ligands irrespective of the nature of the halogen. In contrast, halogenation at position 7 and dihalogenation produced a halogen-size-dependent decay in affinity. Eight novel A2BAR ligands exhibited remarkable affinity (Ki < 10 nM), exquisite subtype selectivity, and enantioselective recognition, with some eutomers eliciting sub-nanomolar affinity. The pharmacokinetic profile of representative derivatives showed enhanced solubility and microsomal stability. Finally, two compounds showed the capacity of reversing the antiproliferative effect of adenosine in activated primary human peripheral blood mononuclear cells.
Assuntos
Halogenação , Antagonistas de Receptores Purinérgicos P1 , Cricetinae , Animais , Humanos , Células CHO , Leucócitos Mononucleares/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Receptor A2B de Adenosina/metabolismo , Ligantes , HalogêniosRESUMO
In rheumatoid arthritis (RA), the identification of biomarkers to adjust treatment intensity and to correctly diagnose the disease in early stages still constitutes a challenge and, as such, novel biomarkers are needed. We proposed that autoantibodies (aAbs) against CD26 (DPP4) might have both etiological importance and clinical value. Here, we perform a prospective study of the potential diagnostic power of Anti-CD26 aAbs through their quantification in plasmas from 106 treatment-naïve early and undifferentiated AR. Clinical antibodies, Anti-CD26 aAbs, and other disease-related biomarkers were measured in plasmas obtained in the first visit from patients, which were later classified as RA and non-RA according to the American College of Rheumatology criteria. Two different isotype signatures were found among ten groups of patients, one for Anti-CD26 IgA and other for Anti-CD26 IgG and IgM isotypes, both converging in patients with arthritis (RA and Unresolved Undifferentiated Arthritis: UUA), who present elevated levels of all three isotypes. The four UUA patients, unresolved after two years, were ACPA and rheumatic factor (RF) negatives. In the whole cohort, 51.3% of ACPA/RF seronegatives were Anti-CD26 positives, and a similar frequency was observed in the seropositive RA patients. Only weak associations of the three isotypes with ESR, CRP and disease activity parameters were observed. Anti-CD26 aAbs are present in treatment-naïve early arthritis patients, including ACPA and RF seronegative individuals, suggestive of a potential pathogenic and/or biomarker role of Anti-CD26 aAbs in the development of rheumatic diseases.
Assuntos
Artrite Reumatoide , Dipeptidil Peptidase 4 , Autoanticorpos , Humanos , Estudos Prospectivos , Fator ReumatoideRESUMO
Galectins are a group of ß-galactoside-binding mammalian lectins that play important roles in the regulation of the immune response by promoting T cell tolerance, blunting Th1 and Th17 responses and suppressing autoimmune inflammation. However, the synthesis of these molecules by different T helper (Th) subsets and in the context of human type 1 diabetes (T1D) has not yet been studied. Our results show that Th17 polarising conditions induce the synthesis of higher levels of galectin-1 compared to Th1-polarised lymphocytes. In the context of human diabetes, peripheral blood mononuclear cells (PBMCs) from T1D patients, either unstimulated or after stimulation, secreted significantly lower amounts of galectin-1 in vitro compared to healthy donors. The reduced galectin-1 synthesis observed in this autoimmune disease occurs in a dominant pro-inflammatory cytokine milieu and it is mainly due to the lower synthesis by monocytes. Surprisingly, CD4(+) T helper cells from these patients secreted similar levels of galectin-1 compared to healthy donors, probably mediated by Th17 cytokines. In conclusion, CD4(+) T helper lymphocytes from T1D patients produce normal levels of the immunoregulator galectin-1 but its reduced synthesis by monocytes helps to maintain a skewed pro-inflammatory response.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Galectina 1/biossíntese , Monócitos/imunologia , Células Th1/imunologia , Sequência de Bases , Estudos de Casos e Controles , Membrana Celular/imunologia , Membrana Celular/metabolismo , Proliferação de Células , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Galectina 1/genética , Galectina 1/imunologia , Humanos , Técnicas In Vitro , Interleucinas/farmacologia , Masculino , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Defects in T cell receptor (TCR) repertoire are proposed to predispose to autoimmunity. Here we show, by analyzing >2 × 108 TCRB sequences of circulating naive, central memory, regulatory and stem cell-like memory CD4+ T cell subsets from patients with type 1 diabetes and healthy donors, that patients have shorter TCRB complementarity-determining region 3s (CDR3), in all cell subsets, introduced by increased deletions/reduced insertions during VDJ rearrangement. High frequency of short CDR3s is also observed in unproductive TCRB sequences, which are not subjected to thymic culling, suggesting that the shorter CDR3s arise independently of positive/negative selection. Moreover, TCRB CDR3 clonotypes expressed by autoantigen-specific CD4+ T cells are shorter compared with anti-viral T cells, and with those from healthy donors. Thus, early events in thymic T cell development and repertoire generation are abnormal in type 1 diabetes, which suggest that short CDR3s increase the potential for self-recognition, conferring heightened risk of autoimmune disease.
Assuntos
Autoantígenos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Recombinação V(D)J/imunologia , Adulto , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Separação Celular/métodos , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Diabetes Mellitus Tipo 1/genética , Feminino , Citometria de Fluxo/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Recombinação V(D)J/genética , Adulto JovemRESUMO
Type 1 diabetes (T1D) is one of the most studied archetypal organ-specific autoimmune diseases. Although many clinical, epidemiological, and pathological characteristics have been described, there are still important issues which need to be resolved as these will have a major impact on the development of future antigen-specific immunotherapies. An important question relates to T lymphocytes in the development of the disease, in particular their role in the destruction of insulin-producing beta cells. Since the discovery that certain class II histocompatibility complex molecules (HLA) are linked to the development of T1D, much research has focused on CD4+ helper T lymphocytes; however, recent studies highlight class I HLA molecules as an independent risk factor; hence, research into the role played by CD8+ cytotoxic T lymphocytes has gained momentum. In this review, we summarize recent studies clarifying the role played by both sets of autoreactive T lymphocytes in T1D, discuss the targets recognized by these cells and their phenotype in T1D patients. Finally, we will examine the possible generation of regulatory CD8+ T lymphocytes upon different immuno-intervention strategies.
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Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígenos HLA/genética , Células Secretoras de Insulina/imunologia , Insulina/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/genética , Humanos , Imunomodulação , Polimorfismo GenéticoRESUMO
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
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
The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.