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1.
Proc Natl Acad Sci U S A ; 119(15): e2110987119, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35385352

RESUMO

Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to reestablish tolerance. The Endotope platform supports the optimal presentation of endogenously expressed epitopes on appropriate major histocompatibility complex (MHC) class I and II molecules. Using specific epitopes that are disease-relevant (including neoepitopes and mimotopes) and restricted to the subject's MHC haplotypes provides a more focused and tailored way of targeting autoreactive T cells. We evaluated the efficacy of an Endotope DNA vaccine tailored to the nonobese diabetic (NOD) mouse in parallel to one expressing the Proinsulin protein, a central autoantigen in NOD mice, and assessed the influence of several parameters (e.g., route, dosing frequency, disease stage) on diabetes prevention. Secretion of encoded peptides and intradermal delivery of DNA offered more effective disease prevention. Long-term weekly treatments were needed to achieve protection that can persist after discontinuation, likely mediated by regulatory T cells induced by at least one epitope. Although epitopes were presented for at least 2 wk, weekly treatments were needed, at least initially, to achieve significant protection. While Endotope and Proinsulin DNA vaccines were effective at both the prediabetic normoglycemic and dysglycemic stages of disease, Proinsulin provided better protection in the latter stage, particularly in animals with slower progression of disease, and Endotope limited insulitis the most in the earlier stage. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.


Assuntos
Diabetes Mellitus Tipo 1 , Proinsulina , Vacinas de DNA , Animais , Diabetes Mellitus Tipo 1/prevenção & controle , Epitopos de Linfócito T/imunologia , Camundongos , Camundongos Endogâmicos NOD , Medicina de Precisão , Proinsulina/genética , Proinsulina/imunologia , Vacinação , Vacinas de DNA/imunologia
2.
Cytometry A ; 99(9): 866-874, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34145721

RESUMO

As the optimized multicolor immunofluorescence panel (OMIP) platform entered its 10th anniversary since its launch, the multicolor flow cytometry landscape has changed significantly. Likewise, OMIPs have continuously evolved to cover larger panel sizes, increasing number of subpopulations profiled in a single panel, and new species. After a decade of contributions to the OMIP platform, a review of this collection, summarizing its content and purpose for the research community, is timely and due. This review provides an overview of OMIPs and a presentation of the depth and diversity of this collection of validated panels, with the expectation that readers will take advantage of them to empower and accelerate their research.


Assuntos
Pesquisa Biomédica , Citometria de Fluxo , Imunofluorescência
3.
Nat Immunol ; 10(9): 1026-33, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19668219

RESUMO

Type 1 diabetes may result from a breakdown in peripheral tolerance that is partially controlled by the expression of peripheral tissue antigens (PTAs) in lymph nodes. Here we show that the transcriptional regulator Deaf1 controls the expression of genes encoding PTAs in the pancreatic lymph nodes (PLNs). The expression of canonical Deaf1 was lower, whereas that of an alternatively spliced variant was higher, during the onset of destructive insulitis in the PLNs of nonobese diabetic (NOD) mice. We identified an equivalent variant Deaf1 isoform in the PLNs of patients with type 1 diabetes. Both the NOD mouse and human Deaf1 variant isoforms suppressed PTA expression by inhibiting the transcriptional activity of canonical Deaf1. Lower PTA expression resulting from the alternative splicing of DEAF1 may contribute to the pathogenesis of type 1 diabetes.


Assuntos
Antígenos/genética , Diabetes Mellitus Tipo 1/imunologia , Linfonodos/imunologia , Proteínas Nucleares/fisiologia , Pâncreas/imunologia , Processamento Alternativo , Animais , Sequência de Bases , Proteínas de Ligação a DNA , Feminino , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Isoformas de Proteínas , Fatores de Transcrição
4.
J Autoimmun ; 119: 102612, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33611150

RESUMO

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.


Assuntos
Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Autoimunidade , Suscetibilidade a Doenças/imunologia , Timo/imunologia , Timo/metabolismo , Animais , Antígenos , Doenças Autoimunes/patologia , Biomarcadores , Seleção Clonal Mediada por Antígeno/imunologia , Modelos Animais de Doenças , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Imunofenotipagem , Linfopoese/genética , Linfopoese/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Especificidade de Órgãos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
5.
Diabetologia ; 62(11): 2040-2051, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31486854

RESUMO

AIMS/HYPOTHESIS: Tolerance induction in lymph nodes can be mediated by both haematopoietic cells (e.g. specific dendritic cells subsets) and by non-haematopoietic cells (e.g. lymph node stromal cells [LNSCs]) when they present peripheral tissue antigens to autoreactive T cells. LNSCs normally regulate T cell trafficking and survival and help to maintain peripheral tolerance by exerting immunosuppressive effects. However, whether autoimmunity can be associated with defective tolerogenic functions of LNSCs is unknown and studies aimed at characterising LNSCs in humans are lacking. We hypothesised that dysregulated T cell responses in pancreatic lymph nodes (PLNs) from donors with type 1 diabetes and from NOD mice may be associated with altered LNSC function. METHODS: We analysed PLNs from donors with type 1 diabetes and NOD mice for LNSC distribution and phenotype using flow cytometry. We assessed the expression of tolerance-related genes in different subsets of LNSCs from human donors, as well as in a population of dendritic cells enriched in autoimmune regulator (AIRE)+ cells and identified as HLA-DRhigh CD45low. RESULTS: The relative frequency of different LNSC subsets was altered in both donors with type 1 diabetes and NOD mice, and both MHC class II and programmed death-ligand 1 (PD-L1) expression were upregulated in human type 1 diabetes. Tolerance-related genes showed similar expression profiles between mouse and human LNSCs at steady state but were generally upregulated in the context of human type 1 diabetes, while, at the same time, many such genes were downregulated in the AIRE-enriched dendritic cell population. CONCLUSION/INTERPRETATION: Our study shows that LNSCs are substantially altered in type 1 diabetes, but, surprisingly, they exhibit an enhanced tolerogenic phenotype along with increased antigen-presenting potential, which may indicate an attempt to offset dendritic cell-related tolerogenic defects in tolerance. Thus, LNSCs could constitute alternative therapeutic targets in which to deliver antigens to help re-establish tolerance and prevent or treat type 1 diabetes. DATA AVAILABILITY: All data generated or analysed during this study are included in the published article (and its online supplementary files). Biomark gene expression data were deposited on the Mendeley repository at https://data.mendeley.com/datasets/d9rdzdmvyf/1 . Any other raw datasets are available from the corresponding author on reasonable request. No applicable resources were generated or analysed during the current study.


Assuntos
Diabetes Mellitus Tipo 1/patologia , Linfonodos/patologia , Pâncreas/patologia , Células Estromais/citologia , Animais , Apresentação de Antígeno , Antígenos , Autoimunidade , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Tolerância Imunológica/imunologia , Terapia de Imunossupressão , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Fenótipo , Linfócitos T/citologia
6.
EMBO J ; 34(7): 841-55, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25733347

RESUMO

The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes.


Assuntos
Reprogramação Celular/imunologia , Terapia de Imunossupressão , Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas/imunologia , Imunologia de Transplantes , Animais , Autoenxertos , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Humanos , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/transplante , Transplante de Rim
7.
J Autoimmun ; 98: 13-23, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30454875

RESUMO

Type 1 diabetes (T1D) is caused by diabetogenic T cells that evaded tolerance mechanisms and react against multiple ß-cell antigens. Antigen-specific therapy to reinstate tolerance (typically using a single ß-cell antigen) has so far proved unsuccessful in T1D patients. Plasmid DNA (pDNA)-mediated expression of proinsulin has demonstrated transient protection in clinical trials, but long-lasting tolerance is yet to be achieved. We aimed to address whether pDNA delivery of multiple epitopes/mimotopes from several ß-cell antigens efficiently presented to CD4+ and CD8+ T cells could also induce tolerance. This approach significantly delayed T1D development, while co-delivery of pDNA vectors expressing four full antigens protected more mice. Delivery of multiple epitopes resulted in a broad engagement of specific T cells, eliciting a response distinct from endogenous epitopes draining from islets. T-cell phenotypes also varied with antigen specificity. Unexpectedly, the repertoire of T cells reactive to the same epitope was highly polyclonal. Despite induction of some CD25+ Foxp3+ regulatory T cells, protection from disease did not persist after treatment discontinuation. These data demonstrate that epitope-based tolerogenic DNA vaccines constitute effective precision medicine tools to target a broad range of specific CD4+ and CD8+ diabetogenic T-cell populations for prevention or treatment of T1D.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Células Secretoras de Insulina/metabolismo , Vacinas de DNA/imunologia , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Modelos Animais de Doenças , Epitopos de Linfócito T/genética , Humanos , Tolerância Imunológica , Células Secretoras de Insulina/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Vacinas de DNA/genética
8.
Stem Cells ; 34(4): 809-19, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26840009

RESUMO

The evolution of Type 1 diabetes (T1D) therapy has been marked by consecutive shifts, from insulin replacement to immunosuppressive drugs and targeted biologics (following the understanding that T1D is an autoimmune disease), and to more disease-specific or patient-oriented approaches such as antigen-specific and cell-based therapies, with a goal to provide efficacy, safety, and long-term protection. At the same time, another important paradigm shift from treatment of new onset T1D patients to prevention in high-risk individuals has taken place, based on the hypothesis that therapeutic approaches deemed sufficiently safe may show better efficacy if applied early enough to maintain endogenous ß cell function, a concept supported by many preclinical studies. This new strategy has been made possible by capitalizing on a variety of biomarkers that can more reliably estimate the risk and rate of progression of the disease. More advanced ("omic"-based) biomarkers that also shed light on the underlying contributors of disease for each individual will be helpful to guide the choice of the most appropriate therapies, or combinations thereof. In this review, we present current efforts to stratify patients according to biomarkers and current alternatives to conventional drug-based therapies for T1D, with a special emphasis on cell-based therapies, their status in the clinic and potential for treatment and/or prevention.


Assuntos
Autoimunidade , Terapia Baseada em Transplante de Células e Tecidos , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/patologia , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/transplante , Transplante de Células-Tronco Mesenquimais , Linfócitos T/imunologia , Linfócitos T/metabolismo
9.
Nat Chem Biol ; 8(12): 990-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23103943

RESUMO

Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.


Assuntos
Citocinas/fisiologia , Interleucina-4/análogos & derivados , Interleucina-4/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/farmacologia , Interleucina-4/química , Mutação/fisiologia , Fenótipo , Fosforilação , Engenharia de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-4/efeitos dos fármacos , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
10.
Front Immunol ; 15: 1258369, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38933266

RESUMO

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but clinical implementation has been challenging. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA used (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Over time, the peptides induced an IgE-independent anaphylactic reaction, the incidence of which was significantly delayed when peptides were in SAgA form rather than in free form. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Immunologic anaphylaxis occurred in NOD mice in a dose-dependent manner but not in C57BL/6 or BALB/c mice; however, its incidence did not correlate with the level of anti-peptide antibodies. We provide evidence that SAgAs significantly improve the efficacy of peptides to induce tolerance and prevent autoimmune diabetes while at the same time reducing their anaphylactogenic potential.


Assuntos
Diabetes Mellitus Tipo 1 , Tolerância Imunológica , Camundongos Endogâmicos NOD , Peptídeos , Animais , Camundongos , Diabetes Mellitus Tipo 1/imunologia , Peptídeos/imunologia , Peptídeos/administração & dosagem , Feminino , Autoantígenos/imunologia , Linfócitos T Reguladores/imunologia , Imunoterapia/métodos , Anafilaxia/prevenção & controle , Anafilaxia/imunologia , Dessensibilização Imunológica/métodos , Dessensibilização Imunológica/efeitos adversos
11.
Nat Cancer ; 5(3): 433-447, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38286827

RESUMO

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.


Assuntos
Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Humanos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Insulina , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
12.
Hum Vaccin Immunother ; 19(1): 2154098, 2023 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-36656048

RESUMO

Antigen-specific immunotherapies (ASITs) address important clinical needs in treating autoimmune diseases. However, Type 1 diabetes is a heterogeneous disease wherein patient characteristics influence responsiveness to ASITs. Targeting not only disease-relevant T cell populations, but also specific groups of patients using precision medicine is a new goal toward achieving effective treatment. HLA-restricted peptides provide advantages over protein as antigens, however, methods for profiling antigen-specific T cells need to improve in sensitivity, depth, and throughput to facilitate epitope selection. Delivery approaches are highly diverse, illustrating the many ways relevant antigen-presenting cell populations and anatomical locations can be targeted for tolerance induction. The role of persistence of antigen presentation in promoting durable antigen-specific tolerance requires further investigation. Based on the outcome of ASIT trials, the field is moving toward using patient-specific variations to improve efficacy, but challenges still lie on the path to delivering more effective and safer treatment to the T1D patient population.


Assuntos
Doenças Autoimunes , Diabetes Mellitus Tipo 1 , Humanos , Diabetes Mellitus Tipo 1/terapia , Epitopos , Antígenos , Imunoterapia/métodos
13.
bioRxiv ; 2023 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-37205572

RESUMO

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but the limited in vivo stability and uptake of peptides impedes clinical implementation. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Both SAgA variants significantly delayed anaphylaxis compared to their respective free peptides. The anaphylaxis, which occurred in NOD mice but not in C57BL/6 mice, was dose-dependent but did not correlate with the production of IgG1 or IgE against the peptides. We provide evidence that SAgAs significantly improve the efficacy and safety of peptide-based immunotherapy.

14.
JCI Insight ; 8(3)2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-36752206

RESUMO

Patients with nonalcoholic steatohepatitis (NASH) have increased expression of liver monocyte chemoattractant protein-1 (MCP-1), but its cellular source and contribution to various aspects of NASH pathophysiology remain debated. We demonstrated increased liver CCL2 (which encodes MCP-1) expression in patients with NASH, and commensurately, a 100-fold increase in hepatocyte Ccl2 expression in a mouse model of NASH, accompanied by increased liver monocyte-derived macrophage (MoMF) infiltrate and liver fibrosis. To test repercussions of increased hepatocyte-derived MCP-1, we generated hepatocyte-specific Ccl2-knockout mice, which showed reduced liver MoMF infiltrate as well as decreased liver fibrosis. Forced hepatocyte MCP-1 expression provoked the opposite phenotype in chow-fed wild-type mice. Consistent with increased hepatocyte Notch signaling in NASH, we observed a close correlation between markers of Notch activation and CCL2 expression in patients with NASH. We found that an evolutionarily conserved Notch/recombination signal binding protein for immunoglobulin kappa J region binding site in the Ccl2 promoter mediated transactivation of the Ccl2 promoter in NASH diet-fed mice. Increased liver MoMF infiltrate and liver fibrosis seen in opposite gain-of-function mice was ameliorated with concomitant hepatocyte Ccl2 knockout or CCR2 inhibitor treatment. Hepatocyte Notch activation prompts MCP-1-dependent increase in liver MoMF infiltration and fibrosis.


Assuntos
Quimiocina CCL2 , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Quimiocina CCL2/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo
15.
Sci Rep ; 12(1): 559, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017558

RESUMO

Identification of cognate interactions between antigen-specific T cells and dendritic cells (DCs) is essential to understanding immunity and tolerance, and for developing therapies for cancer and autoimmune diseases. Conventional techniques for selecting antigen-specific T cells are time-consuming and limited to pre-defined antigenic peptide sequences. Here, we demonstrate the ability to use deep learning to rapidly classify videos of antigen-specific CD8+ T cells. The trained model distinguishes distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and DCs over 20 to 80 min. The model classified high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalized well to other types of high and low affinity CD8+ T cells. The classification accuracy achieved by the model was consistently higher than simple image analysis techniques, and conventional metrics used to differentiate between cognate and non-cognate T cells, such as speed. Also, we demonstrated that experimental addition of anti-CD40 antibodies improved model prediction. Overall, this method demonstrates the potential of video-based deep learning to rapidly classify cognate T cell-DC interactions, which may also be potentially integrated into high-throughput methods for selecting antigen-specific T cells in the future.


Assuntos
Linfócitos T CD8-Positivos
16.
Mol Metab ; 56: 101417, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34902607

RESUMO

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease characterized by impaired immune tolerance to ß-cell antigens and progressive destruction of insulin-producing ß-cells. Animal models have provided valuable insights for understanding the etiology and pathogenesis of this disease, but they fall short of reflecting the extensive heterogeneity of the disease in humans, which is contributed by various combinations of risk gene alleles and unique environmental factors. Collectively, these factors have been used to define subgroups of patients, termed endotypes, with distinct predominating disease characteristics. SCOPE OF REVIEW: Here, we review the gaps filled by these models in understanding the intricate involvement and regulation of the immune system in human T1D pathogenesis. We describe the various models developed so far and the scientific questions that have been addressed using them. Finally, we discuss the limitations of these models, primarily ascribed to hosting a human immune system (HIS) in a xenogeneic recipient, and what remains to be done to improve their physiological relevance. MAJOR CONCLUSIONS: To understand the role of genetic and environmental factors or evaluate immune-modifying therapies in humans, it is critical to develop and apply models in which human cells can be manipulated and their functions studied under conditions that recapitulate as closely as possible the physiological conditions of the human body. While microphysiological systems and living tissue slices provide some of these conditions, HIS mice enable more extensive analyses using in vivo systems.


Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Animais , Diabetes Mellitus Tipo 1/genética , Humanos , Sistema Imunitário/patologia , Células Secretoras de Insulina/patologia , Camundongos
17.
J Clin Invest ; 132(24)2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36282594

RESUMO

As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic ß cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate ß-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-ß that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.


Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Camundongos , Animais , Proteína Forkhead Box O1/genética , Fatores de Transcrição Forkhead/genética , Camundongos Endogâmicos NOD , Insulina/genética
18.
Blood ; 113(26): 6638-47, 2009 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-19363220

RESUMO

Because of their potent immunoregulatory capacity, dendritic cells (DCs) have been exploited as therapeutic tools to boost immune responses against tumors or pathogens, or dampen autoimmune or allergic responses. Murine bone marrow-derived DCs (BM-DCs) are the closest known equivalent of the blood monocyte-derived DCs that have been used for human therapy. Current imaging methods have proven unable to properly address the migration of injected DCs to small and deep tissues in mice and humans. This study presents the first extensive analysis of BM-DC homing to lymph nodes (and other selected tissues) after intravenous and intraperitoneal inoculation. After intravenous delivery, DCs accumulated in the spleen, and preferentially in the pancreatic and lung-draining lymph nodes. In contrast, DCs injected intraperitoneally were found predominantly in peritoneal lymph nodes (pancreatic in particular), and in omentum-associated lymphoid tissue. This uneven distribution of BM-DCs, independent of the mouse strain and also observed within pancreatic lymph nodes, resulted in the uneven induction of immune response in different lymphoid tissues. These data have important implications for the design of systemic cellular therapy with DCs, and in particular underlie a previously unsuspected potential for specific treatment of diseases such as autoimmune diabetes and pancreatic cancer.


Assuntos
Células Dendríticas/citologia , Tecido Linfoide/citologia , Animais , Células da Medula Óssea/citologia , Movimento Celular/fisiologia , Células Dendríticas/transplante , Feminino , Genes Reporter , Imunoterapia Adotiva , Injeções Intraperitoneais , Injeções Intravenosas , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Pulmão , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Omento , Especificidade de Órgãos , Pâncreas , Baço
19.
Mol Ther ; 18(12): 2112-20, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20628358

RESUMO

Bone marrow-derived dendritic cells (DCs) are cells of the immune system that have been used as a tool to boost, modulate, or dampen immune responses. In the context of autoimmunity, DCs can be modified to express immunoregulatory products encoded by transgenes, and used therapeutically in adoptive cellular therapy. DCs that were lentivirally transduced (lt) to express interleukin 4 (IL-4) can significantly delay or prevent the onset of autoimmune diabetes in nonobese diabetic (NOD) mice. However, modifying cells using viral vectors carries the dual risk of oncogenicity or immunogenicity. This study demonstrates that NOD DCs, electroporated with "translationally enhanced" IL-4 mRNA (eDC/IL-4), can be equally efficient therapeutically, despite the reduced amount and shorter duration of IL-4 secretion. Moreover, a single injection of eDC/IL-4 in NOD mice shortly after the onset of hyperglycemia was able to maintain stable glycemia for up to several months in a significant fraction of treated mice. Treatment with eDC/IL-4 boosted regulatory T (Tregs) cell functions and modulated T helper responses to reduce pathogenicity. Thus, treatment with DCs, electroporated with modified IL-4 mRNA to express IL-4 for up to 24 hours, constitutes a viable cellular therapy approach for the regulation of autoimmune diabetes, as a preferred alternative to the use of viral vectors.


Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Diabetes Mellitus Tipo 1/terapia , Interleucina-4/uso terapêutico , Animais , Células Dendríticas/transplante , Eletroporação , Interleucina-4/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , RNA Mensageiro/genética
20.
Diabetes ; 70(6): 1334-1346, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33468513

RESUMO

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address the limited clinical efficacy of ASIT. Soluble antigen arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using either hydrolysable linkers (hSAgAs) or stable click chemistry linkers (cSAgAs). They were evaluated for the ability to block spontaneous development of disease in a nonobese diabetic mouse model of type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgAs) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers, such as CD73, interleukin-10, programmed death-1, and KLRG-1. Anaphylaxis caused by free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely nonoverlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to free peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Peptídeos/farmacocinética , Análise Serial de Proteínas , Animais , Autoantígenos/imunologia , Química Click , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/imunologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Feminino , Imunoterapia/instrumentação , Imunoterapia/métodos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacocinética , Peptídeos/administração & dosagem , Indução de Remissão/métodos , Solubilidade , Resultado do Tratamento
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