RESUMO
Adoptive immunotherapy with Tregs is a promising approach for preventing or treating type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR redirected NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Cotransfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In WT NOD mice, InsB-g7 CAR Tregs prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising therapeutic approach for the prevention of autoimmune diabetes.
Assuntos
Diabetes Mellitus Tipo 1 , Receptores de Antígenos Quiméricos , Camundongos , Animais , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevenção & controle , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos Endogâmicos NOD , Insulina/metabolismo , Linfócitos T ReguladoresRESUMO
BACKGROUND: Type 1 diabetes is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include the use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). METHODS: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4 + and CD8 + T cells and tested their ability to reject HLA-A2 + islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T-cell engraftment, islet function, and xGVHD were assessed longitudinally. RESULTS: The speed and consistency of A2-CAR T-cell-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of coinjected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, coinjection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2 + human islets within 1 wk and without xGVHD for 12 wk. CONCLUSIONS: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of islet-replacement therapies.
Assuntos
Doença Enxerto-Hospedeiro , Insulinas , Transplante das Ilhotas Pancreáticas , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Antígeno HLA-A2 , Leucócitos Mononucleares , Rejeição de Enxerto/prevenção & controleRESUMO
Background: Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- versus -host disease (xGVHD). Methods: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4+ and CD8+ T cells and tested their ability to reject HLA-A2+ islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T cell engraftment, islet function and xGVHD were assessed longitudinally. Results: The speed and consistency of A2-CAR T cells-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of co-injected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, co-injection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2+ human islets within 1 week and without xGVHD for 12 weeks. Conclusions: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of isletreplacement therapies.
RESUMO
Adoptive immunotherapy with Tregs is a promising approach for prevention or treatment of type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B-chain 10-23 peptide presented in the context of the IA g7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR re-directed NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Co-transfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In wild type NOD mice, InsB-g7 CAR Tregs stably expressed Foxp3 and prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising new therapeutic approach for the prevention of autoimmune diabetes. Brief Summary: Chimeric antigen receptor Tregs specific for an insulin B-chain peptide presented by MHC class II prevent autoimmune diabetes.
RESUMO
Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. This protocol was validated in: Sci Transl Med (2020), DOI: 10.1126/scitranslmed.aaz3866 Graphical abstract.
RESUMO
Regulatory T-cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signaling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T-cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.
Assuntos
Doenças Autoimunes , PTEN Fosfo-Hidrolase , Linfócitos T Reguladores , Fatores de Transcrição Forkhead/metabolismo , Humanos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Up to 6% of diabetes has a monogenic cause including mutations in the insulin gene, and patients are candidates for a gene therapy. Using a mouse model of permanent neonatal diabetes, we assessed the efficacy of an adeno-associated virus (AAV)-mediated gene therapy. We used AAVs with a rat insulin 1 promoter (Ins1) regulating a human insulin gene (INS; AAV Ins1-INS) or native mouse insulin 1 (Ins1; AAV Ins-Ins1) to deliver an insulin gene to ß-cells of constitutive insulin null mice (Ins1-/-Ins2-/-) and adult inducible insulin-deficient mice [Ins1-/-Ins2f/f PdxCreER and Ins1-/-Ins2f/f mice administered AAV Ins1-Cre)]. Although AAV Ins1-INS could successfully infect and confer insulin expression to ß-cells, insulin null ß-cells had a prohormone processing defect. Secretion of abundant proinsulin transiently reversed diabetes. We reattempted therapy with AAV Ins1-Ins1, but Ins1-/-Ins2-/- ß-cells still had a processing defect of both replaced Ins1 and pro-islet amyloid polypeptide (proIAPP). In adult inducible models, ß-cells that lost insulin expression developed a processing defect that resulted in impaired proIAPP processing and elevated circulating proIAPP, and cells infected with AAV Ins1-Ins1 to rescue insulin expression secreted proinsulin. We assessed the subcellular localization of prohormone convertase 1/3 (PC1/3) and detected defective sorting of PC1/3 to glycogen-containing vacuoles and retention in the endoplasmic reticulum as a potential mechanism underlying defective processing. We provide evidence that persistent production of endogenous proinsulin within ß-cells is necessary for ß-cells to be able to properly store and process proinsulin.
Assuntos
Células Secretoras de Insulina , Proinsulina , Animais , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Proinsulina/genética , Proinsulina/metabolismo , RatosRESUMO
Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery.
Assuntos
Glicocálix , Rejeição de Enxerto , Aloenxertos , Animais , Rejeição de Enxerto/prevenção & controle , Imunossupressores , Camundongos , PolímerosRESUMO
Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal Treg therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs To date, most of the CAR Treg studies used second-generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs Using a human leukocyte antigen-A2-specific CAR platform and human Tregs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. Tregs expressing a CAR encoding CD28wt were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and an ability to suppress CD80 expression on dendritic cells as key in vitro predictors of in vivo function. This comprehensive study of CAR signaling domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.
Assuntos
Receptores de Antígenos Quiméricos , Antígenos CD28 , Humanos , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T ReguladoresRESUMO
In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on ß-cell function and organism physiology. Cre-LoxP is a system wherein LoxP sites flanking a gene are recognized by Cre recombinase. Cre transgenic mice are the most prevalent technology used to deliver Cre but many models have caveats of off-target recombination, impaired ß-cell function, and high cost of animal production. Inducible estrogen receptor conjugated Cre models face leaky recombination and confounding effects of tamoxifen. As an alternative, we characterize an adeno associated virus (AAV) with a rat insulin 1 promoter driving Cre recombinase (AAV8 Ins1-Cre) that is economical and rapid to implement, and has limited caveats. Intraperitoneal AAV8 Ins1-Cre produced efficient ß-cell recombination, alongside some hepatic, exocrine pancreas, α-cell, δ-cell, and hypothalamic recombination. Delivery of lower doses via the pancreatic duct retained good rates of ß-cell recombination and limited rates of off-target recombination. Unlike inducible Cre in transgenic mice, AAV8 Ins1-Cre required no tamoxifen and premature recombination was avoided. We demonstrate the utility of this technology by inducing hyperglycemia in inducible insulin knockout mice (Ins1-/-;Ins2f/f). AAV-mediated expression of Cre in ß-cells provides an effective alternative to transgenic approaches for inducible knockout studies.
Assuntos
Dependovirus , Células Secretoras de Insulina/metabolismo , Insulina/genética , Regiões Promotoras Genéticas , Recombinação Genética , Animais , Insulina/metabolismo , Integrases , Camundongos , Camundongos TransgênicosRESUMO
Chimeric antigen receptor (CAR) technology can be used to engineer the antigen specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from nonhuman antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain variable fragment (Fv) derived from a mouse antibody, we developed a panel of 20 humanized A2-CARs (hA2-CARs). Systematic testing demonstrated variations in expression, and ability to bind HLA-A*02:01 and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen specificity of CARs, revealing that humanization reduced HLA-A cross-reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell-mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs that can be used to engineer specificity and homing of therapeutic Tregs.
Assuntos
Isoantígenos/imunologia , Isoantígenos/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Aloenxertos , Animais , Feminino , Antígenos HLA-A , Antígeno HLA-A2/imunologia , Humanos , Tolerância Imunológica , Imunoterapia , Imunoterapia Adotiva , Células K562 , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única , Pele/patologia , Transplante de Pele , Imunologia de Transplantes , Transplante Homólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Insulin receptor (IR) insufficiency in ß-cells leads to impaired insulin secretion and reduced ß-cell hyperplasia in response to hyperglycemia. Selective IR deficiency in ß-cells in later embryological development may lead to compensatory ß-cell hyperplasia. Although these findings suggest insulin signaling on the ß-cell is important for ß-cell function, they are confounded by loss of signaling by the insulinlike growth factors through the IR. To determine whether insulin itself is necessary for ß-cell development and maturation, we performed a characterization of pancreatic islets in mice with deletions of both nonallelic insulin genes (Ins1-/-Ins2-/-). We immunostained neonatal Ins1-/-Ins2-/- and Ins1+/+Ins2+/+ pancreata and performed quantitative polymerase chain reaction on isolated neonatal islets. Insulin-deficient islets had reduced expression of factors normally expressed in maturing ß-cells, including muscoloaponeurotic fibrosarcoma oncogene homolog A, homeodomain transcription factor 6.1, and glucose transporter 2. Ins1-/-Ins2-/-ß-cells expressed progenitor factors associated with stem cells or dedifferentiated ß-cells, including v-myc avian myolocytomatosis viral oncogene lung carcinoma derived and homeobox protein NANOG. We replaced insulin by injection or islet transplantation to keep mice alive into adulthood to determine whether insulin replacement was sufficient for the completed maturation of insulin-deficient ß-cells. Short-term insulin glargine (Lantus®) injections partially rescued the ß-cell phenotype, whereas long-term replacement of insulin by isogenic islet transplantation supported the formation of more mature ß-cells. Our findings suggest that tightly regulated glycemia, insulin species, or other islet factors are necessary for ß-cell maturation.
Assuntos
Hiperglicemia/cirurgia , Células Secretoras de Insulina/metabolismo , Insulina/deficiência , Transplante das Ilhotas Pancreáticas , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Feminino , Fibrose , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Terapia de Reposição Hormonal/efeitos adversos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/etiologia , Hiperglicemia/patologia , Injeções Subcutâneas , Insulina/genética , Insulina/metabolismo , Insulina Glargina/administração & dosagem , Insulina Glargina/efeitos adversos , Insulina Glargina/uso terapêutico , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de TecidosRESUMO
The gut epithelium's large surface area, its direct exposure to ingested nutrients, its vast stem cell population and its immunotolerogenic environment make it an excellent candidate for therapeutic cells to treat diabetes. Thus, several attempts have been made to coax immature gut cells to differentiate into insulin-producing cells by altering the expression patterns of specific transcription factors. Furthermore, because of similarities in enteroendocrine and pancreatic endocrine cell differentiation pathways, other approaches have used genetically engineered enteroendocrine cells to produce insulin in addition to their endogenous secreted hormones. Several studies support the utility of both of these approaches for the treatment of diabetes. Converting a patient's own gut cells into meal-regulated insulin factories in a safe and immunotolerogenic environment is an attractive approach to treat and potentially cure diabetes. Here, we review work on these approaches and indicate where we feel further advancements are required.
Assuntos
Engenharia Celular , Diabetes Mellitus/terapia , Células Enteroendócrinas/fisiologia , Células Secretoras de Insulina/fisiologia , Insulina/uso terapêutico , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Células Enteroendócrinas/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Transplante das Ilhotas Pancreáticas , Camundongos , Ratos , Transplante de Células-TroncoRESUMO
Pancreatic progenitors derived from human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating diabetes and are currently being tested in clinical trials. Yet, how the milieu of pancreatic progenitor cells, including exposure to different factors after transplant, may influence their maturation remains unclear. Here, we examined the effect of thyroid dysregulation on the development of hESC-derived progenitor cells in vivo. Hypothyroidism was generated in SCID-beige mice using an iodine-deficient diet containing 0.15% propyl-2-thiouracil, and hyperthyroidism was generated by addition of L-thyroxine (T4) to drinking water. All mice received macroencapsulated hESC-derived progenitor cells, and thyroid dysfunction was maintained for the duration of the study ("chronic") or for 4 weeks posttransplant ("acute"). Acute hyperthyroidism did not affect graft function, but acute hypothyroidism transiently impaired human C-peptide secretion at 16 weeks posttransplant. Chronic hypothyroidism resulted in severely blunted basal human C-peptide secretion, impaired glucose-stimulated insulin secretion, and elevated plasma glucagon levels. Grafts from chronic hypothyroid mice contained fewer ß-cells, heterogenous MAFA expression, and increased glucagon(+) and ghrelin(+) cells compared to grafts from euthyroid mice. Taken together, these data suggest that long-term thyroid hormone deficiency may drive the differentiation of human pancreatic progenitor cells toward α- and ε-cell lineages at the expense of ß-cell formation.
Assuntos
Diferenciação Celular , Diabetes Mellitus Tipo 1/cirurgia , Modelos Animais de Doenças , Xenoenxertos/patologia , Células-Tronco Embrionárias Humanas/transplante , Hipotireoidismo/complicações , Células Secretoras de Insulina/transplante , Animais , Antitireóideos/intoxicação , Biomarcadores/sangue , Biomarcadores/metabolismo , Linhagem Celular , Células Imobilizadas/citologia , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Xenoenxertos/citologia , Xenoenxertos/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Hipertireoidismo/induzido quimicamente , Hipertireoidismo/complicações , Hipotireoidismo/etiologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Iodo/deficiência , Masculino , Camundongos SCID , Propiltiouracila/intoxicação , Distribuição Aleatória , Tiroxina/intoxicação , Transplante Heterólogo , Transplante HeterotópicoRESUMO
Leptin can reverse hyperglycemia in rodent models of type 1 diabetes. However, these models have used chemical or immune mediated ß-cell destruction where insulin depletion is incomplete. Thus it is unknown which actions of leptin are entirely insulin independent, versus those which require insulin. To directly assess this we maximized blockage of insulin action using an insulin receptor antagonist in combination with streptozotocin-diabetic mice; leptin treatment was still able to reduce blood glucose. Next, we leptin-treated adult insulin knockout (InsKO) mice. Remarkably, leptin-treated InsKO mice were viable for up to 3 weeks without insulin therapy. Leptin treatment reduced plasma corticosterone, glucagon, ß-hydroxybutyrate, triglycerides, cholesterol, fatty acids and glycerol. However, leptin-treated InsKO mice exhibited overt fed hyperglycemia and severe fasting hypoglycemia. Therefore, leptin can normalize many metabolic parameters in the complete absence of insulin, but blood glucose levels are volatile and the length of survival finite.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/genética , Leptina/farmacologia , Peptídeos/farmacologia , Receptor de Insulina/antagonistas & inibidores , Ácido 3-Hidroxibutírico/sangue , Animais , Glicemia/metabolismo , Colesterol/sangue , Corticosterona/sangue , Ácidos Graxos/sangue , Glucagon/sangue , Glucagon/efeitos dos fármacos , Glicerol/sangue , Hiperglicemia , Hipoglicemia , Camundongos , Camundongos Knockout , Triglicerídeos/sangueRESUMO
Human embryonic stem cell (hESC)-derived pancreatic progenitor cells effectively reverse hyperglycemia in rodent models of type 1 diabetes, but their capacity to treat type 2 diabetes has not been reported. An immunodeficient model of type 2 diabetes was generated by high-fat diet (HFD) feeding in SCID-beige mice. Exposure to HFDs did not impact the maturation of macroencapsulated pancreatic progenitor cells into glucose-responsive insulin-secreting cells following transplantation, and the cell therapy improved glucose tolerance in HFD-fed transplant recipients after 24 weeks. However, since diet-induced hyperglycemia and obesity were not fully ameliorated by transplantation alone, a second cohort of HFD-fed mice was treated with pancreatic progenitor cells combined with one of three antidiabetic drugs. All combination therapies rapidly improved body weight and co-treatment with either sitagliptin or metformin improved hyperglycemia after only 12 weeks. Therefore, a stem cell-based therapy may be effective for treating type 2 diabetes, particularly in combination with antidiabetic drugs.
Assuntos
Diferenciação Celular , Diabetes Mellitus Tipo 2/etiologia , Dieta/efeitos adversos , Células-Tronco Embrionárias Humanas/citologia , Hipoglicemiantes/farmacologia , Obesidade/etiologia , Pâncreas/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Terapia Baseada em Transplante de Células e Tecidos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/terapia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glucose/metabolismo , Humanos , Hiperglicemia , Resistência à Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Fígado/anatomia & histologia , Fígado/metabolismo , Camundongos , Camundongos SCID , Obesidade/metabolismo , Obesidade/terapia , Tamanho do Órgão , FenótipoRESUMO
Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Diabetes Mellitus/terapia , Insulina/metabolismo , Células-Tronco Pluripotentes/transplante , Animais , Diferenciação Celular , Diabetes Mellitus/patologia , Células-Tronco Embrionárias/transplante , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/transplante , Camundongos , Pâncreas/metabolismo , Pâncreas/patologiaRESUMO
BACKGROUND & AIMS: Type 1 diabetes is caused by an aberrant response against pancreatic ß cells. Intestinal K cells are glucose-responsive endocrine cells that might be engineered to secrete insulin. We generated diabetes-prone non-obese diabetic (NOD) mice that express insulin, via a transgene, in K cells. We assessed the effects on immunogenicity and diabetes development. METHODS: Diabetes incidence and glucose homeostasis were assessed in NOD mice that expressed mouse preproinsulin II from a transgene in K cells and nontransgenic NOD mice (controls); pancreas and duodenum tissues were collected and analyzed by histology. We evaluated T cell responses to insulin, levels of circulating autoantibodies against insulin, and the percentage of circulating antigen-specific T cells. Inflammation of mesenteric and pancreatic lymph node cells was also evaluated. RESULTS: The transgenic mice tended to have lower blood levels of glucose than control mice, associated with increased plasma levels of immunoreactive insulin and proinsulin. Fewer transgenic mice developed diabetes than controls. In analyses of pancreas and intestine tissues from the transgenic mice, insulin-producing K cells were not affected by the immune response and the mice had reduced destruction of endogenous ß cells. Fewer transgenic mice were positive for insulin autoantibodies compared with controls. Cells isolated from mesenteric lymph nodes of the transgenic mice had significantly lower rates of proliferation and T cells from transgenic mice tended to secrete lower levels of inflammatory cytokines than from controls. At 15 weeks, transgenic mice had fewer peripheral CD8(+) T cells specific for NRP-V7 than control mice. CONCLUSIONS: NOD mice with intestinal K cells engineered to express insulin have reduced blood levels of glucose, are less likely to develop diabetes, and have reduced immunity against pancreatic ß cells compared with control NOD mice. This approach might be developed to treat patients with type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/prevenção & controle , Células Enteroendócrinas/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/imunologia , Insulina/metabolismo , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Duodeno/metabolismo , Duodeno/patologia , Células Enteroendócrinas/patologia , Feminino , Homeostase/fisiologia , Insulina/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pâncreas/metabolismo , Pâncreas/patologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Antibodies against the wheat storage globulin Glo-3A from a patient with both type 1 diabetes (T1D) and celiac disease were enriched to identify potential molecular mimicry between wheat antigens and T1D target tissues. Recombinant Glo-3A was used to enrich anti-Glo-3A immunoglobulin G antibodies from plasma by batch affinity chromatography. Rat jejunum and pancreas, as well as human duodenum and monocytes were probed, and binding was evaluated by immunohistochemistry and confocal microscopy. Glo-3A-enriched antibodies bound to a specific subset of cells in the lamina propria of rat jejunum that co-localized mostly with a marker of resident, alternatively activated CD163-positive (CD163âº) macrophages. Blood monocytes and macrophage-like cells in human duodenum were also labelled with the enriched antibodies. Blocking studies revealed that binding to CD163⺠macrophages was not due to cross-reactivity with anti-Glo-3A antibodies, but rather to non-Glo-3A antibodies co-purified during antibody enrichment. The novel finding of putative autoantibodies against tolerogenic intestinal CD163⺠macrophages suggests that regulatory macrophages were targeted in this patient with celiac disease and T1D.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Doença Celíaca/imunologia , Diabetes Mellitus Tipo 1/imunologia , Macrófagos/imunologia , Receptores de Superfície Celular/metabolismo , Animais , Formação de Anticorpos , Autoimunidade , Biomarcadores , Doença Celíaca/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Proteínas da Matriz Extracelular/imunologia , Humanos , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Monócitos/imunologia , Ratos , Ratos Endogâmicos BB , Proteínas de Armazenamento de Sementes/imunologia , Hipersensibilidade a TrigoRESUMO
Type 1 diabetes (T1D) is an autoimmune disorder that results from the destruction of insulin-producing beta-cells in the islets of Langerhans. To date, autoimmune T-cell response and antibody reactivity to more than 20 autoantigens have been linked to this disease. Some studies have described the intermediate filament protein peripherin (PRPH) as an autoantigen associated with T1D in non-obese diabetic (NOD) mice. We evaluated immune reactivity of mouse and rabbit sera and human plasma to a 58 kDa protein expressed in RIN-m5F rat insulinoma cells. The protein was isolated using 2-DE and identified by mass spectrometry as PRPH. Antibodies from healthy humans and T1D patients, CD-1 mice, C57BL/6 mice, NOR (non-obese diabetes resistant) mice, and NOD mice reacted with PRPH on Western blots. However, antibody response to PRPH was stronger in NOD than non-autoimmune prone C57BL/6 mice. We conclude that immune reactivity to PRPH is not exclusively associated with NOD mice or human patients with T1D. Furthermore, the frequent occurrence of PRPH-reactive antibodies in mouse and human blood suggests that binding may be non-specific or could reflect the presence of natural autoantibodies against PRPH. These findings point to the need for a re-evaluation of PRPH as a T1D autoantigen in NOD mice and raise the question of the physiological relevance of such widespread immune reactivity against this peripheral nervous system protein.