RESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Inmunoterapia/métodos , Células TH1/inmunología , Células Th2/inmunología , Línea Celular , Criopreservación , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores de Antígenos de Linfocitos T/metabolismo , Células TH1/metabolismo , Células Th2/metabolismoRESUMEN
The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G2/M phases of the cell-cycle (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without protocol refinement, TDS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of TDS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV+ carriers; and recently-diagnosed T1D patients. TDS were reproducibly more abundant among CD8+ T cells and a defined subset of T-regulatory CD4+ T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive TDS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking TDS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.
Asunto(s)
Puntos de Control del Ciclo Celular/inmunología , Monitorización Inmunológica/métodos , Linfocitos T/inmunología , Animales , Citometría de Flujo/métodos , Humanos , Ratones , Ratones TransgénicosRESUMEN
Autoreactive B lymphocytes play a key role as APCs in diaebetogenesis. However, it remains unclear whether B-cell tolerance is compromised in NOD mice. Here, we describe a new B lymphocyte transgenic NOD mouse model, the 116C-NOD mouse, where the transgenes derive from an islet-infiltrating B lymphocyte of a (8.3-NODxNOR) F1 mouse. The 116C-NOD mouse produces clonal B lymphocytes with pancreatic islet beta cell specificity. The incidence of T1D in 116C-NOD mice is decreased in both genders when compared with NOD mice. Moreover, several immune selection mechanisms (including clonal deletion and anergy) acting on the development, phenotype, and function of autoreactive B lymphocytes during T1D development have been identified in the 116C-NOD mouse. Surprisingly, a more accurate analysis revealed that, despite their anergic phenotype, 116C B cells express some costimulatory molecules after activation, and induce a T-cell shift toward a Th17 phenotype. Furthermore, this shift on T lymphocytes seems to occur not only when both T and B cells contact, but also when helper T (Th) lineage is established. The 116C-NOD mouse model could be useful to elucidate the mechanisms involved in the generation of Th-cell lineages.
Asunto(s)
Linfocitos B/inmunología , Anergia Clonal , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Tolerancia Inmunológica/genética , Activación de Linfocitos , Células Th17/inmunología , Animales , Supresión Clonal , Citocinas/genética , Citocinas/inmunología , Tolerancia Inmunológica/inmunología , Ratones Endogámicos NOD , Ratones Transgénicos , Fenotipo , Bazo/anatomía & histología , Bazo/citología , Bazo/inmunología , TransgenesRESUMEN
Type 1 diabetes (T1D) is a metabolic disease that results from the autoimmune attack against insulin-producing ß-cells in the pancreatic islets of Langerhans. Currently, there is no treatment to restore endogenous insulin secretion in patients with autoimmune diabetes. In the last years, the development of new therapies to induce long-term tolerance has been an important medical health challenge. Apoptosis is a physiological mechanism that contributes to the maintenance of immune tolerance. Apoptotic cells are a source of autoantigens that induce tolerance after their removal by antigen presenting cells (APCs) through a process called efferocytosis. Efferocytosis will not cause maturation in dendritic cells, one of the most powerful APCs, and this process could induce tolerance rather than autoimmunity. However, failure of this mechanism due to an increase in the rate of ß-cells apoptosis and/or defects in efferocytosis results in activation of APCs, contributing to inflammation and to the loss of tolerance to self. In fact, T1D and other autoimmune diseases are associated to enhanced apoptosis of target cells and defective apoptotic cell clearance. Although further research is needed, the clinical relevance of immunotherapies based on apoptosis could prove to be very important, as it has translational potential in situations that require the reestablishment of immunological tolerance, such as autoimmune diseases. This review summarizes the effects of apoptosis of ß-cells towards autoimmunity or tolerance and its application in the field of emerging immunotherapies.
Asunto(s)
Apoptosis/inmunología , Autoinmunidad , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/inmunología , Tolerancia Inmunológica , Células Secretoras de Insulina/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Autoantígenos/genética , Autoantígenos/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Células Dendríticas/patología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Regulación de la Expresión Génica , Humanos , Inmunoterapia , Células Secretoras de Insulina/patología , Ratones , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Celiac disease (CD) is an autoimmune disorder, which damages the small intestine and is caused by ingestion of gluten in genetically susceptible individuals. The only known effective treatment is a lifelong gluten-free diet. Genetic risk factors have been identified and nearly all patients are HLA-DQ2 and/or HLA-DQ8 positive. Specific autoantibodies, IgA antitissue transglutaminase-2, antiendomysium, and antideaminated forms of gliadin peptide antibodies, are widely used as diagnostic aids in celiac patients. However, the discovery of new biomarkers may help in the diagnosis and follow-up of the disease. Recently, the molecule REG Iα, involved in tissue regeneration, has been proposed as a new biomarker of CD. REG Iα expression is increased in the target tissue and in the sera of celiac patients during damage and inflammation, decreasing after gluten-free diet. In this article we review the main biomarkers for diagnosis and monitoring of CD, focusing on the immune response-related mechanisms.
Asunto(s)
Autoanticuerpos/sangre , Enfermedad Celíaca/diagnóstico , Antígenos HLA-DQ/genética , Litostatina/sangre , Animales , Autoinmunidad , Biomarcadores/sangre , Enfermedad Celíaca/sangre , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Dieta Sin Gluten , Marcadores Genéticos , Humanos , Valor Predictivo de las Pruebas , Pronóstico , Índice de Severidad de la EnfermedadRESUMEN
Celiac disease is an autoimmune disorder induced by gluten in genetically predisposed people. The discovery of new biomarkers may help in the diagnosis and follow-up of celiac patients. Regenerating islet-derived 1 alpha (REGIα)--a biomarker related to tissue regeneration--is increased in serum at the onset of the disease, decreasing after gluten-free diet (GFD). As REGIα is a 18 kDa soluble glycoprotein, it may be detected in urine samples, increasing in celiac patients. We have determined REGIα levels by ELISA. No differences were found among patients (onset or after GFD) and controls and no correlation exists among REGIα in sera and urine.
Asunto(s)
Enfermedad Celíaca/sangre , Enfermedad Celíaca/orina , Litostatina/sangre , Litostatina/orina , Adolescente , Adulto , Biomarcadores/sangre , Biomarcadores/orina , Estudios de Casos y Controles , Enfermedad Celíaca/diagnóstico , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , MasculinoRESUMEN
Aims: Recent studies highlight the potentially important role of neoepitopes in breaking immune tolerance in type 1 diabetes. T cell reactivity to these neoepitopes has been reported, but how this response compares quantitatively and phenotypically with previous reports on native epitopes is not known. Thus, an understanding of the relationship between native and neoepitopes and their role as tolerance breakers or disease drivers in type 1 diabetes is required. We set out to compare T cell reactivity and phenotype against a panel of neo- and native islet autoantigenic epitopes to examine how this relates to stages of type 1 diabetes development. Methods: Fifty-four subjects comprising patients with T1D, and autoantibody-positive unaffected family members were tested against a panel of neo- and native epitopes by ELISPOT (IFN-γ, IL-10, and IL-17). A further subset of two patients was analyzed by Single Cell Immune Profiling (RNAseq and TCR α/ß) after stimulation with pools of native and neoepitope peptides. Results: T cell responses to native and neoepitopes were present in patients with type 1 diabetes and at-risk subjects, and overall, there were no significant differences in the frequency, magnitude, or phenotype between the two sets of peptide stimuli. Single cell RNAseq on responder T cells revealed a similar profile in T1D patients stimulated with either neo- or native epitopes. A pro-inflammatory gene expression profile (TNF-α, IFN-γ) was dominant in both native and neoepitope stimulated T cells. TCRs with identical clonotypes were found in T cell responding to both native and neoepitopes. Conclusion/Interpretation: These data suggest that in peripheral blood, T cell responses to both native and neoepitopes are similar in terms of frequency and phenotype in patients with type 1 diabetes and high-risk unaffected family members. Furthermore, using a combination of transcriptomic and clonotypic analyses, albeit using a limited panel of peptides, we show that neoepitopes are comparable to native epitopes currently in use for immune-monitoring studies.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Epítopos/inmunología , Células Secretoras de Insulina/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Tolerancia Inmunológica , Lactante , Masculino , Receptores de Antígenos de Linfocitos T/inmunología , Adulto JovenRESUMEN
PURPOSE OF REVIEW: The role of T cells specific for islet autoantigens is proven in pathogenesis of type 1 diabetes. Recently, there has been rapid expansion in the number of T-cell subsets identified, this has coincided with an increase in the repertoire of reported islet antigens mainly through the discovery of novel epitopes. A discussion of how these marry together is now warranted and timely. RECENT FINDINGS: In this review, we will discuss the autoreactivity against neo-epitopes. We then explore the growing array of T-cell subsets for both CD4 T cells, including follicular and peripheral T helper cells, and CD8 T cells, discussing evolution from naïve to exhausted phenotypes. Finally, we detail how subsets correlate with disease stage and loss of ß-cell function and are impacted by immunotherapy. SUMMARY: The expanding list of T-cell subsets may be potentially encouraging in terms of elucidating disease mechanisms and have a role as biomarkers for disease progression. Furthermore, T-cell subsets can be used in stratifying patients for clinical trials and for monitoring immunotherapy outcomes. However, the definition of subsets needs to be refined in order to ensure that there is a uniform approach in designating T-cell subset attributes that is globally applied.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Subgrupos de Linfocitos T/fisiología , Autoantígenos/inmunología , Autoantígenos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Diabetes Mellitus Tipo 1/patología , Progresión de la Enfermedad , Humanos , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Islotes Pancreáticos/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Type 1 diabetes is an autoimmune disease caused by the destruction of the insulin-producing ß-cells. To revert type 1 diabetes, the suppression of the autoimmune attack should be combined with a ß-cell replacement strategy. It has been previously demonstrated that liraglutide, a glucagon-like peptide-1 receptor agonist, restores ß-cell mass in type 1 diabetes, via α-cell transdifferentiation and neogenesis. We report here that treatment with liraglutide does not prevent type 1 diabetes in the spontaneous non-obese diabetic (NOD) mouse model, but it tends to reduce leukocytic islet infiltration. However, in combination with an immunotherapy based on tolerogenic liposomes, it is effective in ameliorating hyperglycaemia in diabetic NOD mice. Importantly, liraglutide is not detrimental for the tolerogenic effect that liposomes exert on dendritic cells from patients with type 1 diabetes in terms of membrane expression of molecules involved in antigen presentation, immunoregulation and activation. Moreover, the in vivo effect of the combined therapy was tested in mice humanised with peripheral blood mononuclear cells from patients with type 1 diabetes, showing no adverse effects in leukocyte subsets. In conclusion, the combination therapy with liraglutide and a liposome-based immunotherapy is a promising candidate strategy for type 1 diabetes.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Células Secretoras de Insulina/citología , Insulina/administración & dosificación , Liraglutida/administración & dosificación , Adulto , Animales , Terapia Combinada , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Femenino , Humanos , Inmunoterapia , Insulina/química , Insulina/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Liposomas , Liraglutida/farmacología , Masculino , Ratones , Ratones Endogámicos NOD , Resultado del Tratamiento , Adulto JovenRESUMEN
Type 1 diabetes is an autoimmune disease caused by the destruction of the insulin-producing ß-cells. An ideal immunotherapy should combine the blockade of the autoimmune response with the recovery of functional target cell mass. With the aim to develop new therapies for type 1 diabetes that could contribute to ß-cell mass restoration, a drug repositioning analysis based on systems biology was performed to identify the ß-cell regenerative potential of commercially available compounds. Drug repositioning is a strategy used for identifying new uses for approved drugs that are outside the scope of the medical indication. A list of 28 non-synonymous repurposed drug candidates was obtained, and 16 were selected as diabetes mellitus type 1 treatment candidates regarding pancreatic ß-cell regeneration. Drugs with poor safety profile were further filtered out. Lastly, we selected liraglutide for its predictive efficacy values for neogenesis, transdifferentiation of α-cells, and/or replication of pre-existing ß-cells. Liraglutide is an analog of glucagon-like peptide-1, a drug used in patients with type 2 diabetes. Liraglutide was tested in immunodeficient NOD-Scid IL2rg-/- (NSG) mice with type 1 diabetes. Liraglutide significantly improved the blood glucose levels in diabetic NSG mice. During the treatment, a significant increase in ß-cell mass was observed due to a boost in ß-cell number. Both parameters were reduced after withdrawal. Interestingly, islet bihormonal glucagon+insulin+ cells and insulin+ ductal cells arose during treatment. In vitro experiments showed an increase of insulin and glucagon gene expression in islets cultured with liraglutide in normoglycemia conditions. These results point to ß-cell replacement, including transdifferentiation and neogenesis, as aiding factors and support the role of liraglutide in ß-cell mass restoration in type 1 diabetes. Understanding the mechanism of action of this drug could have potential clinical relevance in this autoimmune disease.
Asunto(s)
Reprogramación Celular , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Péptido 1 Similar al Glucagón/análogos & derivados , Hiperglucemia/prevención & control , Células Secretoras de Insulina/efectos de los fármacos , Liraglutida/farmacología , Animales , Péptido 1 Similar al Glucagón/administración & dosificación , Hiperglucemia/etiología , Hiperglucemia/metabolismo , Hiperglucemia/patología , Hipoglucemiantes/farmacología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Non-genetic factors are crucial in the pathogenesis of type 1 diabetes (T1D), a disease caused by autoimmunity against insulin-producing ß-cells. Exposure to medications in the prenatal period may influence the immune system maturation, thus altering self-tolerance. Prenatal administration of betamethasone -a synthetic glucocorticoid given to women at risk of preterm delivery- may affect the development of T1D. It has been previously demonstrated that prenatal betamethasone administration protects offspring from T1D development in nonobese diabetic (NOD) mice. The direct effect of betamethasone on the immature and mature immune system of NOD mice and on target ß-cells is analysed in this paper. In vitro, betamethasone decreased lymphocyte viability and induced maturation-resistant dendritic cells, which in turn impaired γδ T cell proliferation and decreased IL-17 production. Prenatal betamethasone exposure caused thymus hypotrophy in newborn mice as well as alterations in immune cells subsets. Furthermore, betamethasone decreased ß-cell growth, reduced C-peptide secretion and altered the expression of genes related to autoimmunity, metabolism and islet mass in T1D target tissue. These results support the protection against T1D in the betamethasone-treated offspring and demonstrate that this drug alters the developing immune system and ß-cells. Understanding how betamethasone generates self-tolerance could have potential clinical relevance in T1D.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Betametasona/administración & dosificación , Diabetes Mellitus Tipo 1/prevención & control , Glucocorticoides/administración & dosificación , Tolerancia Inmunológica/efectos de los fármacos , Animales , Péptido C/inmunología , Péptido C/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Cuerpos de Inclusión/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Activación de Linfocitos , Exposición Materna , Ratones , Ratones Endogámicos NOD , Trabajo de Parto Prematuro/prevención & control , EmbarazoRESUMEN
In type 1 diabetes, cytotoxic CD8+ T cells with specificity for ß cell autoantigens are found in the pancreatic islets, where they are implicated in the destruction of insulin-secreting ß cells. In contrast, the disease relevance of ß cell-reactive CD8+ T cells that are detectable in the circulation, and their relationship to ß cell function, are not known. Here, we tracked multiple, circulating ß cell-reactive CD8+ T cell subsets and measured ß cell function longitudinally for 2 years, starting immediately after diagnosis of type 1 diabetes. We found that change in ß cell-specific effector memory CD8+ T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8+ T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer-specific protein of 37 kDa, and CD16, and reduced expression of CD28) compared with their CD57- counterparts, and network association modeling indicated that the dynamics of ß cell-reactive CD57+ effector memory CD8+ T cell subsets were strongly linked. Thus, coordinated changes in circulating ß cell-specific CD8+ T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Memoria Inmunológica , Células Secretoras de Insulina/inmunología , Adulto , Linfocitos T CD8-positivos/patología , Niño , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Femenino , Humanos , Células Secretoras de Insulina/patología , MasculinoRESUMEN
Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing ß-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow ß-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic ß-cells arrested autoimmunity to ß-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)-the main signal of the apoptotic cell membrane-and ß-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.
Asunto(s)
Apoptosis/inmunología , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/inmunología , Tolerancia Inmunológica/inmunología , Fosfatidilserinas/inmunología , Adolescente , Adulto , Autoantígenos/inmunología , Células Cultivadas , Femenino , Humanos , Inmunoterapia/métodos , Liposomas , Masculino , Persona de Mediana Edad , Imitación Molecular/inmunología , Fagocitosis , Adulto JovenRESUMEN
Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34+ population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34+ controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγnull mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×106 CD34+ cells committed to the granulocytic lineage and 3.9×109 neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Sangre Fetal/citología , Anemia Aplásica/sangre , Anemia Aplásica/etiología , Anemia Aplásica/prevención & control , Animales , Antígenos CD34/sangre , Biotecnología , Diferenciación Celular , Linaje de la Célula , Ensayo de Unidades Formadoras de Colonias , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Femenino , Sangre Fetal/inmunología , Refuerzo Inmunológico de Injertos/métodos , Granulocitos/citología , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neutrófilos/citologíaRESUMEN
The pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members have been reported to be expressed in Crohn's disease (CD) and ulcerative colitis (UC) and to be involved as proliferative mucosal factors in IBD. However, expression of all REG family genes in IBD is still unclear. Here, we analyzed expression of all REG family genes (REG Iα, REG Iß, REG III, HIP/PAP, and REG IV) in biopsy specimens of UC and CD by real-time RT-PCR. REG Iα, REG Iß, and REG IV genes were overexpressed in CD samples. REG IV gene was also overexpressed in UC samples. We further analyzed the expression mechanisms of REG Iα, REG Iß, and REG IV genes in human colon cells. The expression of REG Iα was significantly induced by IL-6 or IL-22, and REG Iß was induced by IL-22. Deletion analyses revealed that three regions (- 220 to - 211, - 179 to - 156, and - 146 to - 130) in REG Iα and the region (- 274 to- 260) in REG Iß promoter were responsible for the activation by IL-22/IL-6. The promoters contain consensus transcription factor binding sequences for MZF1, RTEF1/TEAD4, and STAT3 in REG Iα, and HLTF/FOXN2F in REG Iß, respectively. The introduction of siRNAs for MZF1, RTEF1/TEAD4, STAT3, and HLTF/FOXN2F abolished the transcription of REG Iα and REG Iß. The gene activation mechanisms of REG Iα/REG Iß may play a role in colon mucosal regeneration in IBD.
RESUMEN
Cell-based tolerogenic therapy is a promising approach for the treatment of autoimmune diseases and transplant rejection. Regulatory T cells and tolerogenic dendritic cells have been particularly explored in the treatment of various autoimmune disorders in experimental models of disease. Although some of these cells have already been tested in a limited number of clinical trials, there is still a need for preclinical research on tolerogenic cells in animal models of autoimmunity. This review will focus on the relevance of data obtained from studies in experimental animal models for the use of tolerogenic cell-based therapy in humans. Also, perspectives for further improvement of tolerogenic cell preparation towards enhanced suppressive activity and stability of the cells will be discussed.
Asunto(s)
Artritis Reumatoide/terapia , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Diabetes Mellitus Tipo 1/terapia , Modelos Animales de Enfermedad , Esclerosis Múltiple/terapia , Animales , Artritis Reumatoide/inmunología , Autoinmunidad/efectos de los fármacos , Autoinmunidad/inmunología , Diabetes Mellitus Tipo 1/inmunología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/inmunología , Inmunosupresores/administración & dosificación , Esclerosis Múltiple/inmunología , Compuestos Orgánicos/administración & dosificación , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
AIM: Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. MATERIALS & METHODS: Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed in vitro and in vivo. RESULTS: Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25+ FoxP3- CD4+ T-cell subset. CONCLUSION: This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.
Asunto(s)
Autoantígenos/administración & dosificación , Inmunoterapia/métodos , Liposomas/química , Esclerosis Múltiple/terapia , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Péptidos/administración & dosificación , Fosfatidilserinas/química , Animales , Autoantígenos/inmunología , Autoantígenos/uso terapéutico , Femenino , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/uso terapéutico , Péptidos/inmunología , Péptidos/uso terapéutico , Linfocitos T Reguladores/inmunologíaRESUMEN
Immunotherapy using short immunogenic peptides of disease-related autoantigens restores immune tolerance in preclinical disease models. We studied safety and mechanistic effects of injecting human leukocyte antigen-DR4(DRB1*0401)-restricted immunodominant proinsulin peptide intradermally every 2 or 4 weeks for 6 months in newly diagnosed type 1 diabetes patients. Treatment was well tolerated with no systemic or local hypersensitivity. Placebo subjects showed a significant decline in stimulated C-peptide (measuring insulin reserve) at 3, 6, 9, and 12 months versus baseline, whereas no significant change was seen in the 4-weekly peptide group at these time points or the 2-weekly group at 3, 6, and 9 months. The placebo group's daily insulin use increased by 50% over 12 months but remained unchanged in the intervention groups. C-peptide retention in treated subjects was associated with proinsulin-stimulated interleukin-10 production, increased FoxP3 expression by regulatory T cells, low baseline levels of activated ß cell-specific CD8 T cells, and favorable ß cell stress markers (proinsulin/C-peptide ratio). Thus, proinsulin peptide immunotherapy is safe, does not accelerate decline in ß cell function, and is associated with antigen-specific and nonspecific immune modulation.
Asunto(s)
Diabetes Mellitus Tipo 1/terapia , Inmunoterapia/métodos , Péptidos/uso terapéutico , Proinsulina/uso terapéutico , Adolescente , Adulto , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Péptido C/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Método Doble Ciego , Femenino , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Linfocitos T Reguladores/metabolismo , Adulto JovenRESUMEN
The transmembrane glycoprotein CD26 or dipeptidyl peptidase IV (DPPIV) is a multifunctional protein. In immune system, CD26 plays a role in T-cell function and is also involved in thymic maturation and emigration patterns. In preclinical studies, treatment with DPPIV inhibitors reduces insulitis and delays or even reverses the new -onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the specific mechanisms involved in these effects remain unknown. The aim of the present study was to investigate how DPPIV inhibition modifies the expression of genes in the thymus of NOD mice by microarray analysis. Changes in the gene expression of ß-cell autoantigens and Aire in thymic epithelial cells (TECs) were also evaluated by using qRT-PCR. A DPPIV inhibitor, MK626, was orally administered in the diet for 4 and 6 weeks starting at 6-8 weeks of age. Thymic glands from treated and control mice were obtained for each study checkpoint. Thymus transcriptome analysis revealed that 58 genes were significantly over-expressed in MK626-treated mice after 6 weeks of treatment. Changes in gene expression in the thymus were confined mainly to the immune system, including innate immunity, chemotaxis, antigen presentation and immunoregulation. Most of the genes are implicated in central tolerance mechanisms through several pathways. No differences were observed in the expression of Aire and ß-cell autoantigens in TECs. In the current study, we demonstrate that treatment with the DPPIV inhibitor MK626 in NOD mice alters the expression of the immune response-related genes in the thymus, especially those related to immunological central tolerance, and may contribute to the prevention of T1D.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Indinavir/farmacología , Animales , Presentación de Antígeno/genética , Femenino , Regulación de la Expresión Génica/inmunología , Redes Reguladoras de Genes , Inmunomodulación/genética , Ratones Endogámicos NOD , Timo/efectos de los fármacos , Timo/metabolismo , TranscriptomaRESUMEN
INTRODUCTION: The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow ß-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. OBJECTIVE: To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to ß-cells in type 1 diabetes. METHODS: A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. RESULTS: We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. CONCLUSIONS: We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.