Targeting macrophages with phosphatidylserine-rich liposomes as a potential antigen-specific immunotherapy for type 1 diabetes.

Type 1 diabetes (T1D) results from a breakdown in immunological tolerance, with pivotal involvement of antigen-presenting cells. In this context, antigen-specific immunotherapies have been developed to arrest autoimmunity, such as phosphatidylserine (PS)-liposomes. However, the role of certain antigen-presenting cells in immunotherapy, particularly human macrophages (Mφ) in T1D remains elusive. The aim of this study was to determine the role of Mφ in antigen-specific immune tolerance and T1D. To that end, we evaluated Mφ ability to capture apoptotic-body mimicking PS-liposomes in mice and conducted a phenotypic and functional characterisation of four human monocyte-derived Mφ (MoMφ) subpopulations (M0, M1, M2a and M2c) after PS-liposomes uptake. Our findings in mice identified Mφ as the most phagocytic cell subset in the spleen and liver. In humans, while phagocytosis rates were comparable between T1D and control individuals, PS-liposome capture dynamics differed among Mφ subtypes, favouring inflammatory (M1) and deactivated (M2c) Mφ. Notably, high nanoparticle concentrations did not affect macrophage viability. PS-liposome uptake by Mφ induced alterations in membrane molecule expression related to immunoregulation, reduced secretion of IL-6 and IL-12, and diminished autologous T-cell proliferation in the context of autoantigen stimulation. These results underscore the tolerogenic effects of PS-liposomes and emphasize their potential to target human Mφ, providing valuable insights into the mechanism of action of this preclinical immunotherapy.

Immunometabolic biomarkers for partial remission in type 1 diabetes mellitus.

Shortly after diagnosis of type 1 diabetes mellitus (T1DM) and initiation of insulin therapy, many patients experience a transient partial remission (PR) phase, also known as the honeymoon phase. This phase presents a potential therapeutic opportunity due to its association with immunoregulatory and ß cell-protective mechanisms. However, the lack of biomarkers makes its characterization difficult. In this review, we cover the current literature addressing the discovery of new predictive and monitoring biomarkers that contribute to the understanding of the metabolic, epigenetic, and immunological mechanisms underlying PR. We further discuss how these peripheral biomarkers reflect attempts to arrest ß cell autoimmunity and how these can be applied in clinical practice.

NOD mouse dorsal root ganglia display morphological and gene expression defects before and during autoimmune diabetes development.

Introduction: During the development of Autoimmune Diabetes (AD) an autoimmune attack against the Peripheral Nervous System occurs. To gain insight into this topic, analyses of Dorsal Root Ganglia (DRG) from Non-Obese Diabetic (NOD) mice were carried out. Methods: Histopathological analysis by electron and optical microscopy in DRG samples, and mRNA expression analyzes by the microarray technique in DRG and blood leukocyte samples from NOD and C57BL/6 mice were performed. Results: The results showed the formation of cytoplasmic vacuoles in DRG cells early in life that could be related to a neurodegenerative process. In view of these results, mRNA expression analyses were conducted to determine the cause and/or the molecules involved in this suspected disorder. The results showed that DRG cells from NOD mice have alterations in the transcription of a wide range of genes, which explain the previously observed alterations. In addition, differences in the transcription genes in white blood cells were also detected. Discussion: Taken together, these results indicate that functional defects are not only seen in beta cells but also in DRG in NOD mice. These results also indicate that these defects are not a consequence of the autoimmune process that takes place in NOD mice and suggest that they may be involved as triggers for its development.

Immunoregulatory Biomarkers of the Remission Phase in Type 1 Diabetes: miR-30d-5p Modulates PD-1 Expression and Regulatory T Cell Expansion.

The partial remission (PR) phase of type 1 diabetes (T1D) is an underexplored period characterized by endogenous insulin production and downmodulated autoimmunity. To comprehend the mechanisms behind this transitory phase and develop precision medicine strategies, biomarker discovery and patient stratification are unmet needs. MicroRNAs (miRNAs) are small RNA molecules that negatively regulate gene expression and modulate several biological processes, functioning as biomarkers for many diseases. Here, we identify and validate a unique miRNA signature during PR in pediatric patients with T1D by employing small RNA sequencing and RT-qPCR. These miRNAs were mainly related to the immune system, metabolism, stress, and apoptosis pathways. The implication in autoimmunity of the most dysregulated miRNA, miR-30d-5p, was evaluated in vivo in the non-obese diabetic mouse. MiR-30d-5p inhibition resulted in increased regulatory T cell percentages in the pancreatic lymph nodes together with a higher expression of CD200. In the spleen, a decrease in PD-1+ T lymphocytes and reduced PDCD1 expression were observed. Moreover, miR-30d-5p inhibition led to an increased islet leukocytic infiltrate and changes in both effector and memory T lymphocytes. In conclusion, the miRNA signature found during PR shows new putative biomarkers and highlights the immunomodulatory role of miR-30d-5p, elucidating the processes driving this phase.

Fast-screening flow cytometry method for detecting nanoplastics in human peripheral blood.

Plastic pollution is a global problem. Animals and humans can ingest and inhale plastic particles, with uncertain health consequences. Nanoplastics (NPs) are particles ranging from 1 nm to 1000 nm that result from the erosion or breakage of larger plastic debris, and can be highly polydisperse in physical properties and heterogeneous in composition. Potential effects of NPs exposure may be associated with alterations in the xenobiotic metabolism, nutrients absorption, energy metabolism, cytotoxicity, and behavior. In humans, no data on NPs absorptions has been reported previously. Given that their detection relies significantly on environmental exposure, we have prospectively studied the presence of NPs in human peripheral blood (PB). Specifically, we have used fluorescence techniques and nanocytometry, together with the staining of the lipophilic dye Nile Red (NR), to demonstrate that NPs can be accurately detected using flow cytometry.•Potential effects of nanoplastics exposure.•Fluorescence techniques and nanocytometry.•Accurate detection using flow cytometry.

A new platform for autoimmune diseases. Inducing tolerance with liposomes encapsulating autoantigens.

Autoimmune diseases (AIDs) are caused by the loss of self-tolerance and destruction of tissues by the host's immune system. Several antigen-specific immunotherapies, focused on arresting the autoimmune attack, have been tested in clinical trials with discouraging results. Therefore, there is a need for innovative strategies to restore self-tolerance safely and definitively in AIDs. We previously demonstrated the therapeutic efficacy of phosphatidylserine (PS)-liposomes encapsulating autoantigens in experimental type 1 diabetes and multiple sclerosis. Here, we show that PS-liposomes can be adapted to other autoimmune diseases by simply replacing the encapsulated autoantigen. After administration, they are distributed to target organs, captured by phagocytes and interact with several immune cells, thus exerting a tolerogenic and immunoregulatory effect. Specific PS-liposomes demonstrate great preventive and therapeutic efficacy in rheumatoid arthritis and myasthenia gravis. Thus, this work highlights the therapeutic potential of a platform for several autoimmunity settings, which is specific, safe, and with long-term effects.

Impact of Betamethasone Pretreatment on Engrafment of Cord Blood-Derived Hematopoietic Stem Cells.

Hematopoietic stem cell (HSC) transplantation is crucial to cure hematologic malignancies. Umbilical cord blood (UCB) is a source of stem cells, but 90% of UCB units are discarded due to low cellularity. Improving the engraftment capacities of CD34+ stem cells would allow the use of UCB that were so far rejected. Betamethasone induces long-term transcriptomic and epigenomic changes in immune cells through glucocorticoid receptor. We hypothesize that discarded UCB could be used owing to improvements induced by betamethasone. Isolated CD34+ HSC from UCB were exposed to the synthetic glucocorticoids betamethasone and fluticasone for 20 h, and cell phenotype was determined before transplantation. NSG mice were sub-lethally irradiated (1 Gy or 2 Gy) 6 h before intravenously transferring 2-5 × 105 CD34+ HSC. The peripheral blood engraftment levels and the leukocyte subsets were followed up for 20 weeks using flow cytometry. At end point, the engraftment and leukocyte subsets were determined in the spleen and bone marrow. We demonstrated that betamethasone has surprising effects in recovering immune system homeostasis. Betamethasone and fluticasone increase CXCR4 and decrease HLA class II and CD54 expression in CD34+ HSCs. Both glucocorticoids-exposed cells showed a similar engraftment in 2 Gy-irradiated NSG mice. Interestingly, betamethasone-exposed cells showed enhanced engraftment in 1 Gy-irradiated NSG mice, with a trend to increase regulatory T cell percentage when compared to control. Betamethasone induces alterations in CD34+ HSCs and improve the engraftment, leading to a faster immune system recovery, which will contribute to engrafted cells survival.

Prenatal Betamethasone Exposure and its Impact on Pediatric Type 1 Diabetes Mellitus: A Preliminary Study in a Spanish Cohort.

Background: Betamethasone, a glucocorticoid used to induce lung maturation when there is a risk of preterm delivery, can affect the immune system maturation and type 1 diabetes (T1D) incidence in the progeny. It has been described that prenatal betamethasone protects offspring from experimental T1D development. The main aim of this study was to evaluate the possible association between betamethasone prenatal exposure and T1D in humans. Research Design and Methods. A retrospective case-control study with a total of 945 children, including 471 patients with T1D and 474 healthy siblings, was performed. Participants were volunteers from the Germans Trias i Pujol Hospital and DiabetesCero Foundation. Parents of children enrolled in the study completed a questionnaire that included questions about weeks of gestation, preterm delivery risk, weight at birth, and prenatal betamethasone exposure of their children. Multiple logistic regression was used to detect the association between betamethasone exposure and T1D. Results: We compared T1D prevalence between subjects prenatally exposed or unexposed to betamethasone. The percent of children with T1D in the exposed group was 37.5% (21 of 56), and in the unexposed group was 49.52% (410 of 828) (p = 0.139). The percentage of betamethasone-treated subjects with T1D in the preterm group (18.05%, 13 of 72) was significantly higher than that found in the control group (12.5%, 9 of 72) (p = 0.003). The odds ratio for T1D associated with betamethasone in the univariate logistic regression was 0.59 (95% confidence interval, 0.33; 1.03 [p = 0.062]) and in the multivariate logistic regression was 0.83 (95% confidence interval, 0.45; 1.52 [p = 0.389]). Conclusions: The results demonstrate that the prenatal exposure to betamethasone does not increase T1D susceptibility, and may even be associated with a trend towards decreased risk of developing the disease. These preliminary findings require further prospective studies with clinical data to confirm betamethasone exposure effect on T1D risk.

Candidate Biomarkers for the Prediction and Monitoring of Partial Remission in Pediatric Type 1 Diabetes.

The partial remission (PR) phase, a period experienced by most patients with type 1 diabetes (T1D) soon after diagnosis, is characterized by low insulin requirements and improved glycemic control. Given the great potential of this phase as a therapeutic window for immunotherapies because of its association with immunoregulatory mechanisms and ß-cell protection, our objective was to find peripheral immunological biomarkers for its better characterization, monitoring, and prediction. The longitudinal follow-up of 17 pediatric patients with new-onset T1D over one year revealed that, during the PR phase, remitter patients show increased percentages of effector memory (EM) T lymphocytes, terminally differentiated EM T lymphocytes, and neutrophils in comparison to non-remitter patients. On the contrary, remitter patients showed lower percentages of naïve T lymphocytes, regulatory T cells (TREG), and dendritic cells (DCs). After a year of follow-up, these patients also presented increased levels of regulatory B cells and transitional T1 B lymphocytes. On the other hand, although none of the analyzed cytokines (IL-2, IL-6, TGF-ß1, IL-17A, and IL-10) could distinguish or predict remission, IL-17A was increased at T1D diagnosis in comparison to control subjects, and remitter patients tended to maintain lower levels of this cytokine than non-remitters. Therefore, these potential monitoring immunological biomarkers of PR support that this stage is governed by both metabolic and immunological factors and suggest immunoregulatory attempts during this phase. Furthermore, since the percentage of TREG, monocytes, and DCs, and the total daily insulin dose at diagnosis were found to be predictors of the PR phase, we next created an index-based predictive model comprising those immune cell percentages that could potentially predict remission at T1D onset. Although our preliminary study needs further validation, these candidate biomarkers could be useful for the immunological characterization of the PR phase, the stratification of patients with better disease prognosis, and a more personalized therapeutic management.

Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes.

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

A century later, still fighting back: antigen-specific immunotherapies for type 1 diabetes.

Type 1 diabetes (T1D) is a chronic metabolic disease caused by the autoimmune destruction of insulin-producing ß-cells. Ever since the 1920s, the fate of patients suffering from T1D was dramatically improved owing to the isolation and production of insulin, and the scientific field has largely progressed as a result of the evidence gathered about its underpinnings and mechanisms. The last years have seen this knowledge transformed into actual antigen-specific immunotherapies with potential to restore selectively the breach of tolerance to ß-cell autoantigens and halt the autoimmune aggression. However, so far, the results of both prevention and reversion trials in T1D have been rather discouraging, so there is still an urgent need to optimize those immunotherapies and their associated factors, for example, posology and administration patterns, route and timing. In this review, we look back on what has been achieved in the last century and identify the main autoantigens driving the autoimmune attack in T1D. Then, we take a deep dive into the numerous antigen-specific immunotherapies trialed and the ones still at a preclinical phase, ranging from peptides, proteins and agent combinations to gene transfer, nanoparticles, cell-based strategies and novel approaches exploiting naturally occurring tolerogenic processes. Finally, we provide insight into the several features to be considered in a T1D clinical trial, the ideal time point for intervention and the biomarkers needed for monitoring the successful regulatory effect of the antigen-specific immunotherapy. Although further research and optimization remain imperative, the development of a therapeutic armamentarium against T1D autoimmunity is certainly advancing with a confident step.

Antigen-specific immunotherapy combined with a regenerative drug in the treatment of experimental type 1 diabetes.

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.

Immune System Remodelling by Prenatal Betamethasone: Effects on ß-Cells and Type 1 Diabetes.

Type 1 diabetes (T1D) is a multifactorial disease of unknown aetiology. Studies focusing on environment-related prenatal changes, which might have an influence on the development of T1D, are still missing. Drugs, such as betamethasone, are used during this critical period without exploring possible effects later in life. Betamethasone can interact with the development and function of the two main players in T1D, the immune system and the pancreatic ß-cells. Short-term or persistent changes in any of these two players may influence the initiation of the autoimmune reaction against ß-cells. In this review, we focus on the ability of betamethasone to induce alterations in the immune system, impairing the recognition of autoantigens. At the same time, betamethasone affects ß-cell gene expression and apoptosis rate, reducing the danger signals that will attract unwanted attention from the immune system. These effects may synergise to hinder the autoimmune attack. In this review, we compile scattered evidence to provide a better understanding of the basic relationship between betamethasone and T1D, laying the foundation for future studies on human cohorts that will help to fully grasp the role of betamethasone in the development of T1D.

Repurposed Analog of GLP-1 Ameliorates Hyperglycemia in Type 1 Diabetic Mice Through Pancreatic Cell Reprogramming.

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.

NK Cell Subsets Changes in Partial Remission and Early Stages of Pediatric Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic metabolic disease characterized by the autoimmune destruction of ß-cells in the pancreatic islets. T1D is preceded by islet-specific inflammation led by several immune cells. Among them, natural killer (NK) cells are emerging as important players in T1D development. Human NK cells are characterized by CD56 and CD16 expression, which allows classifying NK cells into four subsets: 1) CD56dimCD16+ or effector NK cells (NKeff); 2) CD56brightCD16- or regulatory NK cells (NKreg); 3) intermediate CD56brightCD16+ NK cells; and 4) CD56dimCD16- NK cells, whose function is not well determined. Since many studies have shown that T1D progression is associated with changes in various immune cell types, we hypothesize that the kinetics of NK cell subsets in the blood could correlate with different stages of T1D. To that aim, pediatric patients newly diagnosed with T1D were recruited, and peripheral NK cell subsets were analyzed by flow cytometry at several disease checkpoints: disease onset, partial remission (PR), 8 months (for non-remitters), and 12 months of progression. Our results showed that total NK cells and their four subsets are altered at the early stages of T1D. A decrease in the counts and percentage of total NK cells and NKeff cells at the different disease stages was found when compared to controls. These results suggest the extravasation of these cells into the islets at disease onset, which is maintained throughout the follow-up. By contrast, NKreg cells increased during the early stages after T1D onset, and both intermediate NK cells and CD56dimCD16- NK cells diminished at the PR stage, which might reflect the immunoregulatory attempts and could be candidate biomarkers for this stage. Also, CD56dimCD16- NK cells increased during T1D progression. Finally, changes in CD16 expression were identified in the different T1D stages, highlighting a CD16 expression reduction in total NK cells and NKeff cells 1 year after diagnosis. That may reflect a state of exhaustion after multiple cell-to-cell interactions. Altogether, our preliminary data provide a longitudinal picture of peripheral NK cell subpopulations during the different T1D stages, which could be potential candidate biomarkers indicators of disease progression.

A Role for Oncostatin M in the Impairment of Glucose Homeostasis in Obesity.

CONTEXT: Oncostatin M (OSM) plays a key role in inflammation, but its regulation and function during obesity is not fully understood. OBJECTIVE: The aim of this study was to evaluate the relationship of OSM with the inflammatory state that leads to impaired glucose homeostasis in obesity. We also assessed whether OSM immunoneutralization could revert metabolic disturbances caused by a high-fat diet (HFD) in mice. DESIGN: 28 patients with severe obesity were included and stratified into two groups: (1) glucose levels <100 mg/dL and (2) glucose levels >100 mg/dL. White adipose tissue was obtained to examine OSM gene expression. Human adipocytes were used to evaluate the effect of OSM in the inflammatory response, and HFD-fed C57BL/6J mice were injected with anti-OSM antibody to evaluate its effects. RESULTS: OSM expression was elevated in subcutaneous and visceral fat from patients with obesity and hyperglycemia, and correlated with Glut4 mRNA levels, serum insulin, homeostatic model assessment of insulin resistance, and inflammatory markers. OSM inhibited adipogenesis and induced inflammation in human adipocytes. Finally, OSM receptor knockout mice had increased Glut4 mRNA levels in adipose tissue, and OSM immunoneutralization resulted in a reduction of glucose levels and Ccl2 expression in adipose tissue from HFD-fed mice. CONCLUSIONS: OSM contributes to the inflammatory state during obesity and may be involved in the development of insulin resistance.

Preclinical evaluation of antigen-specific nanotherapy based on phosphatidylserine-liposomes for type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing cells. Due to the ability of apoptotic cells clearance to induce tolerance, we previously generated liposomes rich in phophatidylserine (PS) -a feature of apoptotic cells- loaded with insulin peptides to mimic apoptotic beta-cells. PS-liposomes arrested autoimmunity in experimental T1D through the induction of tolerance. The aim of this study was to investigate the potential of several peptides from different T1D autoantigens encapsulated in (PS)-liposomes for T1D prevention and to assess its safety. T1D autoantigens (Insulin, C-peptide, GAD65 and IA2) were encapsulated in PS-liposomes. Liposomes were administered to the 'gold-standard' model for the study of autoimmune T1D, the Non-Obese Diabetic mouse, that spontaneously develop the disease. Safety and toxicity of liposomes were also determined. Only PS-liposomes encapsulating insulin peptides decrease T1D incidence in the Non-Obese Diabetic mouse model. Disease prevention correlates with a decrease in the severity of the autoimmune islet destruction driven by leukocytes. PS-liposomes neither showed toxic effect nor secondary complications. Among the here referred autoantigens, insulin peptides are the best candidates to be encapsulated in liposomes, like an artificial apoptotic cell, for the arrest of autoimmunity in T1D in a safe manner.

Impaired Phagocytosis in Dendritic Cells From Pediatric Patients With Type 1 Diabetes Does Not Hamper Their Tolerogenic Potential.

Type 1 diabetes (T1D) is prompted by defective immunological tolerance, an event in which dendritic cells (DCs) are crucial as immune response orchestrators. In fact, they contribute to maintaining tolerance to self-antigens, but they can also prompt an immunogenic response against them, leading to autoimmunity. Countless factors can potentially impact on the proper functionality of the DCs, which range from altered subset distribution, impaired phagocytic function to abnormal gene expression. Moreover, in T1D, metabolic dysregulation could impair DC functions as well. Indeed, since T1D clinical course is likely to be more aggressive in children and adolescents and entails severe dysglycemia, the aim of this study was to analyze circulating DCs subpopulations in pediatric T1D at different stages, as well as to characterize their phagocytosis ability and tolerance induction potential. Thus, pediatric patients newly diagnosed with T1D, with established disease and control subjects were recruited. Firstly, DCs subsets from peripheral blood were found quantitatively altered during the first year of disease, but recovered in the second year of progression. Secondly, to study the tolerogenic functionality of DCs, liposomes with phosphatidylserine (PS) were designed to mimic apoptotic beta cells, which are able to induce tolerance, as previously demonstrated by our group in DCs from adult patients with T1D. In this study, monocyte-derived DCs from pediatric patients with T1D and control subjects were assessed in terms of PS-liposomes capture kinetics, and transcriptional and phenotypic changes. DCs from pediatric patients with T1D were found to phagocyte PS-liposomes more slowly and less efficiently than DCs from control subjects, inversely correlating with disease evolution. Nonetheless, the transcription of PS receptors and immunoregulatory genes, cytokine profile, and membrane expression of immunological markers in DCs was consistent with tolerogenic potential after PS-liposomes phagocytosis. In conclusion, T1D progression in childhood entails altered peripheral blood DCs subsets, as well as impaired DCs phagocytosis, although tolerance induction could still function optimally. Therefore, this study provides useful data for patient follow-up and stratification in immunotherapy clinical trials.

B-Lymphocyte Phenotype Determines T-Lymphocyte Subset Differentiation in Autoimmune Diabetes.

Previous studies indicate that B-lymphocytes play a key role activating diabetogenic T-lymphocytes during the development of autoimmune diabetes. Recently, two transgenic NOD mouse models were generated: the NOD-PerIg and the 116C-NOD mice. In NOD-PerIg mice, B-lymphocytes acquire an activated proliferative phenotype and support accelerated autoimmune diabetes development. In contrast, in 116C-NOD mice, B-lymphocytes display an anergic-like phenotype delaying autoimmune diabetes onset and decreasing disease incidence. The present study further evaluates the T- and B-lymphocyte phenotype in both models. In islet-infiltrating B-lymphocytes (IIBLs) from 116C-NOD mice, the expression of H2-Kd and H2-Ag7 is decreased, whereas that of BAFF, BAFF-R, and TACI is increased. In contrast, IIBLs from NOD-PerIg show an increase in CD86 and FAS expression. In addition, islet-infiltrating T-lymphocytes (IITLs) from NOD-PerIg mice exhibit an increase in PD-1 expression. Moreover, proliferation assays indicate a high capacity of B-lymphocytes from NOD-PerIg mice to secrete high amounts of cytokines and induce T-lymphocyte activation compared to 116C B-lymphocytes. This functional variability between 116C and PerIg B-lymphocytes ultimately results in differences in the ability to shape T-lymphocyte phenotype. These results support the role of B-lymphocytes as key regulators of T-lymphocytes in autoimmune diabetes and provide essential information on the phenotypic characteristics of the T- and B-lymphocytes involved in the autoimmune response in autoimmune diabetes.

Partial remission and early stages of pediatric type 1 diabetes display immunoregulatory changes. A pilot study.

Type 1 diabetes (T1D) is a chronic metabolic disease of unknown etiology that results from ß-cell destruction. The onset of the disease, which arises after a long asymptomatic period of autoimmune attack, may be followed by a relapsing and remitting progression, a phenomenon that is most evident during the partial remission phase (PR). This stage lasts for a few months, shows minor requirements of exogenous insulin and could be explained by a recovery of immunological tolerance. This study aims to identify new biomarkers at early stages of pediatric T1D that reflect immunoregulatory changes. To that end, pediatric patients with T1D (n = 52) and age-related control subjects (n = 30) were recruited. Immune response-related molecules and lymphocyte subsets were determined starting at T1D onset and until the second year of progression. Results showed that circulating TGF-ß levels decreased during PR, and that betatrophin concentration was increased in all the considered stages without differing among studied checkpoints. Moreover, an increase of regulatory T, B and NK subsets was found during T1D progression, probably reflecting an attempt to restore self-tolerance. By contrast, a reduction in monocyte levels was observed at the early stages of diabetes. The results reveal significant changes in immunological parameters during the different early stages of T1D in children, which could ultimately serve as potential biomarkers to characterize the progression of T1D.