Corrigendum: NK cell subsets changes in partial remission and early stages of pediatric type 1 diabetes.

[This corrects the article DOI: 10.3389/fimmu.2020.611522.].

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.

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.

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.

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.

Liposome-based immunotherapy against autoimmune diseases: therapeutic effect on multiple sclerosis.

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.

CD26/DPPIV inhibition alters the expression of immune response-related genes in the thymi of NOD mice.

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.

B-cell anergy induces a Th17 shift in a novel B lymphocyte transgenic NOD mouse model, the 116C-NOD mouse.

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.

Efferocytosis promotes suppressive effects on dendritic cells through prostaglandin E2 production in the context of autoimmunity.

INTRODUCTION: Efferocytosis is a crucial process by which apoptotic cells are cleared by phagocytes, maintaining immune tolerance to self in the absence of inflammation. Peripheral tolerance, lost in autoimmune processes, may be restored by the administration of autologous dendritic cells loaded with islet apoptotic cells in experimental type 1 diabetes. OBJECTIVE: To evaluate tolerogenic properties in dendritic cells induced by the clearance of apoptotic islet cells, thus explaining the re-establishment of tolerance in a context of autoimmunity. METHODS: Bone marrow derived dendritic cells from non-obese diabetic mice, a model of autoimmune diabetes, were generated and pulsed with islet apoptotic cells. The ability of these cells to induce autologous T cell proliferation and to suppress mature dendritic cell function was assessed, together with cytokine production. Microarray experiments were performed using dendritic cells to identify differentially expressed genes after efferocytosis. RESULTS: Molecular and functional changes in dendritic cells after the capture of apoptotic cells were observed. 1) Impaired ability of dendritic cells to stimulate autologous T cell proliferation after the capture of apoptotic cells even after proinflammatory stimuli, with a cytokine profile typical for immature dendritic cells. 2) Suppressive ability of mature dendritic cell function. 3) Microarray-based gene expression profiling of dendritic cells showed differential expression of genes involved in antigen processing and presentation after efferocytosis. 4) Prostaglandin E2 increased production was responsible for immunosuppressive mechanism of dendritic cells after the capture of apoptotic cells. CONCLUSIONS: The tolerogenic behaviour of dendritic cells after islet cells efferocytosis points to a mechanism of silencing potential autoreactive T cells in the microenvironment of autoimmunity. Our results suggest that dendritic cells may be programmed to induce specific immune tolerance using apoptotic cells; this is a viable strategy for a variety of autoimmune diseases.

Anti-peripherin B lymphocytes are positively selected during diabetogenesis.

Rearrangement analysis of immunoglobulin genes is an exceptional opportunity to look back at the B lymphocyte differentiation during ontogeny and the subsequent immune response, and thus to study the selective pressures involved in autoimmune disorders. In a recent study to characterize the antigenic specificity of B lymphocytes during T1D progression, we generated hybridomas of islet-infiltrating B lymphocytes from NOD mice and other related strains developing insulitis, but with different degrees of susceptibility to T1D. We found that a sizable proportion of hybridomas produced monoclonal antibodies reactive to peripherin, an intermediate filament protein mainly found in the peripheral nervous system. Moreover, we found that anti-peripherin antibody-producing hybridomas originated from B lymphocytes that had undergone immunoglobulin class switch recombination, a characteristic of secondary immune response. Therefore, in the present study we performed immunoglobulin VL and VH analysis of these hybridomas to ascertain whether they were derived from B lymphocytes that had undergone antigen-driven selection. The results indicated that whereas some anti-peripherin hybridomas showed signs of oligoclonality, somatic hypermutation and/or secondary rearrangements (receptor edition and receptor revision), others seemed to directly derive from the preimmune repertoire. In view of these results, we conclude that anti-peripherin B lymphocytes are positively selected and primed in the course of T1D development in NOD mice, and reinforce the idea that peripherin is a relevant autoantigen targeted during T1D development in this animal model.

Peripherin is a relevant neuroendocrine autoantigen recognized by islet-infiltrating B lymphocytes.

Most of our knowledge of the antigenic repertoire of autoreactive B lymphocytes in type 1 diabetes (T1D) comes from studies on the antigenic specificity of both circulating islet-reactive autoantibodies and peripheral B lymphocyte hybridomas generated from human blood or rodent spleen. In a recent study, we generated hybridoma cell lines of infiltrating B lymphocytes from different mouse strains developing insulitis, but with different degrees of susceptibility to T1D, to characterize the antigenic specificity of islet-infiltrating B lymphocytes during progression of the disease. We found that many hybridomas produced mAbs restricted to the peripheral nervous system (PNS), thus indicating an active B lymphocyte response against PNS elements in the pancreatic islet during disease development. The aim of this study was to identify the autoantigen recognized by these anti-PNS mAbs. Our results showed that peripherin is the autoantigen recognized by all anti-PNS mAbs, and, therefore, a relevant neuroendocrine autoantigen targeted by islet-infiltrating B lymphocytes. Moreover, we discovered that the immune dominant epitope of this B lymphocyte immune response is found at the C-terminal end of Per58 and Per61 isoforms. In conclusion, our study strongly suggests that peripherin is a major autoantigen targeted during T1D development and poses a new question on why peripherin-specific B lymphocytes are mainly attracted to the islet during disease.

Phenotype and functional characteristics of islet-infiltrating B-cells suggest the existence of immune regulatory mechanisms in islet milieu.

B-cells participate in the autoimmune response that precedes the onset of type 1 diabetes, but how these cells contribute to disease progression is unclear. In this study, we analyzed the phenotype and functional characteristics of islet-infiltrating B-cells in the diabetes-prone NOD mouse and in the insulitis-prone but diabetes-resistant (NOD x NOR)F1 mouse. The results indicate that B-cells accumulate in the islets of both mice influenced by sex traits. Phenotypically and functionally, these B-cells are highly affected by the islet inflammatory milieu, which may keep them in a silenced status. Moreover, although islet-infiltrating B-cells seem to be antigen experienced, they can only induce islet-infiltrating T-cell proliferation when they act as accessory cells. Thus, these results strongly suggest that islet-infiltrating B-cells do not activate islet-infiltrating T-cells in situ, although they may affect the progression of the disease otherwise.

Islet-infiltrating B-cells in nonobese diabetic mice predominantly target nervous system elements.

B-cells accumulate in pancreatic islets during the autoimmune response that precedes the onset of type 1 diabetes. However, the role and antigenic specificity of these cells remain a mystery. To elucidate the antigenic repertoire of islet-infiltrating B-cells in type 1 diabetes, we generated hybridoma cell lines of islet-infiltrating B-cells from nonobese diabetic (NOD) mice and NOD mice expressing a diabetogenic T-cell receptor (8.3-NOD). Surprisingly, characterization of the tissue specificity of the antibodies secreted by these cells revealed that a predominant fraction of these hybridomas produce antibodies specific for the pancreatic nervous system. Similar results were obtained with B-cell hybridomas derived from mild insulinic lesions of diabetes-resistant (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice. Immunoglobulin class analyses further indicated that most islet-derived hybridomas had arisen from B-cells that had undergone immunoglobulin class switch recombination, suggesting that islet-associated B-cells are involved in active, T-helper-driven immune responses against local antigenic targets. This is the first evidence showing the existence of a predominant active B-cell response in situ against pancreatic nervous system elements in diabetogenesis. Our data are consistent with the idea that this B-cell response precedes the progression of insulitis to overt diabetes, thus strongly supporting the idea that pancreatic nervous system elements are early targets in type 1 diabetes.