miRNA Regulation of T Cells in Islet Autoimmunity and Type 1 Diabetes.

PURPOSE OF REVIEW: Regulatory T cells (Tregs) are critical contributors to immune homeostasis and their dysregulation can lead to the loss of immune tolerance and autoimmune diseases like type 1 diabetes (T1D). Recent studies have highlighted microRNAs (miRNAs) as important regulators of the immune system, by fine-tuning relevant genes in various immune cell types. In this review article, we discuss recent insights into miRNA regulation of immune tolerance and activation. Specifically, we discuss how the dysregulation of miRNAs in T cells contributes to their aberrant function and the onset of islet autoimmunity, as well as their potential as targets of novel intervention strategies to interfere with autoimmune activation. RECENT FINDINGS: Several studies have shown that the dysregulation of individual miRNAs in T cells can contribute to impaired immune tolerance, contributing to onset and progression of islet autoimmunity. Importantly, the targeting of these miRNAs, including miR-92a, miR-142-3p and miR-181a, resulted in relevant effects on downstream pathways, improved Treg function and reduced islet autoimmunity in murine models. miRNAs are critical regulators of immune homeostasis and the dysregulation of individual miRNAs in T cells contributes to aberrant T cell function and autoimmunity. The specific targeting of individual miRNAs could improve Treg homeostasis and therefore limit overshooting T cell activation and islet autoimmunity.

miRNA92a targets KLF2 and the phosphatase PTEN signaling to promote human T follicular helper precursors in T1D islet autoimmunity.

Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)+CD4+ TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.

Adipose-tissue regulatory T cells: Critical players in adipose-immune crosstalk.

Obesity and type-2 diabetes (T2D) are associated with metabolic defects and inflammatory processes in fat depots. FoxP3+ regulatory T cells (Tregs) control immune tolerance, and have an important role in controlling tissue-specific inflammation. In this mini-review we will discuss current insights into how cross-talk between T cells and adipose tissue shapes the inflammatory environment in obesity-associated metabolic diseases, focusing on the role of CD4+ T cells and Tregs. We will also highlight potential opportunities for how the immunoregulatory properties of Tregs could be harnessed to control inflammation in obesity and T2D and emphasize the critical need for more research on humans to establish mechanisms that are conserved in both mice and humans.

Follicular Helper T Cells in Autoimmunity.

The development of multiple disease-relevant autoantibodies is a hallmark of autoimmune diseases. In autoimmune type 1 diabetes (T1D), a variable time frame of autoimmunity precedes the clinically overt disease. The relevance of T follicular helper (TFH) cells for the immune system is increasingly recognized. Their pivotal contribution to antibody production by providing help to germinal center (GC) B cells facilitates the development of a long-lived humoral immunity. Their complex differentiation process, involving various stages and factors like B cell lymphoma 6 (Bcl6), is strictly controlled, as anomalous regulation of TFH cells is connected with immunopathologies. While the adverse effects of a TFH cell-related insufficient humoral immunity are obvious, the role of increased TFH frequencies in autoimmune diseases like T1D is currently highlighted. High levels of autoantigen trigger an excessive induction of TFH cells, consequently resulting in the production of autoantibodies. Therefore, TFH cells might provide promising approaches for novel therapeutic strategies.

Extra-thymically induced regulatory T cells: do they have potential in disease prevention?

Fopx3(+) Treg safeguard against autoimmune diseases and immune pathology. The extrathymic conversion of naïve T cells into Foxp3(+) regulatory T cells can be achieved in vivo by the delivery of strong-agonist ligands under subimmunogenic conditions. Tolerogenic vaccination with strong-agonist mimetopes of self-antigen to promote self-antigen specific tolerance may represent the most specific and safest means of preventing autoimmunity. This review discusses the requirements for induction of dominant tolerance exerted by Foxp3(+) Tregs in autoimmunity with special emphasis on their impact to interfere with T1D. The future goals are the understanding of self-non-self discrimination at the cellular and molecular level, which should then enable investigators to develop clinical vaccination protocols that specifically interfere with unwanted immune responses.

Nuclear factor of activated T cells - a transcription factor family as critical regulator in lung and colon cancer.

Nuclear factor of activated T cells (NFAT) was first identified as a transcription factor which is activated upon T cell stimulation. Subsequent studies uncovered that a whole family of individual NFAT proteins exists with pleiotropic functions not only in immune but also in nonimmune cells. However, dysregulation of NFAT thereby favors malignant growth and cancer. Summarizing the recent advances in understanding how individual NFAT factors regulate the immune system, this review gives new insights into the critical role of NFAT in cancer development with special focus on inflammation-associated colorectal cancer.

Niche-specific control of tissue function by regulatory T cells-Current challenges and perspectives for targeting metabolic disease.

Tissue regulatory T cells (Tregs) exert pivotal functions in both immune and metabolic regulation, maintaining local tissue homeostasis, integrity, and function. Accordingly, Tregs play a crucial role in controlling obesity-induced inflammation and supporting efficient muscle function and repair. Depending on the tissue context, Tregs are characterized by unique transcriptomes, growth, and survival factors and T cell receptor (TCR) repertoires. This functional specialization offers the potential to selectively target context-specific Treg populations, tailoring therapeutic strategies to specific niches, thereby minimizing potential side effects. Here, we discuss challenges and perspectives for niche-specific Treg targeting, which holds promise for highly efficient and precise medical interventions to combat metabolic disease.

The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Enhancement of antigen-specific Treg vaccination in vivo.

The conversion of naive T cells into Treg can be achieved in vivo by delivery of antigen under subimmunogenic conditions. Here we have examined several drugs for their ability to enhance the conversion process in vivo and have found that the rapamycin analog everolimus potently enhances Treg conversion by interfering with T-cell costimulation, reducing cell division and thereby activation of DNA methyltransferase 1 as well as by reducing T-cell activation through the ATP-gated P2×7 receptor controlling Ca2(+) influx. The resulting Tregs exhibit increased stability of Foxp3 expression even when generated in TGFß-containing media in vitro. Thus the mammalian target of rapamycin (mTOR) inhibitor everolimus in addition to inhibiting immune responses enhances Treg conversion by several distinct pathways. The converted Tregs can be further expanded by injection of IL-2/IL-2ab complexes. These complexes also increase the number of CD25(+)Foxp3(-) cells that, however, do not represent cytokine secreting effector cells but anergic cells, some of which can secrete IL-10 and can themselves be considered regulatory T cells as well. The combined use of everolimus and IL-2/IL-2ab complexes in vivo makes it feasible to achieve highly effective antigen-driven conversion of naive T cells into Treg and their expansion in vivo and thereby the described protocols constitute important tools to achieve immunological tolerance by Treg vaccination.

Regulatory T cells require IL6 receptor alpha signaling to control skeletal muscle function and regeneration.

Muscle-residing regulatory T cells (Tregs) control local tissue integrity and function. However, the molecular interface connecting Treg-based regulation with muscle function and regeneration remains largely unexplored. Here, we show that exercise fosters a stable induction of highly functional muscle-residing Tregs with increased expression of amphiregulin (Areg), EGFR, and ST2. Mechanistically, we find that mice lacking IL6Rα on T cells (TKO) harbor significant reductions in muscle Treg functionality and satellite and fibro-adipogenic progenitor cells, which are required for muscle regeneration. Using exercise and sarcopenia models, IL6Rα TKO mice demonstrate deficits in Tregs, their functional maturation, and a more pronounced decline in muscle mass. Muscle injury models indicate that IL6Rα TKO mice have significant disabilities in muscle regeneration. Treg gain of function restores impaired muscle repair in IL6Rα TKO mice. Of note, pharmacological IL6R blockade in WT mice phenocopies deficits in muscle function identified in IL6Rα TKO mice, thereby highlighting the clinical implications of the findings.

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Treg vaccination with a strong-agonistic insulin mimetope.

Foxp3(+) regulatory T (Treg) cells serve as a vital mechanism of negative regulation to maintain immunological self-tolerance thereby suppressing immune-mediated inflammation. The identification of the transcription factor Foxp3 as the specification factor for the Treg cell lineage facilitated our understanding in the biology of Treg generation and function. In the past, we carefully studied the extrathymic conversion of naive CD4(+) T cells into Foxp3(+) expressing Treg cells and found that this process is most efficient upon subimmunogenic supply of strong-agonistic T cell receptor (TCR) ligands avoiding activation of antigen-presenting and T cells. In contrast, weak-agonistic antigens fail to efficiently induce stable Foxp3(+) Treg cells irrespective of the applied dose. Here, we discuss the specific requirements for the establishment of Treg vaccination protocols to interfere with autoimmunity such as Type 1 diabetes.

Beta cell and immune cell interactions in autoimmune type 1 diabetes: How they meet and talk to each other.

BACKGROUND: The highly complex pathogenesis of Type 1 Diabetes is driven by several immune cell types with both effector and regulatory characteristics, which ultimately ends in the destruction of the insulin-producing beta cells. There are multiple layers of interaction between these immune cell populations and the pancreatic islets. SCOPE OF REVIEW: In this review article, we aim to discuss important recent insights into the multiple layers of interaction between immune cell populations and the pancreatic islets. Specifically, we discuss the environment where immune and beta cell interactions occur, the key cell types and molecules involved, and the outcomes of these interactions. MAJOR CONCLUSIONS: Most of the molecular mechanisms underlying aberrant immune cell activation and impaired immune tolerance remain insufficiently understood, which hinders the development of efficient prevention and treatment strategies. In order to overcome this knowledge gap, a better understanding of the complex interactions of immune cells and beta cells, including both the underlying protective and pathogenic mechanisms is urgently required.

Antigen-Specific Treg Therapy in Type 1 Diabetes - Challenges and Opportunities.

Regulatory T cells (Tregs) are key mediators of peripheral self-tolerance and alterations in their frequencies, stability, and function have been linked to autoimmunity. The antigen-specific induction of Tregs is a long-envisioned goal for the treatment of autoimmune diseases given reduced side effects compared to general immunosuppressive therapies. However, the translation of antigen-specific Treg inducing therapies for the treatment or prevention of autoimmune diseases into the clinic remains challenging. In this mini review, we will discuss promising results for antigen-specific Treg therapies in allergy and specific challenges for such therapies in autoimmune diseases, with a focus on type 1 diabetes (T1D). We will furthermore discuss opportunities for antigen-specific Treg therapies in T1D, including combinatorial strategies and tissue-specific Treg targeting. Specifically, we will highlight recent advances in miRNA-targeting as a means to foster Tregs in autoimmunity. Additionally, we will discuss advances and perspectives of computational strategies for the detailed analysis of tissue-specific Tregs on the single-cell level.

Advances in Human Immune System Mouse Models for Personalized Treg-Based Immunotherapies.

Immunodeficient mice engrafted with a functional human immune system [Human immune system (HIS) mice] have paved the way to major advances for personalized medicine and translation of immune-based therapies. One prerequisite for advancing personalized medicine is modeling the immune system of individuals or disease groups in a preclinical setting. HIS mice engrafted with peripheral blood mononuclear cells have provided fundamental insights in underlying mechanisms guiding immune activation vs. regulation in several diseases including cancer. However, the development of Graft-vs.-host disease restrains relevant long-term studies in HIS mice. Alternatively, engraftment with hematopoietic stem cells (HSCs) enables mimicking different disease stages, however, low frequencies of HSCs in peripheral blood of adults impede engraftment efficacy. One possibility to overcome those limitations is the use of patient-derived induced pluripotent stem cells (iPSCs) reprogrammed into HSCs, a challenging process which has recently seen major advances. Personalized HIS mice bridge research in mice and human diseases thereby facilitating the translation of immunomodulatory therapies. Regulatory T cells (Tregs) are important mediators of immune suppression and thereby contribute to tumor immune evasion, which has made them a central target for cancer immunotherapies. Importantly, studying Tregs in the human immune system in vivo in HIS mice will help to determine requirements for efficient Treg-targeting. In this review article, we discuss advances on personalized HIS models using reprogrammed iPSCs and review the use of HIS mice to study requirements for efficient targeting of human Tregs for personalized cancer immunotherapies.

100 Years of Insulin: Lifesaver, immune target, and potential remedy for prevention.

In this review, we bring our personal experiences to showcase insulin from its breakthrough discovery as a life-saving drug 100 years ago to its uncovering as the autoantigen and potential cause of type 1 diabetes and eventually as an opportunity to prevent autoimmune diabetes. The work covers the birth of insulin to treat patients, which is now 100 years ago, the development of human insulin, insulin analogues, devices, and the way into automated insulin delivery, the realization that insulin is the primary autoimmune target of type 1 diabetes in children, novel approaches of immunotherapy using insulin for immune tolerance induction, the possible limitations of insulin immunotherapy, and an outlook how modern vaccines could remove the need for another 100 years of insulin therapy.

Aging Aggravates Cachexia in Tumor-Bearing Mice.

BACKGROUND: Cancer is primarily a disease of high age in humans, yet most mouse studies on cancer cachexia are conducted using young adolescent mice. Given that metabolism and muscle function change with age, we hypothesized that aging may affect cachexia progression in mouse models. METHODS: We compare tumor and cachexia development in young and old mice of three different strains (C57BL/6J, C57BL/6N, BALB/c) and with two different tumor cell lines (Lewis Lung Cancer, Colon26). Tumor size, body and organ weights, fiber cross-sectional area, circulating cachexia biomarkers, and molecular markers of muscle atrophy and adipose tissue wasting are shown. We correlate inflammatory markers and body weight dependent on age in patients with cancer. RESULTS: We note fundamental differences between mouse strains. Aging aggravates weight loss in LLC-injected C57BL/6J mice, drives it in C57BL/6N mice, and does not influence weight loss in C26-injected BALB/c mice. Glucose tolerance is unchanged in cachectic young and old mice. The stress marker GDF15 is elevated in cachectic BALB/c mice independent of age and increased in old C57BL/6N and J mice. Inflammatory markers correlate significantly with weight loss only in young mice and patients. CONCLUSIONS: Aging affects cachexia development and progression in mice in a strain-dependent manner and influences the inflammatory profile in both mice and patients. Age is an important factor to consider for future cachexia studies.

miRNA-Mediated Immune Regulation in Islet Autoimmunity and Type 1 Diabetes.

The important role of microRNAs as major modulators of various physiological processes, including immune regulation and homeostasis, has been increasingly recognized. Consequently, aberrant miRNA expression contributes to the defective regulation of T cell development, differentiation, and function. This can result in immune activation and impaired tolerance mechanisms, which exert a cardinal function for the onset of islet autoimmunity and the progression to T1D. The specific impact of miRNAs for immune regulation and how miRNAs and their downstream targets are involved in the pathogenesis of islet autoimmunity and T1D has been investigated recently. These studies revealed that increased expression of individual miRNAs is involved in several layers of tolerance impairments, such as inefficient Treg induction and Treg instability. The targeted modulation of miRNAs using specific inhibitors, resulting in improved immune homeostasis, as well as improved methods for the targeting of miRNAs, suggest that miRNAs, especially in T cells, are a promising target for the reestablishment of immune tolerance.

The role of T cell miRNAs for regulatory T cell induction in islet autoimmunity.

BACKGROUND: microRNAs (miRNAs) have emerged as critical contributors to immune regulation and homeostasis, and their dysregulation is involved in the aberrant differentiation and function of T cell subsets. In type 1 diabetes (T1D), the clinically overt disease is preceded by a presymptomatic phase which is marked by the presence of islet autoantibodies while the individual is still normoglycemic. Recent analyses revealed impaired regulatory T (Treg) cell induction from naive CD4+ T cells during this early phase of autoimmunity. SCOPE OF THE REVIEW: In this review article, we aim to discuss important recent insights into miRNA regulation of immune homeostasis and activation. Specifically, we highlight the role of miRNAs as biomarkers in autoimmunity and T1D as well as the contribution of specific miRNAs and their downstream pathways to the onset and progression of islet immunity. Furthermore, we focus on critical next steps required to establish miRNAs as biomarkers to predict disease onset and progression and as novel targets of future prevention and treatment strategies to control autoimmunity. MAJOR CONCLUSIONS: Several recent studies have provided considerable insight into the miRNA regulation of immune homeostasis and how dysregulated miRNAs contribute to onset and progression of islet autoimmunity. Specifically, high levels of individual miRNAs such as miR92a and miR181a are involved in impaired Treg induction during the onset of islet autoimmunity, thereby contributing to disease pathogenesis. The recent advancements in the field suggest miRNAs as potential biomarkers for islet autoimmunity and their direct targeting, especially in a T cell-specific manner, could contribute to the reestablishment of immune homeostasis and ultimately interfere with the onset of islet autoimmunity.

Short-term cold exposure supports human Treg induction in vivo.

OBJECTIVE: Obesity and type-2 diabetes (T2D) are metabolic diseases that represent a critical health problem worldwide. Metabolic disease is differentially associated with fat distribution, while visceral white adipose tissue (VAT) is particularly prone to obesity-associated inflammation. Next to their canonical function of immune suppression, regulatory T cells (Tregs) are key in controlling adipose tissue homeostasis. Towards understanding the molecular underpinnings of metabolic disease, we focus on how environmental-metabolic stimuli impinge on the functional interplay between Tregs and adipose tissue. Here, cold exposure or beta3-adrenergic signaling are a promising tool to increase energy expenditure by activating brown adipose tissue, as well as by reducing local inflammation within fat depots by supporting immunosuppressive Tregs. However, in humans, the underlying mechanisms that enable the environmental-immune crosstalk in the periphery and in the respective tissue remain currently unknown. METHODS: We used combinatorial approaches of next generation humanized mouse models and in vitro and in vivo experiments together with beta3-adrenergic stimulation to dissect the underlying mechanisms of human Treg induction exposed to environmental stimuli such as cold. To test the translational relevance of our findings, we analyzed samples from the FREECE study in which human subjects were exposed to individualized cooling protocols. Samples were analyzed ex vivo and after in vitro Treg induction using qRT-PCR, immunofluorescence, as well as with multicolor flow cytometry and cell sorting. RESULTS: In vivo application of the beta3-adrenergic receptor agonist mirabegron in humanized mice induced thermogenesis and improved the Treg induction capacity of naïve T cells isolated from these animals. Using samples from the human FREECE study, we demonstrate that a short-term cold stimulus supports human Treg induction in vitro and in vivo. Mechanistically, we identify BORCS6 encoding the Ragulator-interacting protein C17orf59 to be significantly induced in human CD4+ T cells upon short-term cold exposure. Strong mTOR signaling is known to limit successful Treg induction and thus likely by interfering with mTOR activation at lysosomal surfaces, C17orf59 improves the Treg induction capacity of human naïve T cells upon cold exposure. CONCLUSIONS: These novel insights into the molecular underpinnings of human Treg induction suggest an important role of Tregs in linking environmental stimuli with adipose tissue function and metabolic diseases. Moreover, these discoveries shed new light on potential approaches towards tailored anti-inflammatory concepts that support human adipose tissue homeostasis by enabling Tregs.