The local microenvironment drives activation of neutrophils in human brain tumors.

Neutrophils are abundant immune cells in the circulation and frequently infiltrate tumors in substantial numbers. However, their precise functions in different cancer types remain incompletely understood, including in the brain microenvironment. We therefore investigated neutrophils in tumor tissue of glioma and brain metastasis patients, with matched peripheral blood, and herein describe the first in-depth analysis of neutrophil phenotypes and functions in these tissues. Orthogonal profiling strategies in humans and mice revealed that brain tumor-associated neutrophils (TANs) differ significantly from blood neutrophils and have a prolonged lifespan and immune-suppressive and pro-angiogenic capacity. TANs exhibit a distinct inflammatory signature, driven by a combination of soluble inflammatory mediators including tumor necrosis factor alpha (TNF-ɑ) and Ceruloplasmin, which is more pronounced in TANs from brain metastasis versus glioma. Myeloid cells, including tumor-associated macrophages, emerge at the core of this network of pro-inflammatory mediators, supporting the concept of a critical myeloid niche regulating overall immune suppression in human brain tumors.

Phenotypic diversity of T cells in human primary and metastatic brain tumors revealed by multiomic interrogation.

The immune-specialized environment of the healthy brain is tightly regulated to prevent excessive neuroinflammation. However, after cancer development, a tissue-specific conflict between brain-preserving immune suppression and tumor-directed immune activation may ensue. To interrogate potential roles of T cells in this process, we profiled these cells from individuals with primary or metastatic brain cancers via integrated analyses on the single-cell and bulk population levels. Our analysis revealed similarities and differences in T cell biology between individuals, with the most pronounced differences observed in a subgroup of individuals with brain metastasis, characterized by accumulation of CXCL13-expressing CD39+ potentially tumor-reactive T (pTRT) cells. In this subgroup, high pTRT cell abundance was comparable to that in primary lung cancer, whereas all other brain tumors had low levels, similar to primary breast cancer. These findings indicate that T cell-mediated tumor reactivity can occur in certain brain metastases and may inform stratification for treatment with immunotherapy.

Immunogenomic analysis of human brain metastases reveals diverse immune landscapes across genetically distinct tumors.

Brain metastases (BrMs) are the most common form of brain tumors in adults and frequently originate from lung and breast primary cancers. BrMs are associated with high mortality, emphasizing the need for more effective therapies. Genetic profiling of primary tumors is increasingly used as part of the effort to guide targeted therapies against BrMs, and immune-based strategies for the treatment of metastatic cancer are gaining momentum. However, the tumor immune microenvironment (TIME) of BrM is extremely heterogeneous, and whether specific genetic profiles are associated with distinct immune states remains unknown. Here, we perform an extensive characterization of the immunogenomic landscape of human BrMs by combining whole-exome/whole-genome sequencing, RNA sequencing of immune cell populations, flow cytometry, immunofluorescence staining, and tissue imaging analyses. This revealed unique TIME phenotypes in genetically distinct lung- and breast-BrMs, thereby enabling the development of personalized immunotherapies tailored by the genetic makeup of the tumors.

Forced expression of the non-coding RNA miR-17∼92 restores activation and function in CD28-deficient CD4+ T cells.

CD28 provides the prototypical costimulatory signal required for productive T-cell activation. Known molecular consequences of CD28 costimulation are mostly based on studies of protein signaling molecules. The microRNA cluster miR-17∼92 is induced by T cell receptor stimulation and further enhanced by combined CD28 costimulation. We demonstrate that transgenic miR-17∼92 cell-intrinsically largely overcomes defects caused by CD28 deficiency. Combining genetics, transcriptomics, bioinformatics, and biochemical miRNA:mRNA interaction maps we empirically validate miR-17∼92 target genes that include several negative regulators of T cell activation. CD28-deficient T cells exhibit derepressed miR-17∼92 target genes during activation. CRISPR/Cas9-mediated ablation of the miR-17∼92 targets Pten and Nrbp1 in naive CD28-/- CD4+ T cells differentially increases proliferation and expression of the activation markers CD25 and CD44, respectively. Thus, we propose that miR-17∼92 constitutes a central mediator for T cell activation, integrating signals by the TCR and CD28 costimulation by dampening multiple brakes that prevent T cell activation.

An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.

Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells.

Brain malignancies encompass a range of primary and metastatic cancers, including low-grade and high-grade gliomas and brain metastases (BrMs) originating from diverse extracranial tumors. Our understanding of the brain tumor microenvironment (TME) remains limited, and it is unknown whether it is sculpted differentially by primary versus metastatic disease. We therefore comprehensively analyzed the brain TME landscape via flow cytometry, RNA sequencing, protein arrays, culture assays, and spatial tissue characterization. This revealed disease-specific enrichment of immune cells with pronounced differences in proportional abundance of tissue-resident microglia, infiltrating monocyte-derived macrophages, neutrophils, and T cells. These integrated analyses also uncovered multifaceted immune cell activation within brain malignancies entailing converging transcriptional trajectories while maintaining disease- and cell-type-specific programs. Given the interest in developing TME-targeted therapies for brain malignancies, this comprehensive resource of the immune landscape offers insights into possible strategies to overcome tumor-supporting TME properties and instead harness the TME to fight cancer.

Re-education of macrophages as a therapeutic strategy in cancer.

Tumor-associated macrophages (TAMs) can be educated within the tumor microenvironment to promote cancer development and progression. While TAM-targeted agents have largely focused on macrophage depletion as an anticancer strategy, it is becoming increasingly evident that TAM re-education may represent a more effective approach. In this perspective, we discuss different means to achieve TAM re-education, and review the beneficial effects of these strategies, particularly when combined with immune checkpoint inhibitors.

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells.

Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

KLRG1 expression identifies short-lived Foxp3+ Treg effector cells with functional plasticity in islets of NOD mice.

A progressive waning in Foxp3+ regulatory T (Treg) cell function provokes autoimmunity in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), a cellular defect rescued by prophylactic IL-2 therapy. We showed that most islet-infiltrating Treg cells express inducible T-cell co-stimulator (ICOS) in pre-diabetic NOD mice, and that ICOS+ Treg cells display enhanced fitness and suppressive function in situ. Moreover, T1D progression is associated with decreased expansion and suppressive activity of ICOS+Foxp3+ Treg cells, in islets, an observation consistent with the exacerbated T1D seen in NOD.BDC2.5 mice in which the ICOS pathway is abrogated. Here, we show that a large proportion of islet-resident Treg cells express the KLRG1 marker of terminally differentiation, in contrast to islet-infiltrating ICOS- Treg or Teff cells. We hypothesized that KLRG1 expression designates a subpopulation of ICOS+ Treg cells in islets that progressively loses function, and contributes to the immune dysregulation observed at T1D onset. Indeed, KLRG1-expressing ICOS+ Treg cells are prone to apoptosis, and have an impaired proliferative capacity and suppressive function in vitro and in vivo. T1D protective low-dose IL-2 treatment in vivo could not rescue the loss of KLRG1-expressing Treg cells in situ. While the global pool of Foxp3+ Treg cells displays some degree of functional plasticity in vivo, the KLRG1+ ICOS+ Treg cell subset is particularly susceptible to lose Foxp3 expression and reprogram into Th1- or Th17-like effector T (Teff) cells in the pancreas microenvironment. Overall, KLRG1 expression delineates a subpopulation of dysfunctional Treg cells during T1D progression in autoantigen-specific TCR transgenic NOD mice.

Th1-Like ICOS+ Foxp3+ Treg Cells Preferentially Express CXCR3 and Home to ß-Islets during Pre-Diabetes in BDC2.5 NOD Mice.

Type 1 diabetes (T1D) occurs through a breakdown of self-tolerance resulting in the autoimmune destruction of the insulin producing ß-islets of the pancreas. A numerical and functional waning of CD4+ Foxp3+ regulatory T (Treg) cells, prompted by a pancreatic IL-2 deficiency, accompanies Th1 autoimmunity and T1D progression in non-obese diabetic (NOD) mice. Recently, we identified a dominant subset of intra-islet Treg cells that expresses the ICOS costimulatory receptor and promotes self-tolerance delaying the onset of T1D. ICOS co-stimulation potently enhances IL-2 induced survival and proliferation, and suppressive activity of Treg cells in situ. Here, we propose an ICOS-dependent mechanism of Treg cell homing to the ß-islets during pre-diabetes in the NOD model via upregulation of the CXCR3 chemokine receptor. The islet-specific ICOS+ Treg cell subset preferentially expresses CXCR3 in the pancreatic lymph nodes (pLN) in response to Teff cell-mediated pancreatic inflammation, an expression correlating with the onset and magnitude of IFN-γ production by Teff cells in pancreatic sites. We also reveal that intra-pancreatic APC populations and insulin-producing ß, but not α nor δ, islet cells secrete the CXCR3 chemokines, CXCL9, 10 and 11, and selectively promote ICOS+ CXCR3+ Treg cell chemotaxis in vitro. Strikingly, islet-derived Treg cells also produce these chemokines suggesting an auto-regulation of homing by this subset. Unlike ICOS- cells, ICOS+ Treg cells adopt a Th1-like Treg phenotype while maintaining their suppressive capacity, characterized by expression of T-bet and CXCR3 and production of IFN-γ in the draining pLNs. Finally, in vivo neutralization of IFN-γ blocked Treg cell CXCR3 upregulation evincing its role in regulating expression of this chemokine receptor by Treg cells. Thus, CXCR3-mediated trafficking of Treg cells could represent a mechanism of homeostatic immunoregulation during diabetogeneesis.

Functional dynamics of Foxp3⁺ regulatory T cells in mice and humans.

Forkhead box protein 3 (Foxp3)(+) regulatory T (Treg) cells are critical mediators for the establishment of self-tolerance and immune homeostasis and for the control of pathology in various inflammatory responses. While Foxp3(+) Treg cells often control immune responses in secondary lymphoid tissues, they must also traffic to and persist within non-lymphoid tissues, where they integrate various environmental cues to coordinate and adapt their effector acitvities in these sites. In recent years, our group has made use of several mouse models, including the non-obese diabetic model of type 1 diabetes, to characterize the factors, which impact the homeostasis, function, and reprogramming potential of Foxp3(+) Treg cells in situ. In addition, our recent work shows that Foxp3(+) Treg cells possess distinct post-transcriptional mechanisms of gene regulation, namely mRNA translation, to modulate tissue-specific inflammatory responses. In humans, there is a pressing need for reliable markers of FOXP3(+) Treg cells and their related function in blood and tissue. Experimental progress in our group has enabled us to discover novel markers of FOXP3(+) Treg cell (dys)function and unique gene signatures that discriminate effector and Treg cells, as well as functional and dysfunctional FOXP3(+) Treg cells.

Immune Regulation in T1D and T2D: Prospective Role of Foxp3+ Treg Cells in Disease Pathogenesis and Treatment.

There is increasing evidence that dysregulated immune responses play key roles in the pathogenesis and complications of type 1 but also type 2 diabetes. Indeed, chronic inflammation and autoimmunity, which are salient features of type 1 diabetes, are now believed to actively contribute to the pathogenesis of type 2 diabetes. The accumulation of activated innate and adaptive immune cells in various metabolic tissues results in the release of inflammatory mediators, which promote insulin resistance and ß-cell damage. Moreover, these dysregulated immune responses can also mutually influence the prevalence of both type 1 and 2 diabetes. In this review article, we discuss the central role of immune responses in the patho-physiology and complications of type 1 and 2 diabetes, and provide evidence that regulation of these responses, particularly through the action of regulatory T cells, may be a possible therapeutic avenue for the treatment of these disease and their respective complications.

Phosphatidylinositol 3-kinase-independent signaling pathways contribute to ICOS-mediated T cell costimulation in acute graft-versus-host disease in mice.

We and others have previously shown that ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models of allogeneic bone marrow transplantation. ICOS potentiates TCR-mediated PI3K activation and intracellular calcium mobilization. However, ICOS signal transduction pathways involved in GVHD remain unknown. In this study, we examined the contribution of ICOS-PI3K signaling in the pathogenic potential of T cells using a knock-in mouse strain, ICOS-YF, which selectively lost the ability to activate PI3K. We found that when total T cells were used as alloreactive T cells, ICOS-YF T cells caused less severe GVHD compared with ICOS wild-type T cells, but they induced much more aggressive disease than ICOS knockout T cells. This intermediate level of pathogenic capacity of ICOS-YF T cells was correlated with similar levels of IFN-γ-producing CD8 T cells that developed in the recipients of ICOS-WT or ICOS-YF T cells. We further evaluated the role of ICOS-PI3K signaling in CD4 versus CD8 T cell compartment using GVHD models that are exclusively driven by CD4 or CD8 T cells. Remarkably, ICOS-YF CD8 T cells caused disease similar to ICOS wild-type CD8 T cells, whereas ICOS-YF CD4 T cells behaved very similarly to their ICOS knockout counterparts. Consistent with their in vivo pathogenic potential, CD8 T cells responded to ICOS ligation in vitro by PI3K-independent calcium flux, T cell activation, and proliferation. Thus, in acute GVHD in mice, CD4 T cells heavily rely on ICOS-PI3K signaling pathways; in contrast, CD8 T cells can use PI3K-independent ICOS signaling pathways, possibly through calcium.

Pancreatic islet cell phenotype and endocrine function throughout diabetes development in non-obese diabetic mice.

Type 1 diabetes (T1D) results from a T cell dependent, autoimmune destruction of insulin producing beta cells in pancreatic islets of Langerhans, which results in insulin deficiency despite attempts at beta cell replacement by the emergence of newly-differentiated beta cells throughout T1D development. The origin of these cells has been difficult to assess as these are rapidly destroyed by the underlying autoimmunity. The identification of islets of Langerhans is typically assessed by either immunochemistry or immunofluorescence using antibodies directed against the different signature hormones and surface markers of various endocrine cells. However, the limited number of markers that can be used simultaneously and the uneven spatial distribution of endocrine cells within islets, limit the use of these histological analyses. To circumvent these caveats, we developed a novel approach using multi-parametric flow cytometry to assess the phenotype and function of pancreatic islet cell populations throughout T1D development. Using such strategy, we show that while beta cells undergo autoimmune destruction, insulin-producing cells arise from trans-differentiated alpha or delta cells, an outcome that was not solely the result of beta cell self-renewal. Moreover, we show that CD4(+)T cell-mediated inflammation correlates with the emergence of this insulin-producing beta cell-like cell.

Functional crosstalk between dendritic cells and Foxp3(+) regulatory T cells in the maintenance of immune tolerance.

Peripheral immune tolerance requires a controlled balance between the maintenance of self-tolerance and the capacity to engage protective immune responses against pathogens. Dendritic cells (DCs) serve as sentinels of the immune system by sensing environmental and inflammatory signals, and play an essential role in the maintenance of immune tolerance. To achieve this, DC play a key role in dictating the outcome of immune responses by influencing the balance between inflammatory or Foxp3(+) regulatory T (T(reg)) cell responses. At the heart of this immunological balance is a finely regulated DC and T(reg) cell crosstalk whereby T(reg) cells modulate DC phenotype and function, and DC drive the differentiation of Foxp3(+) T(reg) cells in order to control immune responses. This review will focus on recent advances, which highlight the importance of this bidirectional DC and T(reg) cell crosstalk during the induction of tolerance and organ-specific autoimmunity. More specifically, we will discuss how T(reg) cells modulate DC function for the suppression of inflammatory responses and how DC subsets employ diverse mechanisms to drive differentiation of T(reg) cells. Finally, we will discuss the therapeutic potential of tolerogenic DCs for the induction of tolerance in autoimmune diseases.

Mesenchymal stromal cells improve salivary function and reduce lymphocytic infiltrates in mice with Sjögren's-like disease.

BACKGROUND: Non-obese diabetic (NOD) mice develop Sjögren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freund's adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45(-)/TER119(-) cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined. METHODOLOGY/PRINCIPAL FINDINGS: Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45(-)/TER119(-) cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3(+) (T(reg)) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-ß), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF). CONCLUSIONS/SIGNIFICANCE: The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

ICOS-dependent homeostasis and function of Foxp3+ regulatory T cells in islets of nonobese diabetic mice.

A progressive waning in Foxp3(+) regulatory T cell (Treg) functions is thought to provoke autoimmunity in the NOD model of type 1 diabetes (T1D). A deficiency in IL-2 is one of the main triggers for the defective function of Tregs in islets. Notably, abrogation of the ICOS pathway in NOD neonates or BDC2.5-NOD (BDC2.5) mice exacerbates T1D, suggesting an important role for this costimulatory pathway in tolerance to islet Ags. Thus, we hypothesize that ICOS selectively promotes Foxp3(+) Treg functions in BDC2.5 mice. We show that ICOS expression discriminates effector Foxp3(-) T cells from Foxp3(+) Tregs and specifically designates a dominant subset of intra-islet Tregs, endowed with an increased potential to expand, secrete IL-10, and mediate suppressive activity in vitro and in vivo. Consistently, Ab-mediated blockade or genetic deficiency of ICOS selectively abrogates Treg-mediated functions and T1D protection and exacerbates disease in BDC2.5 mice. Moreover, T1D progression in BDC2.5 mice is associated with a decline in ICOS expression in and expansion and suppression by intra-islet Foxp3(+) Tregs. We further show that the ICOS(+) Tregs, in contrast to their ICOS(-) counterparts, are more sensitive to IL-2, a critical signal for their survival and functional stability. Lastly, the temporal loss in ICOS(+) Tregs is readily corrected by IL-2 therapy or protective Il2 gene variation. Overall, ICOS is critical for the homeostasis and functional stability of Foxp3(+) Tregs in prediabetic islets and maintenance of T1D protection.

Critical co-stimulatory pathways in the stability of Foxp3+ Treg cell homeostasis in Type I diabetes.

Mechanisms of peripheral tolerance maintain a controlled balance between self-tolerance, protective immunity against a spectrum of non-self antigens, and suppressing pathology in various disorders. CD4(+) regulatory T cells (T(reg)) expressing the Foxp3 transcription factor dominantly control the activity and pathological consequences of a variety of effector T cell lineages in various inflammatory settings. This review will focus on recent advances on the roles of B7 family members in regulating Treg cell development, function and homeostasis during tolerance induction and organ-specific autoimmunity.

IL-2 production by dendritic cells promotes Foxp3(+) regulatory T-cell expansion in autoimmune-resistant NOD congenic mice.

Il2 allelic variation in non-obese diabetic mice imparts marked resistance to type 1 diabetes. IL-2 is pivotal for the fitness and homeostasis of Foxp3(+) regulatory T (T(reg)) cells, and the Idd3(B6) locus augments IL-2 production by effector T cells, which in turn enhances the potency of T(reg) cell functions. Given the important role dendritic cells (DCs) play in T(reg) cell-mediated tolerance induction, we hypothesized that DCs from Idd3(B6) congenic mice contribute to increased T(reg) cell activity. Here, we observed that CD11c(+) DCs, harboring protective Idd3(B6) genes, are endowed with the capacity to secrete IL-2, enabling them to preferentially promote T(reg) cell functions in vitro and in vivo. Our results show that Il2 gene variation may imprint DCs to favor T-cell regulation of autoimmunity.

IL-2 as a therapeutic target for the restoration of Foxp3+ regulatory T cell function in organ-specific autoimmunity: implications in pathophysiology and translation to human disease.

Peripheral immune tolerance requires a finely controlled balance between tolerance to self-antigens and protective immunity against enteric and invading pathogens. Self-reactive T cells sometimes escape thymic clonal deletion, and can subsequently provoke autoimmune diseases such as type 1 diabetes (T1D) unless they are controlled by a network of tolerance mechanisms in the periphery, including CD4+ regulatory T cells (Treg) cells. CD4+ Treg cells are characterized by the constitutive expression of the IL-2Rα chain (CD25) and preferentially express the forkhead winged helix transcriptional regulator Foxp3. These cells have been shown to possess immunosuppressive properties towards various immune cell subsets and their defects are thought to contribute to many autoimmune disorders. Strong evidence shows that IL-2 is one of the important stimulatory signals for the development, function and fitness of Treg cells. The non-obese diabetic (NOD) mouse model, a prototypic model of spontaneous autoimmunity, mimics many features of human T1 D. Using this model, the contribution of the IL-2-IL-2R pathway to the development of T1 D and other autoimmune disorders has been extensively studied. In the past years, strong genetic and molecular evidence has indicated an essential role for the IL-2/IL-2R pathway in autoimmune disorders. Thus, the major role of IL-2 is to maintain immune tolerance by promoting Treg cell development, functional fitness and stability. Here we first summarize the genetic and experimental evidence demonstrating a role for IL-2 in autoimmunity, mainly through the study of the NOD mouse model, and analyze the cellular and molecular mechanisms of its action on Treg cells. We then move on to describe how this data can be translated to applications for human autoimmune diseases by using IL-2 as a therapeutic agent to restore Treg cell fitness, numbers and functions.