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Growing malignant tumors must evade destruction by the immune system, a hurdle some malignancies overcome by attracting immune-suppressive regulatory T-cells (Tregs)1. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Tregs, and IKZF2 deficiency enhances immune responses to tumors in mice2, suggesting IKZF2 may be an attractive target for cancer immunotherapy. Here we describe the discovery and characterization of DKY709, the first molecular glue degrader of IKZF2/4 which spares IKZF1/3. DKY709 was identified through a recruitment-guided medicinal chemistry campaign that redirected the degradation selectivity of CRBN binders towards IKZF2. The IKZF transcription factor selectivity of DKY709 was rationalized by the X-ray structure of the CRBN-DKY709-IKZF2(ZF2) ternary complex. Upon exposure to DKY709, human Tregs showed reduced suppressive activity and exhausted T-effector cells recovered IFNγ production. In vivo, oral treatment with DKY709 drove a rapid and sustained degradation of IKZF2 including in humans and led to delayed tumor growth in mice with humanized immune systems and enhanced immunization responses in monkeys. DKY709 is a first-in-class, potent and selective oral IKZF2/4 degrader currently being investigated in a phase 1 clinical trial as an immune-enhancing agent for cancer immunotherapy.

The C1858T single nucleotide polymorphism (SNP) in PTPN22 (protein tyrosine phosphatase nonreceptor 22) leads to the 620 Arg to Trp polymorphism in its encoded human protein LYP. This allelic variant is associated with multiple autoimmune diseases, including type 1 diabetes (T1D), Crohn's disease, rheumatoid arthritis and systemic lupus erythematosus. However, the underlying mechanisms are poorly understood. To study how this polymorphism influences the immune system, we generated a mouse strain with a knock-in of the Trp allele, imitating the human disease-associated variant. We did not find significant difference between the polymorphic and the wild type mice on the proportion of total CD4 T cell, CD8 T cell, NK cell, memory T lymphocyte, macrophage, dendritic cells in both peripheral lymph nodes and spleen. However, macrophages from Trp/Trp mice showed altered morphology and enhanced function, including higher expression of MHCII and B7 molecules and increased phagocytic ability, which further leads to a higher T-cell activation by specific antigen. Our model shows no alteration in immune cell profile by the Trp allele, but brings up macrophages as an important player to consider in explaining the PTPN22 Trp allele effect on autoimmune disease risk.

According to the hygiene hypothesis, the increasing incidence of autoimmune diseases in western countries may be explained by changes in early microbial exposure, leading to altered immune maturation. We followed gut microbiome development from birth until age three in 222 infants in Northern Europe, where early-onset autoimmune diseases are common in Finland and Estonia but are less prevalent in Russia. We found that Bacteroides species are lowly abundant in Russians but dominate in Finnish and Estonian infants. Therefore, their lipopolysaccharide (LPS) exposures arose primarily from Bacteroides rather than from Escherichia coli, which is a potent innate immune activator. We show that Bacteroides LPS is structurally distinct from E. coli LPS and inhibits innate immune signaling and endotoxin tolerance; furthermore, unlike LPS from E. coli, B. dorei LPS does not decrease incidence of autoimmune diabetes in non-obese diabetic mice. Early colonization by immunologically silencing microbiota may thus preclude aspects of immune education.

CD4+FOXP3+ regulatory T (Treg) cells are critical mediators of immune tolerance, and their deficiency owing to FOXP3 mutations in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) patients results in severe autoimmunity. Different FOXP3 mutations result in a wide range of disease severity, reflecting the relative importance of the affected residues in the integrity of the FOXP3 protein and its various molecular interactions. We characterized the cellular and molecular impact of the most common IPEX mutation, p.A384T, on patient-derived Treg cells. We found that the p.A384T mutation abrogated the suppressive capacity of Treg cells while preserving FOXP3's ability to repress inflammatory cytokine production. This selective functional impairment is partly due to a specific disruption of FOXP3A384T binding to the histone acetyltransferase Tat-interacting protein 60 (TIP60) (KAT5) and can be corrected using allosteric modifiers that enhance FOXP3-TIP60 interaction. These findings reveal the functional impact of TIP60 in FOXP3-driven Treg biology and provide a potential target for therapeutic manipulation of Treg activity.

Summary The type 1 diabetes-associated 16p13 locus contains the CLEC16A gene. Its preferential immune cell expression suggests involvement in autoimmunity. Given its elevated expression in dendritic and B cells – known professional antigen-presenting cells (APCs) – we hypothesize that C-type lectin domain family 16 member A (CLEC16A) may be involved in T cell co-stimulation and consequent activation and proliferation. We also sought to identify CLEC16A's subcellular localization. The effect of the CLEC16A knock-down (KD) on B cell co-stimulation and activation of T cells was tested in human lymphoblastoid cell lines (LCLs) by co-culture with CD4+ T cells. T cell activation and proliferation were determined by flow-cytometric analysis of CD69 and CD25 expression and carboxyfluorescein succinimidyl ester (CFSE) dilution, respectively. CLEC16A subcellular localization in K562 cells was examined by immunofluorescence. We show that the CLEC16A KD did not affect the tested indices of lymphoblastoid cell line (LCL) APC capacity. Additionally, the percentage of activated T cells following LCL co-culture was not affected significantly by the CLEC16A KD. T cells co-cultured with KD or control LCLs also exhibited similar cell division profiles. CLEC16A co-localized with an endoplasmic reticulum (ER) marker, suggesting that it may be an ER protein. In conclusion, CLEC16A may not be involved in T cell co-stimulation. Additional studies on CLEC16A, accounting for its ER localization, are needed to uncover its biological role.

Background: While a central role for the T helper (Th) 1/Th2 axis in food allergy has been established, the Th17 response in food-allergic humans has not been addressed. Methods: Th17 responses in 18 peanut-allergic children, who were also allergic to at least one additional food allergen, were assessed relative to 15 age-matched healthy controls. To account for the atopy background in the allergic children, 7 atopic, but not food-allergic, individuals and their age-matched controls were included in this study. PBMCs were analyzed by flow cytometry ex vivo or were stimulated in vitro with peanut allergens, gliadin, or tetanus toxoid followed by analysis of proliferation and cytokine production in antigen-responsive cells. Results: We observed a significantly lower interleukin (IL) 17 production in CD4+ T cells of food-allergic individuals ex vivo (p < 0.02). In vitro, we found that IL-17 production in CD4+ T cells in response to all antigens tested was significantly impaired in food-allergic subjects compared to healthy controls (Ara: p < 0.005; gliadin: p < 0.004; TT: p < 0.03). No significant differences were observed between atopic and nonatopic individuals with no food allergy. Conclusion: Our results thus reveal a systemic, non-allergen-specific defect in Th17 responses to antigen stimulation in food allergic individuals, suggesting a role for Th17 cells in the control of food allergy and implicating IL-17 as a potential biomarker for tolerance to food antigens.

CD4

To the Editor: The immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, a rare disorder characterized by multiorgan autoimmunity, often results in death in infancy.1 The IPEX syndrome results from mutations in the forkhead box P3 (FOXP3) gene, which encodes a transcriptional repressor considered to be the master regulator of differentiation in CD4+ regulatory T (Treg) cells.2,3 Multiple FOXP3 mutations resulting in a range of clinical outcomes have been characterized.1–3 Since neither genetic sequencing nor protein expression correlates reliably with clinical manifestations in the IPEX syndrome,4 identification of the functional consequences of individual mutations is critical to . . .

Two distinct subsets of CD4+Foxp3+ regulatory T (Treg) cells have been described based on the differential expression of Helios, a transcription factor of the Ikaros family. Efforts to understand the origin and biological roles of these Treg populations in regulating immune responses have, however, been hindered by the lack of reliable surface markers to distinguish and isolate them for subsequent functional studies. Using a single-cell cloning strategy coupled with microarray analysis of different Treg functional subsets in humans, we identify the mRNA and protein expression of TIGIT and FCRL3 as a novel surface marker combination that distinguishes Helios+FOXP3+ from Helios−FOXP3+ memory cells. Unlike conventional markers that are modulated on conventional T cells upon activation, we show that the TIGIT/FCRL3 combination allows reliable identification of Helios+ Treg cells even in highly activated conditions in vitro as well as in PBMCs of autoimmune patients. We also demonstrate that the Helios−FOXP3+ Treg subpopulation harbors a larger proportion of nonsuppressive clones compared with the Helios+FOXP3+ cell subset, which is highly enriched for suppressive clones. Moreover, we find that Helios− cells are exclusively responsible for the productions of the inflammatory cytokines IFN-γ, IL-2, and IL-17 in FOXP3+ cells ex vivo, highlighting important functional differences between Helios+ and Helios− Treg cells. Thus, we identify novel surface markers for the consistent identification and isolation of Helios+ and Helios− memory Treg cells in health and disease, and we further reveal functional differences between these two populations. These new markers should facilitate further elucidation of the functional roles of Helios-based Treg heterogeneity.

Immune dysregulation, polyendocrinopathy, enteropathy, X linked (IPEX) syndrome is a rare disorder in humans caused by germ-line mutations in the FOXP3 gene, a master transcriptional regulator for the development of CD4 regulatory T (Treg) cells. This T cell subset has global inhibitory functions that maintain immune homeostasis and mediate self-tolerance. Treg developmental deficiency or dysfunction is a hallmark of IPEX. It leads to severe, multi-organ, autoimmune phenomena including enteropathy, chronic dermatitis, endocrinopathy and other organ-specific diseases such as anaemia, thrombocytopenia, hepatitis and nephritis. In this review, the genetic, immunological and clinical characteristics of IPEX syndrome are described, and the impact of heritable mutations on the function of Treg cells highlighted.

CD4(+) regulatory T (T(reg)) cells expressing the Foxp3 transcription factor are critical for the induction and maintenance of immune homeostasis and self-tolerance in experimental rodents and humans. Foxp3(+) T(reg) cells constitute a unique CD4(+) T cell subset with potent suppressive properties, and their functional and homeostatic stability is essential to ensure dominant tolerance in a variety of inflammatory settings. Interestingly, recent evidence points to the inherent potential of T(reg) cells to adapt to environmental cues and consequently manifest functional plasticity by downregulating Foxp3 expression, and reprogramming into inflammatory T cells. The potential for suppressive Foxp3(+) T(reg) cells to undergo functional plasticity and gain inflammatory properties is of concern when one considers the ex vivo manipulation or generation of such cells for therapeutic purposes in various autoimmune or chronic inflammatory disorders. Collectively, the experimental evidence accumulated so far on the modalities of this plasticity can provide valuable cues as to strategies that can be implemented to control it, potentially allowing to facilitate the path to efficient and safe T(reg)-based therapy.

Natural FOXP3+CD4+CD25High regulatory T cells are critical in immunological self-tolerance. Their characterization in humans is hindered by the failure to discriminate these cells from activated effector T cells in inflammation. To explore the relationship between FOXP3 expression and regulatory function at the clonal level, we used a single-cell cloning strategy of CD25-expressing CD4+ T cell subsets from healthy human donors. Our approach unveils a functional heterogeneity nested within CD4+CD25HighFOXP3+ T cells, and typically not revealed by conventional bulk assays. Whereas most cells display the canonical regulatory T (Treg) cell characteristics, a significant proportion of FOXP3+ T cells is compromised in its suppressive function, despite the maintenance of other phenotypic and functional regulatory T hallmark features. In addition, these nonsuppressive FOXP3+ T cells preferentially emerge from the CD45RO+ memory pool, and arise as a consequence of a rapid downregulation of FOXP3 expression upon T cell reactivation. Surprisingly, these dysfunctional Treg cells with unstable FOXP3 expression do not manifest overt plasticity in terms of inflammatory cytokine secretion. These results open a path to an extensive study of the functional heterogeneity of CD4+CD25HighFOXP3+ Treg cells and warrant caution in the sole use of FOXP3 as a clinical marker for monitoring of immune regulation in humans.

Naturally occurring regulatory T (nT( Reg )) cells play a critical role in the establishment of immunological self-tolerance in humans. Currently, the analysis of nT( Reg ) cell function from bulk PBMC has led to discrepancies, largely due to the failure to discriminate T( Reg ) cells from other antigen-experienced CD4(+) T cells in states of inflammation. We developed a novel, multiparametric, single-cell strategy approach, which consists of isolating and expanding individual CD4(+)CD25(+) T cells into clones, in turn allowing us to discriminate bona fide T( Reg ) cells from activated, FOXP3(+) T( Eff ) cells, which frequently confound bulk CD25(High) T( Reg ) functional assays. This approach enabled us to compare their phenotype and function at the single-cell level and to uncover the functional heterogeneity that exists among the CD4(+)FOXP3(+) T( Reg ) cell population in human PBMC.

Naturally occurring regulatory T (nT( Reg )) cells play a critical role in the establishment of immunological self-tolerance in humans. Currently, the analysis of nT( Reg ) cell function from bulk PBMC has led to discrepancies, largely due to the failure to discriminate T( Reg ) cells from other antigen-experienced CD4(+) T cells in states of inflammation. We developed a novel, multiparametric, single-cell strategy approach, which consists of isolating and expanding individual CD4(+)CD25(+) T cells into clones, in turn allowing us to discriminate bona fide T( Reg ) cells from activated, FOXP3(+) T( Eff ) cells, which frequently confound bulk CD25(High) T( Reg ) functional assays. This approach enabled us to compare their phenotype and function at the single-cell level and to uncover the functional heterogeneity that exists among the CD4(+)FOXP3(+) T( Reg ) cell population in human PBMC.

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.

The immune system requires a homeostatic equilibrium between the mechanisms that assure self-tolerance, those that control the capacity to mount life-long immunity to pathogenic microbes, and those that attenuate effector mechanisms from inducing immune pathology [Sakaguchi S, Yamaguchi T, Nomura T, Ono M: Regulatory T cells and immune tolerance. Cell 2008, 133 (5):775-87; Piccirillo CA, Thornton AM: Cornerstone of peripheral tolerance: naturally occurring CD4+CD25+regulatory T cells. Trends Immunol 2004, 25:374-80]. In the past decade, an overwhelming body of literature showed that CD4+Foxp3+ regulatory T (Treg) cells are a dominant mechanism regulating the decision fate of these different immunological outcomes. Indeed, CD4+Foxp3+ Treg cells develop largely in the thymus but can be induced in the periphery throughout the course of immune responses [Sakaguchi S, Yamaguchi T, Nomura T, Ono M: Regulatory T cells and immune tolerance. Cell 2008, 133 (5):775-87; Piccirillo CA, Thornton AM: Cornerstone of peripheral tolerance: naturally occurring CD4+CD25+regulatory T cells. Trends Immunol 2004, 25:374-80]. Treg cells have emerged as a central control point in the regulation of autoimmune responses. Despite progress made in various in vitro and in vivo models, much uncertainty exists over their mechanism of action in vivo. Here, we summarize research characterizing the functional dynamics of CD4+Foxp3+ Treg cells in the control of autoimmunity in rodents and humans.

OBJECTIVE—In this study, we asked whether a possible quantitative or qualitative deficiency in naturally occurring Foxp3+CD4+ regulatory T-cells (nTreg), which display potent inhibitory effects on T-cell functions in vitro and in vivo, may predispose to the development of type 1 diabetes.RESEARCH DESIGN AND METHODS—We assessed the frequency and function of Foxp3+ nTreg cells in primary and secondary lymphoid tissues in the NOD animal model of type 1 diabetes.RESULTS—We show that the cellular frequency of Foxp3+ nTreg cells in primary and secondary lymphoid tissues is stable and does not decline relative to type 1 diabetes–resistant mice. We show that thymic and peripheral CD4+CD25+ T-cells are fully functional in vivo. We also examined the functional impact of CD4+Foxp3+ nTreg cells on the development of autoimmune diabetes, and we demonstrate that nTreg cells do not affect the initial priming or expansion of antigen-specific diabetogenic T-cells but impact their differentiation in pancreatic lymph nodes. Moreover, CD4+Foxp3+ nTreg cells also regulate later events of diabetogenesis by preferentially localizing in the pancreatic environment where they suppress the accumulation and function of effector T-cells. Finally, we show that the nTreg cell functional potency and intra-pancreatic proliferative potential declines with age, in turn augmenting diabetogenic responses and disease susceptibility.CONCLUSIONS—This study demonstrates that Foxp3-expressing nTreg cells in NOD mice regulate diabetogenesis, but temporal alterations in nTreg cell function promote immune dysregulation and the onset of spontaneous autoimmunity.

A network of regulatory T (Treg) cells exists to downregulate immune responses in various inflammatory circumstances and ultimately assure peripheral T cell tolerance. Naturally occurring CD4(+)CD25(+) Treg cell represents a major lymphocyte population engaged in the dominant control of self-reactive T responses and maintenance of tolerance within this network. CD4(+)CD25(+) Treg cells differentiate in the normal thymus as a functionally distinct subpopulation of T cells bearing a broad T cell receptor repertoire endowing these cells with the capacity to recognize a wide spectrum of self-Ag and non-self-Ag specificities. The development of CD4(+)CD25(+) Treg cells is genetically determined, influenced by Ag-specific and nonspecific signals, costimulation, and cytokines that control their activation, expansion, and suppressive activity. Functional abrogation of these cells in vivo, or genetic defects that affect their development or function, unequivocally predisposes animals and humans to the onset of autoimmune and other inflammatory diseases. Studies have shed light in our understanding of the cellular and molecular basis of CD4(+)CD25(+) Treg cell-mediated immune regulation. In this chapter, we discuss the contribution of naturally occurring CD4(+)CD25(+) Treg cells in the induction of immunologic self-tolerance in animal models and humans and attempt to provide a comprehensive overview of recent findings regarding the phenotype, functional dynamics, and effector mechanism of these cells in autoimmune diseases.

A network of regulatory T (Treg) cells exists to downregulate immune responses in various inflammatory circumstances and ultimately assure peripheral T cell tolerance. Naturally occurring CD4(+)CD25(+) Treg cell represents a major lymphocyte population engaged in the dominant control of self-reactive T responses and maintenance of tolerance within this network. CD4(+)CD25(+) Treg cells differentiate in the normal thymus as a functionally distinct subpopulation of T cells bearing a broad T cell receptor repertoire endowing these cells with the capacity to recognize a wide spectrum of self-Ag and non-self-Ag specificities. The development of CD4(+)CD25(+) Treg cells is genetically determined, influenced by Ag-specific and nonspecific signals, costimulation, and cytokines that control their activation, expansion, and suppressive activity. Functional abrogation of these cells in vivo, or genetic defects that affect their development or function, unequivocally predisposes animals and humans to the onset of autoimmune and other inflammatory diseases. Studies have shed light in our understanding of the cellular and molecular basis of CD4(+)CD25(+) Treg cell-mediated immune regulation. In this chapter, we discuss the contribution of naturally occurring CD4(+)CD25(+) Treg cells in the induction of immunologic self-tolerance in animal models and humans and attempt to provide a comprehensive overview of recent findings regarding the phenotype, functional dynamics, and effector mechanism of these cells in autoimmune diseases.