Treg-specific Deletion Research Articles

CD177 modulates the function and homeostasis of tumor-infiltrating regulatory T cells

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

The semaphorin 4A–neuropilin 1 axis alleviates kidney ischemia reperfusion injury by promoting the stability and function of regulatory T cells

Previous studies have suggested the role of CD4+Foxp3+ regulatory T cells (Tregs) in protection against kidney ischemia reperfusion injury via their immunosuppressive properties. Unfortunately, the associated mechanisms of Tregs in kidney ischemia reperfusion injury have not been fully elucidated. Semaphorin 4A (Sema4A) is essential for maintaining the immunosuppressive capacity of Tregs in tumors. However, whether Sema4A can alleviate kidney ischemia reperfusion injury through Tregs has not yet been demonstrated. Here, we investigated the effect and mechanism of Sema4A on the development of kidney ischemia reperfusion injury. Administration of recombinant human Sema4A-Fc chimera protein prior to ischemia reperfusion injury promoted the expansion and function of Tregs and decreased the accumulation of neutrophils and proinflammatory macrophages thereby attenuating functional and histological injury of the injured kidneys. Depletion of Tregs abrogated the protective effect of Sema4A on kidney ischemia reperfusion injury, suggesting Tregs as the main target cell type for Sema4A in the development of this injury. Mechanistically, Sema4A bound to neuropilin 1 (Nrp1), a cell surface receptor for Sema4A and other ligands and a key regulator of Tregs, which then promoted recruitment of phosphatase and tensin homologue and suppressed the Akt-mTOR pathway in Foxp3Cre mice but not in Nrp1f/fFoxp3Cre mice. Consistently, Treg-specific deletion of Nrp1 blocked the effect of Sema4A on the expansion and function of Treg cells. Thus, our results demonstrate that the Sema4A-Nrp1 axis alleviates the development of ischemia reperfusion injury by promoting the stability and function of Tregs in mouse kidneys.

Leveraging the Treg-intrinsic CTLA4–PKCη signaling pathway for cancer immunotherapy

BackgroundOur previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These...

The glutathione peroxidase Gpx4 prevents lipid peroxidation and ferroptosis to sustain Treg cell activation and suppression of antitumor immunity.

T regulatory (Treg) cells are crucial to maintain immune tolerance and repress antitumor immunity, but the mechanisms governing their cellular redox homeostasis remain elusive. We report that glutathione peroxidase 4 (Gpx4) prevents Treg cells from lipid peroxidation and ferroptosis in regulating immune homeostasis and antitumor immunity. Treg-specific deletion of Gpx4 impairs immune homeostasis without substantially affecting survival of Treg cells at steady state. Loss of Gpx4 results in excessive accumulation of lipid peroxides and ferroptosis of Treg cells upon Tcell receptor (TCR)/CD28 co-stimulation. Neutralization of lipid peroxides and blockade of iron availability rescue ferroptosis of Gpx4-deficient Treg cells. Moreover, Gpx4-deficient Treg cells elevate generation of mitochondrial superoxide and production of interleukin-1β (IL-1β) that facilitates T helper 17 (TH17) responses. Furthermore, Treg-specific ablation of Gpx4 represses tumor growth and concomitantly potentiates antitumor immunity. Our studies establish a crucial role for Gpx4 in protecting activated Treg cells from lipid peroxidation and ferroptosis and offer a potential therapeutic strategy to improve cancer treatment.

Tregs facilitate obesity and insulin resistance via a Blimp-1-IL-10 axis

Abstract Adipose-resident Tregs protect against systemic inflammation and metabolic disease by limiting expansion of pro-inflammatory cells, preserving insulin sensitivity and maintaining glucose tolerance. Although their basic markers and roles have been studied, less is known about the transcriptional machinery regulating their differentiation and function. B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional regulator known to be involved in development, polarization, and maintenance of various immune cells including CD4+ T cells. Using Blimp-1 reporter mice, we discovered that Blimp-1 is constitutively expressed in a subset of visceral adipose tissue (VAT) Tregs, and that Blimp-1+ VAT Tregs are phenotypically distinct from their Blimp-1-counterparts. We also found that Treg-specific Blimp-1 deletion led to altered differentiation and function of VAT and inguinal adipose tissue Tregs. Surprisingly, during diet-induced obesity, Blimp-1 Treg deficient mice gained less weight, had reduced body fat percentage, and exhibited improved insulin sensitivity compared to wild type mice. Furthermore, this was accompanied by upregulation of thermogenic genes such as Ucp1, Prdm16 and Dio2 in inguinal adipose tissue, and increased overall fatty acid oxidation. It has previously been shown that IL-10 can induce thermogenesis. Therefore, we repeated these experiments utilizing mice with Treg-specific deletion of IL-10 and found that they phenocopied the Blimp-1 Treg deficient mice. Based on these results, we hypothesize that cross-talk between Tregs and adipocytes via a Blimp-1-IL-10 axis suppresses thermogenesis, and that absence of Blimp-1+ Tregs is metabolically protective during diet-induced obesity.

Corepressors in CD4+ T cell development and homeostasis

Abstract Corepressors NCOR1 and NCOR2/SMRT serve as scaffolds for the recruitment of histone deacetylases to transcription factor-DNA complexes. These closely related proteins are functionally non-redundant. Though they were first identified by their interactions with nuclear receptors, NCOR1/2 also interact with non-nuclear receptor transcription factors, including several involved in T cell development and polarization. Previous research identified that NCOR1 promotes survival of double positive thymocytes and protects them from negative selection. We have built upon this work by examining the effect of combined NCOR1/2 deletion in developing thymocytes. NCOR1/2 deletion using a CD4-Cre model results in an 8-fold decrease in the frequency of CD4 single positive T cells, compared to a 2-fold decrease and an insignificant decrease for NCOR1 and NCOR2 deletion alone, respectively. NCOR1/2-deficient T cells demonstrate exquisite sensitivity to TCR stimulation and polarize to Th subsets at different frequencies compared to wild-type CD4+ T cells. Despite a profound loss of CD4 single positive T cells and thymic regulatory T cells (Tregs), the frequency of both Treg progenitor populations remains unchanged. In contrast, the frequency of splenic Tregs is not reduced in NCOR1/2-deficient mice. However, there is a striking increase in activation of conventional T cells, suggesting that T cell tolerance is perturbed. Using an OX40-Cre deletion model which deletes NCOR1/2 in dendritic cells (DCs), activated T cells, and Tregs, we observe a decreased CD4+/CD8+ ratio and high incidence of ulcerative dermatitis. We are currently examining how Treg or DC specific deletion of NCOR1/2 affects T cell development, homeostasis, and tolerance.

The role of tyrosine phosphatase SHP-1 in regulatory T cell function and immune tolerance

Abstract Our immune system relies on multiple levels of controls to balance its response to danger while maintaining self-tolerance. The functional interactions between conventional T cells (Tcon) and regulatory T cells (Treg) are crucial in this process. Previous studies from our lab revealed that the tyrosine phosphatase SHP-1 plays a critical role in immune tolerance, and while its regulatory role in Tcon cells is widely studied, the role of SHP-1 in Treg is much less understood. Here, we investigated the role of SHP-1 in Treg cells by using a genetic mouse model carrying a Treg-specific deletion of SHP-1 to address two questions: (I) how SHP-1 affects the activity of Treg cells; and (II) how SHP-1-deficient Treg cells shape the overall immune response. (I) A characterization of SHP-1-deficient Treg cells demonstrated an increase in the expression of a subset of Treg signature markers compared to SHP-1-sufficient Treg cells. Upon stimulation, SHP-1 deficiency in Tregs increases IL-17 cytokine expression. Moreover, we observed that SHP-1 decreases AKT phosphorylation in Treg cells, consistent with previous studies in Tcon cells from us and others. Finally, we found that SHP-limits Treg suppressive activity in vitro. (II) Next, we asked whether SHP-1 deficiency in Treg cells affects the overall immune system and found a significant increase in CD44hi CD4+ and CD8+ T cells, indicating a secondary in vivo effect of SHP-1-deficient Treg cells on SHP-1-sufficient Tcon cells. Using murine models of autoimmune diseases, we are currently addressing the underlying mechanism of this phenotype. In summary, our studies suggest an important regulatory role of SHP-1 in Treg activity and function thereby additionally contributing to the overall immune balance.

When aging meets tregs: It’s all about ROS stress

The E3 ligase DCAF1 is a key regulator of Treg senescence and protects Tregs from ROS stress. Guo, Z, Wang, G, Wu, B, et al. DCAF1 regulates Treg senescence via the ROS axis during immunological aging. J Clin Invest. 2020; 130: 5893- 5908. Aging affects all organs and systems, including the immune system, and a prominent feature of the aging immune system is a progressive decline in protective responses, a phenomenon called immunosenescence. At a cellular level, aging is associated with a marked expansion of effector memory T cells in the periphery, which is believed to contribute to the age-related chronic inflammatory state in the elderly (inflammaging). However, whether and how aging impacts Foxp3+ regulatory T cells (Tregs), a key cell type dedicated to tolerance and immune homeostasis, remains poorly defined. As the elderly nowadays represent the fastest growing transplant population, studies in this area will undoubtedly have significant clinical ramifications. A recent study in the Journal of Clinical Investigation by Guo et al. reported a surprising finding that the Foxp3+ Tregs are more likely to become senescent than T effector cells in aged mice: Tregs tended to more readily lose proliferative and suppressive capacities. The authors identified the E3 ligase DDB1- and CUL4-associated factor 1 (DCAF1) as a key regulator of Treg senescence and, by partnering with glutathione-S-transferase P (GSTP), plays a critical role in protecting Tregs from oxidative stress induced by reactive oxygen species (ROS). They further showed that Treg-specific deletion of DCAF1 led to accelerated aging of Tregs in young mice and that therapeutic blockade of the DCAF1/GSTP/ROS pathway rejuvenated aged Tregs and restored their suppressive activities. The authors initially took multiple approaches to compare Tregs and T effector cells in aged versus young naïve mice and found that aged Tregs proliferated poorly to T cell receptor stimulation and showed reduced suppressive activities compared with their young , counterparts. Unbiased genome-wide RNA-seq analysis revealed selective enrichment of aging-related programs as well as preferential upregulation of a senescence gene signature in aged Tregs (i.e., p16, p19, and p21). Interestingly, this senescence signature is more pronounced in Tregs than in T effector cells, regardless of age. They provide further evidence that aged, compared with young, Tregs showed normal expression of Treg signature genes (Foxp3, Il2ra, and Tnfrsf18) but a clear tendency of enhanced expression of proinflammatory cytokines. In an irradiation-induced aging model, young, but not aged, Tregs effectively prevented the phenotype of inflammaging. Clearly, these data support the notion that Tregs can readily become senescent and contribute to the inflammaging phenotype in aged mice. Guided by their RNA-seq data, the authors honed their efforts on DCAF1 and, by creating a set of genetically modified animal models, they demonstrated that deletion of Dcaf1 in Tregs produced an autoimmune phenotype in young mice, in which the Tregs resembled the phenotype of aged Tregs, i.e., they proliferate poorly, show deteriorated suppressive functions, and die prematurely. They found that in Dcaf1-deleted Tregs, there was marked ROS accumulation and Erk activation. Mechanistically, they showed that DCAF1 binds GSTP to form a complex that is required for intracellular detoxification of ROS by conjugating glutathione to hydrophobic and electrophilic substances (e.g., ROS). Thus, reduced expression of DCAF1 or its genetic deletion results in the accumulation of ROS in Tregs, which is known to activate the Erk pathway. Erk activation then leads to the expression of the senescence gene signature in Tregs and consequently Treg senescence and Treg dysfunction. This notion is further strengthened by the finding that interfering with the DCAF1/GSTP/ROS axis by genetic and pharmacological means rejuvenated aged Tregs and restored their suppressive functions both in vitro and in vivo. Finally, the authors extended their findings to human Tregs in the elderly, showing that similar mechanisms are also operative in the control of human Treg senescence. This new discovery, combined with the demonstration that the senescent Treg phenotype is reversible and can be therapeutically targeted, should both motivate and direct future inquiries and translational efforts. Xian C. Li, MD, PhD, is professor and director at the Immunobiology and Transplant Science Center and Department of Surgery at Houston Methodist Hospital in Texas.

In situ conversion of defective Treg into SuperTreg cells to treat advanced IPEX-like disorders in mice

Mutations disrupting regulatory T (Treg) cell function can cause IPEX and IPEX-related disorders, but whether established disease can be reversed by correcting these mutations is unclear. Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg cell activation and causes fatal autoimmunity in mice. Here, we show with a reversible knockout model that re-expression of Brg1, in conjunction with the severe endogenous proinflammatory environment, can convert defective Treg cells into powerful, super-activated Treg cells (SuperTreg cells) that can resolve advanced autoimmunity, with Brg1 re-expression in a minor fraction of Treg cells sufficient for the resolution in some cases. SuperTreg cells have enhanced trafficking and regulatory capabilities, but become deactivated as the inflammation subsides, thus avoiding excessive immune suppression. We propose a simple, robust yet safe gene-editing-based therapy for IPEX and IPEX-related disorders that exploits the defective Treg cells and the inflammatory environment pre-existing in the patients.

Rational targeting of Cdc42 GTPase destabilizes Treg to promote anti-tumor T cell immunity

Abstract Targeting of regulatory T cells (Treg) is a long-sought approach to overcome tumor immune evasion. However, systemic depletion of Treg likely causes autoimmune diseases. Indeed, we have recently reported that Treg-specific homozygous deletion of Cdc42 GTPase caused a drastic reduction in Treg numbers and systemic inflammatory diseases. Here we show that heterozygous deletion of Cdc42 (Cdc42+/−) did not affect Treg homeostasis but destabilized them, leading to effector T cell reprogramming in these Treg and impaired function in suppressing effector T cells. Accordingly, Cdc42+/− mice had increased effector T cells. Intriguingly, Cdc42+/− mice showed a remarkable suppression of growth of mouse colon and pancreatic tumors, with no noticeable autoimmunity. Mechanistically, Cdc42+/− Treg exhibited a significantly increased expression of carbonic anhydrase 1 (CA1), reversal of which by CA1 shRNA or a CA inhibitor restored Treg stability and tumor growth. Pharmacological inactivation of Cdc42 by a chemical inhibitor, CASIN, destabilized wild type (WT) Treg in mice and triggered anti-tumor T cell immunity, with no detectable autoimmune responses. CASIN also destabilized WT Treg in vitro and adoptive transfer of CASIN-treated Treg with congenic effector T cells into Rag1−/− mice suppressed tumor growth, demonstrating that CASIN-induced Treg instability caused tumor suppression. Combined CASIN and Anti-PD-1 antibody led to an additive suppression of mouse tumors as well as human tumors in human immune cell-reconstituted NSG-SGM3 mice. Collectively, our data suggest that rational targeting of Cdc42-regulated Treg stability holds promise in counteracting tumor immune evasion.

Protein Kinase C-η Deficiency Does Not Impair Antiviral Immunity and CD8+ T Cell Activation.

We reported that protein kinase C-η (PKCη) forms a novel (to our knowledge) signaling complex with the checkpoint inhibitory protein CTLA-4 in regulatory T cells (Tregs). This complex is required for the contact-dependent suppressive activity of Tregs, including suppression of antitumor immunity. However, the importance of PKCη in protective immunity mediated by T effector cells remains unclear. We used mice with germline or conditional Treg-specific deletion of Prkch, the PKCη-encoding gene, to explore CD8+ T cell-dependent antiviral immunity using the lymphocytic choriomeningitis virus Armstrong strain acute infection model as well as the in vitro activation of murine or human CD8+ T cells. Five days following infection, germline Prkch -/- mice displayed enhanced viral clearance compared with control mice. Similarly, Prkch Treg-specific conditional knockout mice also showed improved viral clearance and displayed enhanced expression of granzyme B and IFN-γ by both virus-specific and total CD8+ T cells, demonstrating that enhanced viral clearance in germline Prkch -/- mice is caused by PKCη deficiency in Tregs and the resulting functional defect of Prkch -/- Tregs. In addition, purified Prkch -/- mouse CD8+ T cells as well as PRKCH knockdown human CD8+ T cells displayed intact, or even enhanced, T cell activation in vitro as measured by proliferation and expression of granzyme B and IFN-γ. Thus, global PKCη deletion does not impair overall CD8+ T cell-mediated immunity, including antiviral immunity, implying that selective pharmacological PKCη inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor-specific and, likely, virus-specific immunity.

Blimp-1 in adipose resident Tregs controls adipocyte beiging and obesity

Abstract Visceral adipose tissue regulatory T cells (VAT Tregs) protect against systemic inflammation and metabolic disease by limiting expansion of pro-inflammatory Th1 cells and M1 macrophages, and by preserving insulin sensitivity and glucose tolerance. Although their basic markers and roles have been studied, less is known about the transcriptional machinery regulating their differentiation and function. B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional regulator known to be involved in the development, polarization, and maintenance of various immune cells including CD4+ T cells. Using Blimp-1 reporter mice, we discovered that Blimp-1 is constitutively expressed in a subset of VAT Tregs compared to lymphoid Tregs, and that Blimp-1+ VAT Tregs are phenotypically distinct from their Blimp-1− counterparts. Blimp-1 is not required for VAT Treg development, however Treg-specific deletion of Blimp-1 led to unique changes in VAT Treg markers in lean versus obese adipose tissue. In addition, Blimp-1 knockout mice fed high fat diet had fewer adipose-resident NK cells and increased CD8 T cells. Surprisingly, loss of Blimp-1+ Tregs led to less adipose tissue IL-10, increased expression of thermogenic genes, reduced body fat, decreased weight, and improved insulin sensitivity. Based on these data, we hypothesize that Blimp-1+ Treg dependent IL-10 production suppresses adipocyte beiging, and that loss of these cells results in increased thermogenesis, greater weight loss and improved insulin sensitivity in obese mice.

IL-33 Signaling Alters Regulatory T Cell Diversity in Support of Tumor Development

Regulatory Tcells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single-cell RNA sequencing (RNA-seq) to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, whereas a specialized effector phenotype characterized by enhanced expression of the interleukin-33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ Tcells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer,highlighting potential paths for therapeutic intervention.

The Transcriptional Regulator Id2 Is Critical for Adipose-Resident Regulatory T Cell Differentiation, Survival, and Function.

Adipose regulatory T cells (aTregs) have emerged as critical cells for the control of local and systemic inflammation. In this study, we show a distinctive role for the transcriptional regulator Id2 in the differentiation, survival, and function of aTregs in mice. Id2 was highly expressed in aTregs compared with high Id3 expression in lymphoid regulatory T cells (Tregs). Treg-specific deletion of Id2 resulted in a substantial decrease in aTregs, whereas Tregs in the spleen and lymph nodes were unaffected. Additionally, loss of Id2 resulted in decreased expression of aTreg-associated markers, including ST2, CCR2, KLRG1, and GATA3. Gene expression analysis revealed that Id2 expression was essential for the survival of aTregs, and loss of Id2 increased cell death in aTregs due to increased Fas expression. Id2-mediated aTreg depletion resulted in increased systemic inflammation, increased inflammatory macrophages and CD8+ effector T cells, and loss of glucose tolerance under standard diet conditions. Thus, we reveal an unexpected and novel function for Id2 in mediating differentiation, survival, and function of aTregs that when lost result in increased metabolic perturbation.

Teamwork by IL-10+ and IL-35+ Tregs

Distinct populations of IL-10– and IL-35–producing Treg cells in the tumor microenvironment cooperatively promote upregulation of BLIMP1 and exhaustion of tumor-infiltrating conventional T cells. Distinct populations of IL-10– and IL-35–producing Treg cells in the tumor microenvironment cooperatively promote upregulation of BLIMP1 and exhaustion of tumor-infiltrating conventional T cells. CITATION Sawant DV, Yano H, Chikina M, et al. Adaptive plasticity of IL-10+ and IL-35+ Treg cells cooperatively promotes tumor T cell exhaustion. Nat Immunol. 2019;20(6):724-735. FoxP3+ regulatory T cells (Tregs) are key mediators of self-tolerance and can therapeutically induce tolerance to transplanted organs. Tregs utilize multiple mechanisms to suppress activity of conventional T cells (Tconvs), including metabolic disruption of target cells, surface expression of inhibitory molecules, and secretion of immunosuppressive cytokines, such as interleukin (IL)-10, transforming growth factor (TGF)-β and IL-35. By suppressing self- or donor-reactive Tconvs, Tregs prevent pathological immune responses. However, these suppressive functions become harmful when Tregs inhibit antitumor immunity. Broadly disrupting Treg function in cancer patients to unleash antitumor immunity carries a risk of severe autoimmune complications. Thus, recent work has sought to identify and disrupt suppressive mechanisms employed by Tregs specifically within the tumor microenvironment (TME). Indeed, Tregs have been shown to use specific suppressive mechanisms in a tissue- and context-dependent manner, and, within a particular tissue, Tregs have heterogeneous functional profiles. Sawant and colleagues reveal that within the TME, IL-10 and IL-35 production are segregated into distinct Treg subtypes and the production of both of these cytokines by Tregs is required to maximally promote BLIMP1 expression and exhaustion in tumor-infiltrating Tconvs. Using mice engineered to indicate the expression of FoxP3, IL-10 and a subunit of IL-35 (Ebi3), the authors determined that distinct populations of FoxP3+ cells produced IL-10 and IL-35, with very few cells producing both cytokines simultaneously. This was evident in Tregs isolated from mouse lymph nodes, tumors and lungs as well as human peripheral blood and tumors. While both IL-10– and IL-35–producing Tregs were enriched in the TME compared with tumor-draining lymph nodes, IL-35–producing T cells were the most enriched, suggesting that targeting IL-35–producing Tregs may specifically disrupt immune evasion by the tumor without promoting severe autoimmunity. Both subsets exhibited the ability to interconvert after stimulation in vitro, although more work is needed to determine whether they exhibit plasticity in vivo and which signals direct cells to develop into either subset. To determine the functional role of each Treg subset, the authors used mice with conditional deletion of IL-10 or Ebi3 in FoxP3+ cells. Treg-specific deletion of either cytokine resulted in similarly reduced tumor growth and a decrease in inhibitory receptor (PD-1, TIM3, LAG3, TIGIT and 2B4) expression in tumor-infiltrating Tconvs. Interestingly, the combined conditional deletion of both cytokines did not further reduce tumor growth, indicating that Treg-derived IL-10 and IL-35 play partially overlapping noncompensatory roles in promoting tumor growth. As for cytokine-specific functions, IL-35 better promoted inhibitory receptor expression and reduced differentiation into central memory cells among tumor-infiltrating CD4+ and CD8+ Tconvs, while only IL-10 modestly suppressed effector cytokine production. Both IL-10 and IL-35 contributed to an exhausted transcriptional signature in tumor-infiltrating CD8+ T cells, including upregulation of Prdm1 (BLIMP1) expression in PD1hi TIM3+ tumor-infiltrating cells. The authors found that mediators of IL-35 signaling (STAT1 and STAT4), and to a lesser extent IL-10 signaling (STAT3), exhibited greater binding to the Prdm1 locus in CD8+ T cells after in vitro stimulation, suggesting that these cytokines promote Tconv exhaustion by cooperatively promoting Prdm1 expression. Prdm1 did appear to play a critical role in Tconv exhaustion, because deletion of one Prdm1 allele in CD8+ T cells was associated with reduced expression of inhibitory receptors in tumor-infiltrating cells as well as slower tumor growth. Whether modulation of BLIMP1 expression is the main mechanism of IL-10 and IL-35 promotion of tumor growth and immune evasion remains to be determined. In transplantation, induction of allospecific Tregs and dysfunction of allospecific Tconvs have been associated with a state of donor-specific tolerance. Adoptive transfer of Tregs is under investigation for use as an immunosuppressive therapy in transplant recipients as well as patients with autoimmune diseases, with studies seeking to optimize the suppressive function of transferred cells. The study of Sawant et al. suggests that multiple subsets of Tregs, including both IL-10– and IL-35–producing cells, may play different roles. Likewise, strategies for allospecific Treg induction may be most beneficial if they induce multiple subsets of Tregs.

A Treg-specific deletion of Helios causes autoimmune lipodystrophy and metabolic syndrome

Abstract Regulatory T (Treg) cells suppress immune activation in a dominant manner and play a critical role in the maintenance of self-tolerance. A subpopulation (60–75%) of Foxp3+T regulatory (Treg) cells express the transcription factor Helios. To examine the function of Helios in Treg cells, we have generated Treg-specific Helios deficient mice (cKO, Heliosflxflx Foxp3cre). Although both Treg development in the cKO mice and their in vitro suppressor function are normal, the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation with a Th1 phenotype, hypergammaglobulinemia, and enhanced germinal center formation. Initially, we observed significant lymphocytic infiltrates only in the salivary gland and not in any other organs typically affected by Treg dysregulation. Strikingly, the mice developed lipodystrophy, hepatic steotosis and insulin resistance. Further analysis revealed a significant lymphocytic infiltrate in both the inguinal and perigonadal adipose tissue, indicating autoimmune mediated destruction of the white adipose tissue (WAT). We have further shown that the lymphocytic infiltrate specific to WAT can be transferred from mice with lipodystrophy to immunodeficient mice, confirming the autoimmune nature of the lipodystrophy. Thus, Helios deficiency in Treg disrupts immune homeostasis in the adipose tissue, leading to the destruction of WAT and redirection of lipids to the liver, and ultimately causes metabolic dysfunction. Supported by the Intramural Research Program of the NIAID, NIH.

The role of Blimp-1 in adipose Treg differentiation and effector function

Abstract Visceral adipose tissue regulatory T cells (VAT Tregs) protect against systemic inflammation and metabolic disease by limiting expansion of pro-inflammatory Th1 cells and M1 macrophages, and by preserving insulin sensitivity and glucose tolerance. Although their basic markers and roles have been studied, less is known about the transcriptional machinery regulating their differentiation and function. B lymphocyte-induced maturation protein-1 (BLIMP-1) is a transcriptional regulator known to be involved in the development, polarization, and maintenance of various immune cells including CD4+ T cells. Using BLIMP-1 reporter mice, we have discovered that BLIMP-1 is constitutively expressed in a subset of VAT Tregs compared to lymphoid Tregs, and that BLIMP-1+ VAT Tregs are phenotypically distinct from their BLIMP-1-counterparts. Blimp-1 is not required for VAT Treg development, however Treg-specific deletion of BLIMP-1 led to unique changes in VAT Treg markers in lean versus obese adipose tissue. Based on these preliminary data, our project aims to elucidate the role of BLIMP-1 in shaping the VAT Treg compartment and to explore a possible role for BLIMP-1+ VAT Tregs in metabolic co-morbidities such as obesity and Type II diabetes.

Regulatory T cells sense effector T-cell activation through synchronized JunB expression.

To maintain immune tolerance, effector T-cell (Teff) responses must be checked by the regulatory T cells (Tregs) in time. It remains incompletely understood how Tregs sense real-time Teff activation. Here, we report that the AP-1 transcription factor JunB, which is induced in Teffs upon T-cell receptor (TCR) activation, is also increased in Tregs by TCR stimuli. Treg-specific deletion of Junb impairs Treg identity, causes uncontrolled inflammatory cytokine production by Teffs and leads to the T-box transcription factor T-bet-dependent spontaneous inflammation. Furthermore, JunB deficiency in Tregs unleashes antitumor Teff responses in a mouse model of melanoma. We conclude that JunB alarms Tregs of the emerging Teff activation and synchronizes immune regulation with the immune reaction in autoimmunity and cancer.

Rbpj expression in regulatory T cells is critical for restraining TH2 responses

The transcriptional regulator Rbpj is involved in T-helper (TH) subset polarization, but its function in Treg cells remains unclear. Here we show that Treg-specific Rbpj deletion leads to splenomegaly and lymphadenopathy despite increased numbers of Treg cells with a polyclonal TCR repertoire. A specific defect of Rbpj-deficient Treg cells in controlling TH2 polarization and B cell responses is observed, leading to the spontaneous formation of germinal centers and a TH2-associated immunoglobulin class switch. The observed phenotype is environment-dependent and can be induced by infection with parasitic nematodes. Rbpj-deficient Treg cells adopt open chromatin landscapes and gene expression profiles reminiscent of tissue-derived TH2-polarized Treg cells, with a prevailing signature of the transcription factor Gata-3. Taken together, our study suggests that Treg cells require Rbpj to specifically restrain TH2 responses, including their own excessive TH2-like differentiation potential.

MicroRNA-142-mediated repression of phosphodiesterase 3B critically regulates peripheral immune tolerance.

Tregs play a fundamental role in immune tolerance via control of self-reactive effector T cells (Teffs). This function is dependent on maintenance of a high intracellular cAMP concentration. A number of microRNAs are implicated in the maintenance of Tregs. In this study, we demonstrate that peripheral immune tolerance is critically dependent on posttranscriptional repression of the cAMP-hydrolyzing enzyme phosphodiesterase-3b (Pde3b) by microRNA-142-5p (miR-142-5p). In this manner, miR-142-5p acts as an immunometabolic regulator of intracellular cAMP, controlling Treg suppressive function. Mir142 was associated with a super enhancer bound by the Treg lineage–determining transcription factor forkhead box P3 (FOXP3), and Treg-specific deletion of miR-142 in mice (TregΔ142) resulted in spontaneous, lethal, multisystem autoimmunity, despite preserved numbers of phenotypically normal Tregs. Pharmacological inhibition and genetic ablation of PDE3B prevented autoimmune disease and reversed the impaired suppressive function of Tregs in TregΔ142 animals. These findings reveal a critical molecular switch, specifying Treg function through the modulation of a highly conserved, cell-intrinsic metabolic pathway. Modulation of this pathway has direct relevance to the pathogenesis and treatment of autoimmunity and cancer.