Regulatory T (T
) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity
. Conversely, T
instability, characterized by loss of ...the master transcription factor Foxp3 and acquisition of proinflammatory properties
, can promote autoimmunity and/or facilitate more effective tumour immunity
. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective T
therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse T
cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. T
-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient T
cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in T
cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for T
immunotherapies for cancer and autoimmune disease.
Regulatory T cells (Tregs) are an immunosuppressive population that are identified based on the stable expression of the fate-determining transcription factor forkhead box P3 (Foxp3). Tregs can be ...divided into distinct subsets based on whether they developed in the thymus (tTregs) or in the periphery (pTregs). Whether there are unique functional roles that distinguish pTregs and tTregs remains largely unclear. To elucidate these functions, efforts have been made to specifically identify and modify individual Treg subsets. Deletion of the conserved non-coding sequence (CNS)1 in the Foxp3 locus leads to selective impairment of pTreg generation without disrupting tTreg generation in the C57BL/6J background. Using CRISPR-Cas9 genome editing technology, we removed the Foxp3 CNS1 region in the non-obese diabetic (NOD) mouse model of spontaneous type 1 diabetes mellitus (T1D) to determine if pTregs contribute to autoimmune regulation. Deletion of CNS1 impaired in vitro induction of Foxp3 in naïve NOD CD4+ T cells, but it did not alter Tregs in most lymphoid and non-lymphoid tissues analyzed except for the large intestine lamina propria, where a small but significant decrease in RORγt+ Tregs and corresponding increase in Helios+ Tregs was observed in NOD CNS1-/- mice. CNS1 deletion also did not alter the development of T1D or glucose tolerance despite increased pancreatic insulitis in pre-diabetic female NOD CNS1-/- mice. Furthermore, the proportions of autoreactive Tregs and conventional T cells (Tconvs) within pancreatic islets were unchanged. These results suggest that pTregs dependent on the Foxp3 CNS1 region are not the dominant regulatory population controlling T1D in the NOD mouse model.
Beyond its well-described role in cellular metabolism, intracellular nicotinamide adenine dinucleotide (NAD) levels have been shown to affect the enzymatic activity of a series of NAD-dependent ...enzymes, influencing biological responses such as cell survival and inflammation. Nicotinamide phosphoribosyl transferase activity has been shown to be essential for maintaining adequate intracellular NAD levels, suggesting that this enzyme may in fact play a central role in modulating the activity of a wide range of NAD-dependent enzymes. Several recent observations concur with this hypothesis and suggest that by regulating NAD availability, Nampt is able to control both cell viability and the inflammatory response. Nampt may thus represent a novel pharmacological target with valuable anti-inflammatory and antitumor properties.
Tumor necrosis factor (TNF) synthesis is known to play a major part in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore ...evolved to dampen the production of this key proinflammatory cytokine. The high expression of nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the nicotinamide-dependent NAD biosynthetic pathway, in cells of the immune system has led us to examine the potential relationship between NAD metabolism and inflammation. We show here that intracellular NAD concentration promotes TNF synthesis by activated immune cells. Using a positive screen, we have identified Sirt6, a member of the sirtuin family, as the NAD-dependent enzyme able to regulate TNF production by acting at a post-transcriptional step. These studies reveal a previously undescribed relationship between metabolism and the inflammatory response and identify Sirt6 and the nicotinamide-dependent NAD biosynthetic pathway as novel candidates for immunointervention in an inflammatory setting.
Regulatory T cells (Tregs) are an immunosuppressive population that are identified based on the stable expression of the fate-determining transcription factor forkhead box P3 (Foxp3). Tregs can be ...divided into distinct subsets based on whether they developed in the thymus (tTregs) or in the periphery (pTregs). Whether there are unique functional roles that distinguish pTregs and tTregs remains largely unclear. To elucidate these functions, efforts have been made to specifically identify and modify individual Treg subsets. Deletion of the conserved non-coding sequence (CNS)1 in the Foxp3 locus leads to selective impairment of pTreg generation without disrupting tTreg generation in the C57BL/6J background. Using CRISPR-Cas9 genome editing technology, we removed the Foxp3 CNS1 region in the non-obese diabetic (NOD) mouse model of spontaneous type 1 diabetes mellitus (T1D) to determine if pTregs contribute to autoimmune regulation. Deletion of CNS1 impaired in vitro induction of Foxp3 in na#239;ve NOD CD4.sup.+ T cells, but it did not alter Tregs in most lymphoid and non-lymphoid tissues analyzed except for the large intestine lamina propria, where a small but significant decrease in RORgammat.sup.+ Tregs and corresponding increase in Helios.sup.+ Tregs was observed in NOD CNS1.sup.-/- mice. CNS1 deletion also did not alter the development of T1D or glucose tolerance despite increased pancreatic insulitis in pre-diabetic female NOD CNS1.sup.-/- mice. Furthermore, the proportions of autoreactive Tregs and conventional T cells (Tconvs) within pancreatic islets were unchanged. These results suggest that pTregs dependent on the Foxp3 CNS1 region are not the dominant regulatory population controlling T1D in the NOD mouse model.
Regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we ...reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8+ and CD4+ effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.
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•EZH2 expression is elevated in tumor-infiltrating (TI) Tregs•Pharmacological inhibition of EZH2 destabilizes FOXP3 expression and slows tumor growth•Genetic disruption of Ezh2 function in Tregs leads to robust anti-tumor immunity•Blockade of EZH2 in Tregs reprograms TI-Tregs to gain pro-inflammatory activity
EZH2 plays an intrinsic role in neoplastic cells as an oncogene, prompting the development of EZH2 inhibitors for cancer therapy. Wang et al. show that disrupting EZH2 function also has immunomodulatory activities and, when blocked in Tregs, promotes potent cancer immunity.
Group 2 innate lymphoid cells (ILC2s) and regulatory T (Treg) cells are systemically induced by helminth infection but also sustain metabolic homeostasis in adipose tissue and contribute to tissue ...repair during injury. Here we show that interleukin-33 (IL-33) mediates activation of ILC2s and Treg cells in resting adipose tissue, but also after helminth infection or treatment with IL-2. Unexpectedly, ILC2-intrinsic IL-33 activation was required for Treg cell accumulation in vivo and was independent of ILC2 type 2 cytokines but partially dependent on direct co-stimulatory interactions via ICOSL-ICOS. IFN-γ inhibited ILC2 activation and Treg cell accumulation by IL-33 in infected tissue, as well as adipose tissue, where repression increased with aging and high-fat diet-induced obesity. IL-33 and ILC2s are central mediators of type 2 immune responses that promote tissue and metabolic homeostasis, and IFN-γ suppresses this pathway, likely to promote inflammatory responses and divert metabolic resources necessary to protect the host.
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•IL-33-activated ILC2s are required for Treg accumulation in type 2 immune responses•IL-33 induction of Tregs depends on interactions between Treg ICOS and ILC2 ICOSL•IFN-γ represses IL-33-mediated ILC2 activation and limits Treg cell accumulation
Group 2 innate lymphoid cells (ILC2s) and regulatory T (Treg) cells are systemically induced by helminth infection but also sustain metabolic homeostasis and contribute to tissue repair. Locksley and colleagues describe how the cytokines IL-33 and IFN-γ counter-regulate ILC2 activation to control Treg cell numbers and type 2 immune responses.
Lysine acetylation/deacetylation has been recognized as an important posttranslational modification regulating numerous cellular processes. Sirtuins represent novel players in these complex ...regulatory circuits. These NAD-dependent lysine-deacetylases have attracted much interest based on their role in the regulation of lifespan in lower organisms, and their capacity to interfere with cell growth, proliferation and survival in response to stress. Their absolute requirement for NAD suggests that these enzymes may represent an important molecular link between metabolism and several human disorders such as diabetes and cancer. More recently, the identification of several transcription factors known to play a role in the immune system as sirtuin substrates has suggested that this family of enzymes may also play an important role in the regulation of inflammation, a pathological situation with clear links to metabolism and aging in humans. We review herein the possible links between nuclear sirtuins and the regulation of an immune response, and discuss the possible strategies that may lead to the development of novel therapeutic approaches to treat inflammation by targeting sirtuin activity.
Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the ...extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG
) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG
-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.
Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX ...(immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.
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•IPEX mutation M370I leads to unrestrained Th2 immune response•M370I mutation generates Th2-like Treg cells expressing GATA3 and Th2 cytokines•M370I mutation induces de novo Foxp3 binding sites in the Th2 locus•M370I Treg cells have increased chromatin interaction at the Th2 locus
Naturally occurring FOXP3 mutations provide unique opportunities to leverage clinical observations to increase our understanding of fundamental Treg cell biology. Van Gool et al. identify a mutation in the domain-swap interface of FOXP3 in IPEX patients and demonstrate a direct role for the mutant FOXP3 in reprogramming Treg cells to acquire a Th2-like phenotype, leading to Th2-mediated autoimmunity.
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