Tregs expressing forkhead box P3 (FOXP3) play crucial roles in maintaining immune tolerance and tissue integrity. EZH2, a histone H3 lysine 27 (H3K27) methyltransferase, is known as a key regulator of Treg identity and suppressive function upon activation. Here, we demonstrate that the H3K27 lysine demethylase KDM6B, which catalyzes the opposing reaction to EZH2, is also required for Treg identity and function after activation. Treg-specific deletion of Kdm6b impaired tissue Treg fate and function. KDM6B was upregulated after T cell antigen receptor signaling in Tregs and contributed to the regulation of Treg-associated gene expression through both direct and indirect mechanisms. A subset of Treg functional genes were direct targets of KDM6B and were co-occupied by FOXP3 at cis -regulatory regions, where KDM6B recruitment limited H3K27me3 accumulation. More broadly, KDM6B-dependent H3K27 demethylation facilitated Treg gene expression programs that supported tissue Treg homeostasis.

Regulatory T cells (Tregs) play a crucial role in mediating recovery from acute lung injury (ALI). However, the complex roles of functionally heterogeneous Treg subsets in the lung during the resolution of acute inflammation remain unclear. To investigate the role of peripherally induced Tregs, we utilized mice lacking conserved noncoding sequence 1 (CNS1) of the Foxp3 locus, a genetic deletion that impairs peripheral Treg induction. We found that CNS1-deficient mice exhibit greater mortality and delayed resolution during ALI. Tregs induced via CNS1 modulated antiviral and proinflammatory immune responses in the lung during influenza. Mechanistically, single-cell RNA sequencing reveals that CNS1-deficient Tregs fail to fully engage the Treg transcriptional program that supports optimal suppressive and reparative function in the lung. Our findings highlight a critical role for CNS1-dependent peripherally induced Tregs in determining ALI severity, providing insight into how distinct Treg subpopulations may be therapeutically harnessed to mitigate tissue damage during respiratory disease.

Chronic graft-versus-host disease (cGVHD) is the leading cause of morbidity and non-relapse associated mortality following allogeneic hematopoietic cell transplantation (aHSCT). Treating steroid resistant/refractory cGVHD remains challenging. Epigenetic regulators can have global transcriptional effects that control donor T-cell responses. We previously showed that inhibiting histone lysine motifs by chromatin-modifying enzymes can ameliorate murine cGVHD. Targeting donor T-cell DNA methyltransferases reduce acute GVHD. Here, we sought to investigate the DNA demethylase Tet (ten-eleven translocase) methylcytosine dioxygenases 2 (Tet2) and Tet3 in T follicular helper cell (TFH) dependent cGVHD. In a clinically relevant model of cGVHD that recapitulates pulmonary fibrosis from bronchiolitis obliterans, recipients of Tet2 deleted donor T-cells did not have improved pulmonary function tests in contrast to the markedly improved pulmonary function in Tet3 deleted donor T-cells. Tet3 deleted donor T-cells did not impair TFH-dependent germinal center (GC) formation. Unexpectedly, TET3 deficiency resulted in elevated GATA3 expression in and IL-4 production by TFH cells. TET3 deficient TFH cells supported GC B-cell immunoglobulin (Ig) class switching to nonpathogenic IgG1 but not pathogenic IgG2c allowing mice to escape cGVHD pulmonary fibrosis. Elevated GATA3 expression and disruption of IgG2c class switching was recapitulated in an in-vitro human GC culture system. These studies provide new insights into the function of Tet3 in TFH driven Ig class switching and suggest a new approach to mitigate cGVHD.

Ten Eleven Translocation (TET) proteins can oxidize 5-methylcytosine to generate in sequential steps oxidized forms of cytosine: 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Through their catalytic activity TET proteins promote active DNA demethylation. There are three TET proteins: TET1, TET2 and TET3. In T cells, TET2 and TET3 are more highly expressed. In the past years we have extensively analyzed the impact of TET proteins and 5-hydroxymethylcytosine in T cell development. In this report, we focus on the impact of TET proteins in the TCR alpha (α) and beta (β) repertoires in thymic CD4 single positive cells and upon migration in the periphery. Our data reveal that both wild type and Tet2/3 DKO CD4 cells in the thymus and the spleen are polyclonal. Then, we focus on Tet2/3 DKO CD4 cells that are serially transplanted in recipient mice. Our TCR sequencing data reveals that expanded Tet2/3 DKO CD4 cells are less diverse and oligoclonal. Overall, this report serves as a resource of TCRα and TCRβ repertoire in both wild type and Tet2/3 DKO murine conventional CD4 T cells and provides insights on how expanded Tet2/3 DKO CD4 cells opt for specific TCRα and β repertoires.

Ten eleven translocation (TET) proteins are tumor suppressors that through their catalytic activity oxidize 5-methylcytosine to 5-hydroxymethylcytosine, to promote DNA demethylation and to regulate gene expression. Notably, TET2 is one of the most frequently mutated genes in hematological malignancies, including T cell lymphomas. However, murine models with deletion of TET2 do not exhibit T cell expansion, presumably due to redundancy with other members of the TET family of proteins. In order to gain insight on the TET mediated molecular events that safeguard T cells from aberrant proliferation we performed serial adoptive transfers of murine CD4 T cells that lack concomitantly TET2 and TET3 to fully immunocompetent congenic mice. Here we show a progressive acquisition of malignant traits upon loss of TET2 and TET3 that is characterized by loss of genomic integrity, acquisition of aneuploidy and upregulation of the protooncogene Myc.

DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As DROSHA is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let-7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression.

Ten-eleven translocation (TET) proteins are DNA dioxygenases that mediate active DNA demethylation. TET3 is the most highly expressed TET protein in thymic developing T cells. TET3, either independently or in cooperation with TET1 or TET2, has been implicated in T cell lineage specification by regulating DNA demethylation. However, TET-deficient mice exhibit complex phenotypes, suggesting that TET3 exerts multifaceted roles, potentially by interacting with other proteins. We performed liquid chromatography with tandem mass spectrometry in primary developing T cells to identify TET3 interacting partners in endogenous, in vivo conditions. We discover TET3 interacting partners. Our data establish that TET3 participates in a plethora of fundamental biological processes, such as transcriptional regulation, RNA polymerase elongation, splicing, DNA repair, and DNA replication. This resource brings in the spotlight emerging functions of TET3 and sets the stage for systematic studies to dissect the precise mechanistic contributions of TET3 in shaping T cell biology.

Human natural killer T (NKT) cells have been proposed as a promising cell platform for chimeric antigen receptor (CAR) therapy in solid tumors. Here we generated murine CAR-NKT cells and compared them with CAR-T cells in immune-competent mice. Both CAR-NKT cells and CAR-T cells showed similar antitumor effects in vitro, but CAR-NKT cells showed superior antitumor activity in vivo via CD1d-dependent immune responses in the tumor microenvironment. Specifically, we show that CAR-NKT cells eliminate CD1d-expressing M2-like macrophages. In addition, CAR-NKT cells promote epitope spreading and activation of endogenous T cell responses against tumor-associated neoantigens. Finally, we observed that CAR-NKT cells can co-express PD1 and TIM3 and show an exhaustion phenotype in a model of high tumor burden. PD1 blockade as well as vaccination augmented the antitumor activity of CAR-NKT cells. In summary, our results demonstrate the multimodal function of CAR-NKT cells in solid tumors, further supporting the rationale for developing CAR-NKT therapies in the clinic. Dotti and colleagues show that chimeric antigen receptor (CAR) natural killer T cells have superior antitumor activity compared with CAR-T cells, mediated through the elimination of CD1d-expressing tumor-associated macrophages, activation of dendritic cells and promotion of endogenous T cell responses.

This article discusses methods to assess invariant natural killer T (iNKT) cell subsets isolated from the thymus, as well as the spleen, the liver, and the lung. iNKT cells can be subdivided in distinct, functional subsets based on the transcription factors they express and the cytokines they produce to regulate the immune response. Basic Protocol 1 focuses on characterizing murine iNKT subsets ex vivo by flow cytometry by evaluating the expression of lineage-specifying transcription factors such as PLZF and ROR gamma t. The Alternate Protocol describes a detailed approach to define subsets based on expression of surface markers. This approach can be very useful for maintaining the subsets alive, without fixing them, in order to isolate them for downstream molecular assays such as DNA/RNA isolation, genome-wide analysis to assess gene expression (such as RNA-seq), assessment of chromatin accessibility (for instance, by ATAC-seq), and assessment of DNA methylation by whole-genome bisulfite sequencing. Basic Protocol 2 describes the functional characterization of iNKT cells, which are activated in vitro with PMA and ionomycin for a short period of time and subsequently stained and characterized for production of cytokines, such as IFN gamma and IL-4, by flow cytometry. Basic Protocol 3 describes the process of activating iNKT cells in vivo using alpha-galactosyl-ceramide, a lipid that can be recognized specifically by iNKT cells, allowing assessment of their functionality in vivo. Cells are then isolated and directly stained for cytokine secretion. (c) 2023 Wiley Periodicals LLC.Basic Protocol 1: Identifying iNKT cell subsets based on transcription factor expression by flow cytometryAlternate Protocol: Identifying iNKT cell subsets based on surface marker expression by flow cytometryBasic Protocol 2: iNKT cell functional characterization based on in vitro activation and assessment of cytokine secretionBasic Protocol 3: iNKT cell in vivo activation and assessment of cytokine secretion by flow cytometry

Ten-eleven translocation (TET) proteins are dioxygenases that oxidize 5-methylcytosine to form 5-hydroxymethylcytosine and downstream oxidized modified cytosines. In the past decade, intensive research established that TET-mediated DNA demethylation is critical for immune cell development and function. In this study, we discuss major advances regarding the role of TET proteins in regulating gene expression in the context of T cell lineage specification, function, and proliferation. Then, we focus on open questions in the field. We discuss recent findings regarding the diverse roles of TET proteins in other systems, and we ask how these findings might relate to T cell biology. Finally, we ask how this tremendous progress on understanding the multifaceted roles of TET proteins in shaping T cell identity and function can be translated to improve outcomes of human disease, such as hematological malignancies and immune response to cancer.

Here, we describe steps to isolate mature thymic T cell subsets, namely CD4 single positive (SP), CD8 SP, and invariant natural killer T (iNKT) cells starting from murine total thymocytes using fluorescence-activated cell sorting. We detail protocols to study gene expression by RNA-seq and assess binding of transcription factors across the genome using CUT & RUN. This approach deciphers the molecular principles that govern T cell lineage specification and function. This protocol works well with limited starting material.For complete details on the use and execution of this protocol, please refer to Aijoet al. (2022).1

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells.

TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. The oxidized methylcytosines (oxi-mCs) facilitate DNA demethylation and are also novel epigenetic marks. TET loss-of-function is strongly associated with cancer; TET2 loss-of-function mutations are frequently observed in hematological malignancies that are resistant to conventional therapies. Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we seek to provide a conceptual framework of the mechanisms that fine tune TET activity. Then, we specifically focus on the multifaceted roles of TET proteins in regulating gene expression in immune cell development, function, and disease.

TET proteins are DNA demethylases that can oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) and other oxidized mC bases (oxi-mCs). Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we focus on the role of TET proteins in T-cell lineage specification. We explore the multifaceted roles of TET proteins in regulating gene expression in the contexts of T-cell development, lineage specification, function, and disease. Finally, we discuss the future directions and experimental strategies required to decipher the precise mechanisms employed by TET proteins to fine-tune gene expression and safeguard cell identity.

Cancer genomes are characterized by focal increases in DNA methylation, co-occurring with widespread hypomethylation. Here, we show that TET loss of function results in a similar genomic footprint. Both 5hmC in wild-type (WT) genomes and DNA hypermethylation in TET-deficient genomes are largely confined to the active euchromatic compartment, consistent with the known functions of TET proteins in DNA demethylation and the known distribution of 5hmC at transcribed genes and active enhancers. In contrast, an unexpected DNA hypomethylation noted in multiple TET-deficient genomes is primarily observed in the heterochromatin compartment. In a mouse model of T cell lymphoma driven by TET deficiency (Tet2/3 DKO T cells), genomic analysis of malignant T cells revealed DNA hypomethylation in the heterochromatic genomic compartment, as well as reactivation of repeat elements and enrichment for single-nucleotide alterations, primarily in heterochromatic regions of the genome. Moreover, hematopoietic stem/precursor cells (HSPCs) doubly deficient for Tet2 and Dnmt3a displayed greater losses of DNA methylation than HSPCs singly deficient for Tet2 or Dnmt3a alone, potentially explaining the unexpected synergy between DNMT3A and TET2 mutations in myeloid and lymphoid malignancies. Tet1-deficient cells showed decreased localization of DNMT3A in the heterochromatin compartment compared with WT cells, pointing to a functional interaction between TET and DNMT proteins and providing a potential explanation for the hypomethylation observed in TET-deficient genomes. Our data suggest that TET loss of function may at least partially underlie the characteristic pattern of global hypomethylation coupled to regional hypermethylation observed in diverse cancer genomes, and highlight the potential contribution of heterochromatin hypomethylation to oncogenesis.

Invariant natural killer T cells (iNKTs) are distinct from conventional T cells. iNKT cells express a semi-invariant T cell receptor (TCR) that can specifically recognize lipid antigens presented by CD1d, an MHC class I-like antigen-presenting molecule. Currently, iNKT cells are distinguished in three functionally distinct subsets. Each subset is defined by lineage-specifying factors: T-bet shapes the fate of NKT1 subset that mainly secretes IFNγ, Gata3 specifies the NKT2 subset that produces robustly IL-4 whereas RORγt seals the differentiation of NKT17 subset that secretes IL-17. In the present review, the focus is placed on the regulation of NKT17 specification and their function.

During the last years, intensive research has shed light in the transcriptional networks that shape the invariant NKT (iNKT) cell lineage and guide the choices towards functionally distinct iNKT cell subsets (Constantinides and Bendelac, 2013; Engel and Kronenberg, 2014; Gapin, 2016; Kim et al., 2015). However, the epigenetic players that regulate gene expression and orchestrate the iNKT cell lineage choices remain poorly understood. Here, we summarize recent advances in our understanding of epigenetic regulation of iNKT cell development and lineage choice. Particular emphasis is placed on DNA modifications and the Ten Eleven Translocation (TET) family of DNA demethylases.

DNA methylation is established by DNA methyltransferases and is a key epigenetic mark. Ten-eleven translocation (TET) proteins are enzymes that oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidization products (oxi-mCs), which indirectly promote DNA demethylation. Here, we provide an overview of the effect of TET proteins and altered DNA modification status in T and B cell development and function. We summarize current advances in our understanding of the role of TET proteins and 5hmC in T and B cells in both physiological and pathological contexts. We describe how TET proteins and 5hmC regulate DNA modification, chromatin accessibility, gene expression, and transcriptional networks and discuss potential underlying mechanisms and open questions in the field.

Abstract TET proteins are 2-oxoglutarate- and Fe(II) dependent dioxygenases that catalyze the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products (5-formylcytosine and 5-carboxylcytosine) in DNA. They can act as mediators of “active” (replication-independent) DNA demethylation, achieved through excision of 5fC and 5caC by thymine DNA glycosylase (TDG) followed by replacement with an unmethylated cytosine by base excision repair. They are also novel epigenetic marks that are recognized specifically by readers. The role of TET proteins in developing thymocytes remains elusive. In the present study, we show that simultaneous deletion of TET2 and TET3 in mice (DKO) results in aberrant development of invariant NKT cells that are skewed towards the NKT17 lineage. We also found upregulation of proliferation controlling genes as well as genes that are normally expressed in precursor cells. This results in an unprecedented expansion of iNKT cells, leading to lethality. The expansion is driven by antigen recognition, since the disease is effectively transmitted to fully immunocompetent recipient mice only if they express CD1d, a non-classical MHC protein that presents lipid antigens to iNKT cells. Molecular analysis revealed that TET2 and TET3 drive iNKT lineage specific DNA demethylation and control gene expression via DNA demethylation and/or chromatin accessibility of key lineage specifying factors such as RORgt, Tbet and ThPOK. Collectively, TET2 and TET3 are fundamental regulators that seal iNKT lineage fate, ensure proper development and maturation and safeguard aberrant TCR mediated expansion.