Deciphering the developmental trajectory of tissue-resident Foxp3+ regulatory T cells

Foxp3+ TREG cells have been at the focus of intense investigation for their recognized roles in preventing autoimmunity, facilitating tissue recuperation following injury, and orchestrating a tolerance to innocuous non-self-antigens. To perform these critical tasks, TREG cells undergo deep epigenetic, transcriptional, and post-transcriptional changes that allow them to adapt to conditions found in tissues both at steady-state and during inflammation. The path leading TREG cells to express these tissue-specialized phenotypes begins during thymic development, and is further driven by epigenetic and transcriptional modifications following TCR engagement and polarizing signals in the periphery. However, this process is highly regulated and requires TREG cells to adopt strategies to avoid losing their regulatory program altogether. Here, we review the origins of tissue-resident TREG cells, from their thymic and peripheral development to the transcriptional regulators involved in their tissue residency program. In addition, we discuss the distinct signalling pathways that engage the inflammatory adaptation of tissue-resident TREG cells, and how they relate to their ability to recognize tissue and pathogen-derived danger signals.


Introduction
The immune system is capable of both effectively eliminating internal and external dangers and preventing exacerbated immunemediated tissue pathology.These biological properties, coined disease resistance and disease tolerance, respectively, are complementary and require a controlled balance between proinflammatory and regulatory immune responses (1).This is particularly the case in mammalian hosts, where adaptive immunity allows antigen specificity to sustain long-lasting effector and memory responses that can become a potential threat to the function and homeostasis of an affected tissue long after the elimination of the danger.Amongst the mechanisms capable of controlling inflammation-generated pathology, a lymphocyte of thymic origin, a suppressor or regulatory T cell (T REG ), first described in the late 1960s (2), was shown to be particularly adept at immune suppression.These CD4 + T cells express the Forkhead-Box P3 (Foxp3), a lineage-defining transcription factor that governs a large part of their transcriptional program through the repression of pro-inflammatory genes (e.g.Il2, Ifng) and the activation of genes essential for their suppressive functions (e.g.Il2ra (CD25), Ctla4, Lag3, Entpd1 (CD39), Nt5a (CD73), Il10, Tgfb1, Gzmb) (3,4).In addition, some key signature genes are prominently expressed by these cells when compared to conventional T cells, including Ikzf2 (Helios), Tnfrsf18 (GITR), Nrp1 (Neuropilin 1), and Itgae (CD103) (5).In their capacity, T REG cells occupy a central position in the immune response, and are required to ensure tolerance to self-antigens (6,7), innocuous allergens (8,9), and commensal microflora (10), promote tissue function and regeneration (11), and prevent and control immunopathology (12).
In a mature immune system, T REG cells isolated from tissues encompass a pool of antigen-experienced CD45RA − CD69 + CD45RO + cells that differ in developmental origin, possess unique functions, and display distinct stages of activation (13).A prominent population of T REG cells found in all organs are tissueresident T REG (TR-T REG ) cells that differ from effector memory T REG cells (emT REG ) in that they display higher levels of the alpha E integrin (CD103) (14), lose CCR7 expression, and lose the ability to re-circulate to lymphoid organs (15).Despite the lack of a consensus on the markers to distinguish TR-T REG and emT REG cells in tissues, recent studies have been able to capture the high degree of transcriptional and post-transcriptional modifications that "precursor" TR-T REG cells acquire to localize to nonlymphoid organs, survive, and adjust their specialized functions in situ amidst unfavorable inflammatory, osmotic, or metabolic conditions (16).This program involves the expression of a set of core genes that are typically upregulated, albeit at different levels, by TR-T REG isolated from distinct organs, including the expression of the IL-33 receptor ST2 (17), RORa (18,19), Icos (20, 21) and Gata3 (22)(23)(24).Amongst these differentially expressed proteins, ST2 was recently proposed to distinguish TR-T REG from emT REG (17).Moreover, while there is evidence TR-T REG cells seed nonlymphoid organs, such as the lungs, as early as 8 days of life (25,26), other TR-T REG cells, like visceral adipose tissue T REG (VAT-T REG ), accumulate progressively with age (27), suggesting a highly dynamic and developmental path that is largely organ-specific.Critically, there is novel evidence on the developmental trajectory that lead TR-T REG cells to fully establish in the tissue.For example, recent evidence highlights how the TCR repertoire is a central determinant of TR-T REG localisation (16,28).
Currently, much remains to be understood regarding the origin of TR-T REG cells.Can TR-T REG cells be generated from emT REG cells after the resolution of inflammatory events (29), or do they constitute stable and distinct populations of T REG cells?Seeing that T REG cells found in tissues can originate from the thymic selection process (thymic-derived; tT REG ) or be generated from the induction of Foxp3 in naïve CD4 + T cells in the periphery (peripherallyinduced; pT REG ), can both subsets be considered TR-T REG cells?Thus, a better understanding of the origin, function, and fate of TR-T REG cells is required before we can harness their therapeutic potential.
In this review, we describe the steps required for the generation of TR-T REG , starting from thymic selection and spanning to TCR engagement in the periphery, the switch to distinct metabolic strategies, and the modulation of Foxp3 expression that enables the adoption of key epigenetic and transcriptional changes, which, in turn, lead to the expression of a program that is highly adapted to the target tissue (Figure 1).These processes involve signaling pathways that can, when in excess, hinder, either temporarily or permanently, the stability of their core transcriptional program, revealing mechanisms by which local inflammation guides the timing and potency of immune suppression.Finally, we attempt to guide the reader through the unique signaling events that can lead tissue-resident T REG cells to control type 1, type 2, and type 3driven inflammation.

Origin of tissue-resident T REG cells
Commitment of the T REG cell lineage can occur at various stages of the T cell life cycle.During their development in the thymus, immature thymocytes are selected for the establishment of a functional TCR repertoire.Subsequently, self-reactive thymocytes are either clonally deleted or diverged into a regulatory cell fate as part of a process known as central tolerance.Despite this, a very small fraction of thymocytes escape central tolerance stochastically and must be kept in check by self-reactive thymic-derived T REG cells (tT REG ), making them critical mediators of peripheral tolerance.Importantly, the events giving rise to tT REG cells require optimal TCR signals and a unique combination of cytokines.However, the peptide pool to which thymocytes are exposed to during this selection process does not ensure complete tolerance towards innocuous non-self-antigens such as commensal bacterial peptides or allergens.
This type of peripheral tolerance often requires the in situ induction of peripheral T REG cells (pT REG ) that possess unique nonself TCR repertoires (30)(31)(32) and confer them with non-redundant roles in maintaining homeostatic conditions at barrier sites like the lung and colon.In adoptive transfer models, pT REG cells are capable of suppressing local inflammation in both the colon and the lungs (32-34), but are less efficient at suppressing systemic inflammation (31).Indeed, the distinct transcriptional profiles of tT REG and pT REG cells indicate they favour different suppressive mechanisms that vary in effectiveness in a context-dependent manner (31).Yet, despite these potential differences, attempts at identifying markers in pT REG cells that are distinct from tT REG have so far failed (35,36), rendering them mostly undistinguishable at barrier sites.While Helios and Neuropilin 1 (Nrp1) are highly expressed by tT REG cells (30,37), neither Helios (36) nor Nrp1 (38), were found to be exclusively expressed by these cells.Thus, despite their distinct origin, TCR repertoire, and functions, pT REG cells cannot be distinguished from the pool of tT REG cells in mucosal tissues, and further investigation into features that demarcate each subset is warranted.

Thymic development of T REG cells
Thymic-derived T REG cells undergo the same early core processes of thymic selection as conventional CD4 + T cells (39,40).Namely, newly seeded thymocytes undergo V(D)J recombination in the thymic cortex to generate productive TCR chains capable of self-MHC recognition.Upon successful TCR signaling, committed thymocytes migrate into the thymic medulla where they encounter medullary thymic epithelial cells (mTECs) that express promiscuous transcription factors AIRE and Fezf2, allowing them to transcribe and present tissue-restricted antigens (TRAs) to developing thymocytes (41,42).Here, thymocytes that are strongly reactive toward TRAs and other self-antigens are deleted, while weaker stimulation and the presence of certain cytokines such as TGF-b and IL-2 can skew their fate toward T REG cell differentiation (43)(44)(45)(46)(47).
Optimal TCR signaling is the predominant factor driving T REG cell lineage commitment in the thymus.TCR:peptide-MHC interaction triggers a series of phosphorylation events resulting in downstream activation of NFAT, AP-1, and NF-kB family transcription factors (48,49).Together, these events lead to different T cell lineage specification in the thymus, as well as T cell survival, expansion, and effector function in the periphery.Expression level of the orphan nuclear receptor Nur77 (Nr4a1) has been directly linked to TCR signaling strength, and its expression level is elevated in T REG cells compared to conventional T cells in a TCR-dependent manner (50).Unsurprisingly, since co-stimulatory molecules such as CD28 profoundly augment TCR signaling strength via NF-kB activation, they were found to play an essential role in tT REG cell differentiation (46,(51)(52)(53).Foxp3 transcription is intricately regulated by transcription factor complexes binding at its promoter and four conserved noncoding sequences (CNS), termed CNS0 to CNS3.Upon TCR stimulation, downstream activation of the NF-kB pathway results in the recruitment of c-Rel to the Foxp3 locus at CNS3, which acts as a Foxp3 transcriptional enhancer that is responsive to TCR signaling alone (54,55).By dissecting each CNS region through targeted mutations, Zheng and colleagues demonstrated that CNS3 is the region that acts as a pioneer element to the generation of tT REG cells, while CNS1, a region known to bind TGF-b-induced SMAD factors, and CNS2, a region targeted by CREB and STAT5 signals, were not essential to the induction of Foxp3 in tT REG precursors (55), which still require cytokine signaling to become mature and functional tT REG cells (43)(44)(45)(46)(47).The developmental trajectory of tissue-resident T REG cells involves a series of events starting from thymic selection to peripheral TCR engagement.In this figure, the trajectory of peripheral regulatory T (T REG ) cells is depicted, as currently defined by recent multi-omics approaches conducted in various lymphoid and non-lymphoid tissues.During thymic selection, precursor regulatory T cells (T REG P) expressing self-reactive T-cell receptors (TCR) give rise to a pool of naive CD45RA + CCR7 hi regulatory T cells (T REG ).Once in circulation, these T REG cells encounter their specific antigen, triggering an activation cascade that results in a metabolic shift and chromatin remodeling.Subsequently, CD45RO + CD69 hi effector regulatory T cells (eT REG ) can either stay in lymph nodes as central memory (cmT REG ) or migrate to tissues, where they become tissue-resident (TR-T REG ) or effector memory regulatory T cells (emT REG ).While thymic-derived TR-T REG cells comprise a large portion of T REG cells in tissues, T REG located in the gut, for example, include peripherally-induced regulatory T cells (pT REG ).The absence of clear markers poses a challenge in distinguishing between these two populations in situ.In addition, while TR-T REG cells isolated from various tissues typically display a conserved phenotype marked by the expression of ICOS, ST2, Helios, and GATA3, a significant portion of T REG cells in the gut exhibit a distinctive RORgT-driven phenotype.Interestingly, there is cumulating evidence that T REG cells lacking Helios expression may be more driven to express RORgT, suggesting a possible segregation between TR-T REG cells derived from the thymus or induced in the periphery.
Cytokines, particularly common g (gc) cytokines, are critical for T REG cell development.IL-2 is known to be essential for commitment to the T REG cell lineage (56,57), as well as its maintenance (58).IL-2 signaling mediates STAT5 binding to the distal enhancer CNS0 as well as the promoter of Foxp3 (56,59), and sustains the constitutive expression of Foxp3 through CNS2 binding (57, 59).Not only does STAT5 directly facilitate Foxp3 transcription, Foxp3 also binds to the IL-2 receptor alpha chain (IL2Ra) as a transcriptional activator (60).Completion of this feedforward loop via paracrine IL-2 signaling is obligatory for T REG cell development and homeostasis.Other STAT5-activating gc cytokines have also been linked to T REG cell development, albeit mostly as a compensatory mechanism for impaired IL-2 signaling (45).In addition, TGF-b has also been implicated in tTreg development.While either of its downstream transcription factors, SMAD2 or SMAD3, can directly regulate Foxp3 transcription (61, 62), deletion of the SMAD binding site in the Foxp3 locus predominantly affects the induction of pT REG, but not tT REG cells (62,63).Yet, deletion of the TGF-b receptor TbRI during thymocyte development results in severely reduced T REG cell numbers and defective T REG cell function (64).Nonetheless, a recent study might reconcile the paradoxical discoveries.SMAD3/ 4 can trigger a PKA-dependent signaling cascade that causes the cessation of TCR signaling (65).Thus, the role of TGF-b in tT REG differentiation could most likely be attributed to its effects on TCR signaling rather than direct transcriptional regulation of Foxp3.

2.2
The role for thymic selection events in the genesis of tT REG and pT REG cells.
In recent years, accumulating evidence shows that the nature of TCR signaling during thymic selection influences T REG cell response to signals long after thymus egress.Notably, TCR engagement during thymic selection is a critical step in the establishment of a CpG hypomethylation pattern that characterises the epigenetic background of tT REG cells (66).Numerous studies have identified two distinct tT REG precursor (T REG P) populations thought to develop into CD25 + Foxp3 + tT REG cells (47,(67)(68)(69).The more common CD25 -Foxp3 low and less abundant CD25 + Foxp3 -T REG P cells were shown to have distinct TCR repertoires with affinity to auto-antigens (67).In the thymus, the two T REG P populations display different cytokine and TCRsignaling requirements (47).Importantly, CD25 + T REG P-derived T REG cells are specifically capable of suppressing experimental autoimmune encephalitis (EAE), whereas Foxp3 low T REG P-derived T REG cells cannot (67), suggesting a functional bias within the T REG population.For example, murine T REG cells from the colonic lamina propria that express the same TCRa/b sequence have related transcriptional programs (70), illustrating the close relationship between TCR and the transcriptional fate of antigen-experienced memory T REG cells.
Interestingly, while the relationship between TCR specificity and the establishment of TR-T REG cells is not entirely understood, there are experimental examples that suggest the TCR repertoire generated during thymic selection is critical to the destination of both tT REG and naïve T cells.For example, T REG cells transgenic for a VAT-T REG -derived TCRa/TCRb gene arrangement will preferentially migrate to adipose tissue and differentiate into VAT T REG cells (28).Yet, while these observations suggest TR-T REG cells possess a largely self-specific TCR repertoire, earlier work in viral infection mouse models demonstrated that antigen-experienced T REG cells with predominantly non-self TCR repertoires are generated during tissue injury and activate during re-infection (13, 71), suggesting they also contribute to the TR-T REG pool.In addition, in transgenic mice possessing a fixed TCR-b sequence isolated from a Foxp3 + RORgT + colonic T REG cell, T cells upregulate Foxp3 in the mesenteric lymph node prior to expressing RORgT in the colon (72).As such, both self and non-self-reactive TCR repertoires are key drivers in the generation of TR-T REG cells.

The role of IL-2 and TGF-b
While the strength of TCR signaling acts as the predominant driving force for tT REG cell differentiation, cytokines play a more influential role in the periphery both in maintaining tT REG homeostasis and generating pT REG cells.The signals that lead to the generation of pT REG cells involve chronic suboptimal TCR signaling (73-75) and cytokines such as TGF-b and IL-2 to generate Foxp3-expressing T REG cells in vitro (76, 77) and in tissues (78-80).In addition, TGF-b has been shown to strongly promote Foxp3 induction through its downstream transcription factors (SMAD2 or SMAD3) that target CNS1 (61).Consequently, a deletion of CNS1 predominantly affects the induction of pT REG , but not tT REG cells (63).Lastly, pT REG cell induction via TGF-b can be further augmented by DC-derived retinoic acid in the lamina propria as well as short chain fatty acid metabolites of commensal bacteria (81,82), ensuring the establishment of tolerance at mucosal surfaces.While these examples of signals that promote pT REG induction are part of a complex signaling system that merits its own review, they share the common outcome of facilitating Foxp3 expression in tissue-resident T cells, and further the importance of this transcription factor in forming the regulatory program of tissueresident CCR7 low CD69 + CD45RO + T REG cells.

The epigenetic and transcriptional trajectory of T REG cells
The factors that regulate the differentiation of TR-T REG remain to be fully understood.Miragaia and colleagues demonstrated through single-cell RNA-seq analysis of lymphoid and nonlymphoid (colon and skin) T REG cells that these tissue-specific adaptations originate from events happening in their respective draining lymph node (19).By tracing TCR clonotypes from draining lymph nodes to their respective tissue, the authors were able to establish a pseudo-space relationship detailing the series of events that drive the generation of specialized T REG cells.They were able to establish that T REG cells are activated, switch to a glycolytic metabolism, and cycle rapidly prior to acquiring genes involved in migration to the tissue (19), revealing conserved stages involved in the generation of TR-T REG cells.As such, this seminal work provided confirmation that progressive transcriptional changes guide the generation of eT REG cells that become TR-T REG cells and highlighted how, despite tissue-specific differences, these cells share a series of epigenetic modifications that allow them to migrate, survive, and function at specific non-lymphoid sites.

The importance of peripheral TCR engagement in the generation of TR-T REG cells
The engagement of the TCR of naïve T REG cells is an important prerequisite for the development of tissue-specialized T REG cells (83,84), as it promotes a signaling cascade that elicits the expression of key regulatory genes leading to the suppressive activity of T REG cells (85).Additionally, TCR engagement can induce epigenetic and transcriptional changes in T REG cells, some of which are directly influenced by Foxp3, while others act independently (66).People affected by a loss-of-function mutations in STIM1 or ORAI1, proteins involved in store-operated calcium entry (SOCE), encounter a loss of peripheral tolerance despite some cases displaying normal T REG numbers in circulation (86,87).Similarly, impairing the normal Ca 2+ influx during TCR engagement by deleting proteins that form the Ca 2+ releaseactivated Ca 2+ (CRAC) channels (STIM1 and STIM2) in mice specifically prevents the differentiation of activated T REG cells into follicular and tissue-resident memory T REG cells and generates a cascade of inflammation leading to multiorgan autoimmunity (88).

Aerobic glycolysis in the activation and clonal expansion of T REG cells
Another critical factor involved in the differentiation and clonal expansion of activated T REG cells is the adoption of aerobic glycolysis.This was notably demonstrated in the skin, as aerobic glycolysis by activated T REG cells is required prior to their migration (89).This may, at first glance, seem counter-intuitive, as there is ample evidence that mature T REG cells adopt fatty-acid oxidation (FAO) as a critical metabolic strategy to survive and suppress immune responses in tissues (90).Yet, while less efficient than oxidative phosphorylation (OXPHOS), adopting aerobic glycolysis is a critical step that occurs during T cell activation by rapidly providing the needed energy for expansion and migration, all-thewhile maintaining fatty acid and amino acid reserves for cell division and protein synthesis (91).This is further evidenced by the fact that the mammalian target of rapamycin 1 (mTORC1) which is required for aerobic glycolysis, is not necessary for the thymic or peripheral development of T REG cells, but essential to the function and activation of T REG cells (92).Indeed, to avoid losing their suppressive program, T REG cells balance the intensity of the mTORC1 and mTORC2 pathways (93), a process that is critical during their differentiation.Importantly, however, increasing glycolytic metabolism in T REG cells temporarily deprives them of their suppressive capacity (90, 94), providing further evidence that the differentiation and clonal expansion of T REG cells is contained within a short window of time.As such, the maturational process leading T REG to become eT REG cells requires both TCR engagement and a shift in their metabolic strategy (Figure 1).

The role of Foxp3 in the specialization of memory T REG cells
The Foxp3-driven transcriptome of T REG cells is comprised of a T REG -specific gene signature and a gene set associated with an activation program which is shared with conventional T cells (95).A lymphoproliferative pathology had been previously observed in "Scurfy" mice where the X-linked Foxp3 gene encountered a frameshift mutation that completely disrupts the transcription of Foxp3 (96), confirming the key role of Foxp3 in establishing the suppressive program of T REG cells.Point mutations in Foxp3 that interfere with its function are the cause of a frequently fatal pediatric hereditary syndrome called immune dysregulation, polyendocrinopathy, enteropathy X-linked (IPEX) syndrome (97), featuring early onset diabetes, severe diarrhea, and eczema, which highly reflects the pathology of 'Scurfy" mice.Restoring Foxp3 transcription in mice whose T REG cells were genetically engineered to block Foxp3 expression rescues them from severe autoimmunity as it effectively reinstates their suppressive function (12).However, while Foxp3 is essential for the establishment of T REG cells, it does not determine, by itself, the entire epigenetic and transcriptional identity of mature T REG cells (5,98,99).Rather, Foxp3 ensures that inflammatory and non-inflammatory signals encountered in the periphery do not destabilise the core suppressive program of T REG cells (98,100).
Evidence for the unique roles of Foxp3 in non-lymphoid tissues comes from the observation that functional single nucleotide polymorphisms (SNPs) in the human Foxp3 gene do not generate a homogeneous pathology (97), with multiple accounts of IPEXrelated mutations having distinct functional consequences on T REG cells (101).By transposing human-isolated Foxp3 mutations in conserved murine Foxp3 motifs, Leon and colleagues confirmed that spontaneous multiorgan autoimmunity is largely attributed to mutations in the DNA-binding motifs, while mutations outside these motifs, notably in the N-terminal regions, lead to organspecific dysregulation of T REG cell function (101).In particular, a K199del mutation situated in the zing-finger (ZF) domain or mutations R51Q or C168Y in the N-terminal regions are prone to generating symptoms of enteropathy and skin disorders, while a R337Q mutation in the DNA-binding Fork-head domain can, in addition to these symptoms, lead to the development of diabetes mellitus (101).In addition, a murine model mimicking an A384 mutation in Foxp3 was shown to specifically impair T REG cell function in the periphery, directly impairing the ability of Foxp3 to recognize target genes and altering BATF expression (102), a key transcription factor required for TR-T REG generation (103).As such, the ability of Foxp3 to interact with multiple partners is required to preserve the functional integrity of T REG cells in peripheral tissues.
Although there are elements that suggest protein-protein interactions are critical to this process, we are currently limited in our understanding of how the different molecular complexes that partner with the N-terminal region of Foxp3 (104, 105), such as Tip60, Hdac7, Hdac9, Gata3, c-Rel, Foxp3, Runx1 or Eos, influence the specialization of T REG cells.This is imparted by the fact that it is particularly difficult to dissociate their functions during the early events leading to the differentiation of these cells and the events that happen later in the tissues.One such example is the interaction of Foxp3 with the chromatin remodeling transcription factors TCF1 (encoded by Tcf7) and lymphoid enhancer binding factor 1 (Lef1) of the high-mobility group (HMG) family.In mice, the combined knock-out of both Tcf7 and Lef1 (Foxp3 CRE Tcf7 fl/fl Lef1 fl/fl ) does not perturb lymphoid T REG cells but hinders the capacity of colonic T REG cells to suppress DSS-mediated colitis (106).Mechanistically, the molecular complexes TCF1 and Lef1 form with Foxp3 allow T REG cells to control inflammation by repressing genes associated with excessive cycling and cytotoxic function (GzmB, Prf1, Ifng) and promoting genes associated to a T REG suppressive program (106).Bulk RNAseq of murine mesenteric T REG cells deficient in TCF1 (Foxp3 CRE Tcf7 fl/fl ) show enhanced expression of core genes (including Il2ra, Foxp3, Tgfb1 and Lef1), and a concomitant increase in both pro-inflammatory genes (including Il6ra, Ifngr2, Stat3) and genes involved in TCR activity compared to T REG cells from control mice (107).These data suggest that TCF1 helps maintain a core T REG program and suppress the expression of pro-inflammatory genes during TCR engagement.Similarly, Lef1 is part of an activated T REG program (108), and in vitro gain-offunction experiments reveal it reinforces the expression of Foxp3 target genes (108).As such, these observations indicate that when Foxp3 is abundantly expressed, it interacts with both TFs to suppress pro-inflammatory gene expression and reinforce its own transcriptional profile (109).Yet, both murine and human activated (CD45RO + ) T REG cells display lower Tcf7 and Lef1 expression than conventional T cells (T CONV ) (110) as Foxp3 directly suppresses Tcf7 transcription and protein production, and reduces chromatin accessibility in regions targeted by TCF1 (95).As such, the highlyregulated chromatin-remodelling effect of TCF1 and Lef1 on T REG cells are likely required for their further differentiation and effector function.Furthermore, pseudo-time analysis from single cell RNAseq data of lymphoid and non-lymphoid activated T REG cells reveals Tcf7 and Lef1 to be particularly expressed by lymphoid T REG cells prior to their tissue migration (19), reinforcing the notion that TCF1 and Lef1 are involved during the early specialization events of T REG cells.For example, a T REG -specific depletion of Lef1 abolishes the generation of follicular T REG (T FR ) (107), suggesting Lef1 promotes the generation of these cells in a process similar to what is observed in follicular helper T cells (T FH ) (111).In addition, when compared to murine activated TCF1 -T REG cells, TCF1 + T REG cells display higher mRNA expression of transcription factors associated to helper T cells, including Gata3, Tbx21 and Rorc (107).Collectively, these examples highlight how changes in chromatin accessibility in T REG cells happen mostly after TCR engagement in the lymph node.Nonetheless, Lef1 and TCF1 are but a part of a wide network of known Foxp3-binding partners (104) whose role in defining the specialisation of T REG cells remain ill-defined.

Epigenetic control of T REG differentiation
To effectively reach the tissue, T REG must undergo a series of epigenetic and transcriptional changes that ensure chromatin accessibility in key genes (112).Interestingly, direct comparison between human and murine T REG cells reveal evolutionarily conserved epigenetic mechanisms involved in defining a T REG cell program (110).Histone methylation is an important component in the processes that govern DNA accessibility and, ultimately, a T REG cell transcriptional signature.Importantly, while T REG cells undergo a series of chromatin remodeling events, they actively maintain CpG motif demethylation within the intronic enhancer CNS2 of the Foxp3 locus (55,113,114).Maintaining an open chromatin structure in the CNS2 allows for the robust transcription of Foxp3 by multi-molecular complexes including Foxp3 itself, NFAT, c-Rel, STAT5, Runx1-CBFb, CREB/ATFx and Ets1 (114)(115)(116)(117)(118). Incidentally, a loss of any of these transcription factors or the methylation of CNS2 impairs the transcription of Foxp3 and, ultimately, the suppressive function of T REG cells in the periphery (114)(115)(116)(117)(118)(119), confirming that Foxp3 is critical for the stability of the transcriptional program of tissue-localised T REG cells.
Tagmentation-based whole-genome bisulfide sequencing of lymph node and tissue-isolated murine T REG reveals these cells undergo multiple rounds of DNA alterations before adopting a tissue-residency program, with up to 4000 genes involved in either gain or loss of methylation (120).The processes that govern the establishment of a T REG program by histone modifications have been elegantly reviewed by Joudi and colleagues (121).Globally, a delicate balance between DNA methyltransferases (DNMTs), teneleven translocation dioxygenases (TET), histone acetyltransferases (HATs), and histone deacetylases (HDAC) govern the stability of the T REG cell transcriptional program (119,121), but can be directly influenced by polarizing signals provided during TCR engagement.
Methylation of cytosines located in CpG-rich regions are largely governed by Dnmt1, Dnmt3a and Dnmt3b (122,123).Interestingly, the conditional deletion of Dnmt1, but not Dnmt3a, in murine T REG cells causes a loss of peripheral tolerance by 3 to 4 weeks of life, yet the cells maintain their expression of Foxp3 (124).However, these T REG cells display enhanced expression of pro-inflammatory cytokines (IFNg, IL-6, IL-12, IL-17, IL-22), chemokine receptors (CCR1, CXCR6), and transcription factors (Runx2, Stat3), highlighting the role of Dnmt1 as a non-redundant epigenetic silencer (124).During the S phase, Dnmt1 acts in partnership with the epigenetic regulator ubiquitin-like with plant homeodomain and RING finger domains 1 (Uhrf1) to govern the suppression of these gene loci (125,126), making both Dnmt1 and Uhrf1 important therapeutical targets for the control of T REG stability and function.Yet, because of the necessity of T REG cells to acquire a set of genes associated with pro-inflammatory T cells, it remains to be understood how both regulators act during T REG cell generation.For example, pharmacological inhibition of PI3K through its PIP4K-associated kinase results in a specific decrease in Uhrf1 in human T REG cells but not TCONV cells (127), suggesting that the strength of TCR signaling plays a role in the way T REG cells govern DNA accessibility of pro-inflammatory genes.In addition, signaling by TGF-b leads to the phosphorylation and subsequent sequestration of Uhrf1 outside the nucleus (128), possibly preventing its partnering with Dnmt1.
On the other hand, the modulation of histone acetylation and deacetylation on the epigenetic adaptation of T REG cells remains illdefined.Foxp3 + T REG cells have been found to express histone acetyltransferases (HAT), including p300, Tip60 and CBP, as well as most members of the histone deacetylase family (HDAC) (129).Pan-HDAC inhibitors, for example, promote the acetylation of Foxp3 and the suppressive functions of T REG cells (130), confirming the importance of regulating histone acetylation to maintain a T REG transcriptional program.Interestingly, HATs and HDACs are clearly involved in the helper differentiation of T CONV cells (131), and further investigation is required to understand how they govern the differentiation of T REG cells.

The roles of BATF and Irf4 in the generation of TR-T REG cells
During these early differentiating steps, some transcriptional regulators are found to be particularly critical for the generation of TR-T REG over other emT REG subsets.At its core, the acquisition of a tissue residency program of TR-T REG cells is closely matched to the expression of basic leucine zipper ATF-like transcription factor (BATF) and its downstream targets (16).Delacher and colleagues identified a BATF-dependent transcriptional program that drives, notably, the expression of the IL-33 receptor ST2 (120), a receptor specifically found in TR-T REG (17).A T REG -specific BATF deficiency in mice (Foxp3 CRE Batf fl/fl ; BATF -/-), results in a multiorgan autoimmune disease with death initiating at 6 weeks of age (103).BATF -/-T REG cells fail to accumulate in the lungs, colon, liver, and spleen, and display reduced chromatin accessibility to genes involved in T REG survival in tissue, including Gata3, Irf4, Ikzf4, Ets1 and Icos (103).In addition, Foxp3 CRE Batf fl/fl mice generate exT REG cells that lose T REG -associated genes (Ctla4, Tgfb1, Foxp3) and adopt inflammatory genes (Rorc, Il6ra, Stat3) (103).Specifically, ATAC-seq of murine BATF WT and BATF -/- T REG cells reveals BATF acts as a chromatin regulator, facilitating the expression of TR-T REG -associated genes, including Ctla4, Icos, Gata3, and Irf4, and preserving the demethylated state of the CNS2 region of Foxp3 (103), positioning BATF as the epigenetic guardian of T REG cells as they undergo their differentiation into specialized memory T REG cells.
Another transcription factor (TF) observed to be highly expressed by T REG cells following TCR engagement is the interferon regulatory factor 4 (Irf4) (132).Foxp3 can directly promote the transcription of Irf4 (133) and the BATF-JUN complex (134).In turn, Irf4 collaborates with BATF to further promote T REG activation, proliferation, and transcriptional differentiation (135).Ding and colleagues demonstrated that upon TCR engagement, T REG cells express the SUMO-conjugating enzyme UBC9 to specifically stabilise Irf4 function (136).While not affecting thymic development of murine T REG cells, a T REGspecific deletion of UBC9 causes an early and fatal inflammatory disorder at 3 weeks of age (136), mimicking the dynamics observed in scurfy mice (96).These T REG cells show defects in TCR activation, migration, and peripheral accumulation (136).However, we do not observe such dramatic outcomes when knocking out Irf4 in murine T REG cells, suggesting other factors may compensate for the loss of Irf4.Mice harboring a conditional knock-down (Foxp3 CRE Irf4 fl/fl ) develop spontaneous dermatitis, blepharitis, and lymphadenopathy disease by 5-6 weeks, and die by 3-4 months from a mostly T H 2mediated autoimmune disease (133).Co-immunoprecipitation of Irf4 and Foxp3 shows that both TF interact to, facilitate the transcription of genes such as Icos, Il1rl1, Maf and Ccr8 (133).In addition, Irf4 allows T REG cells to exert their suppressive functions.For example, a knock-out or a disruption of Irf4 expression in murine or human T REG cells, impacts the expression of key suppressive genes, including Il10 (137).Moreover, while there is evidence Irf4 is an important contributor during the early transcriptional events involved in the specialisation of activated T REG cells, this TF is also readily detected in some populations of memory T REG cells in the tissue, suggesting its expression is maintained long after TCR engagement.Finally, BATF and Irf4 are particularly upregulated in relation to the strength of the TCR signal (138,139), and, together, directly suppress Foxp3 transcription in T REG cells induced in vitro (139).Collectively, these observations imply that BATF and Irf4 hinder Foxp3 transcription during the early events that define eT REG formation (Figure 2).

The unique properties of TR-T REG cells
As discussed above, the pool of T REG cells residing in tissues is highly dependent on the organ and is composed in adults of both TR-T REG and emT REG cells whose fate remains ill-defined.Moreover, while the establishment of a peripheral T REG population in mucosal tissues happens in a relatively short amount of time after birth, this is not the case for VAT T REG cells that follow a more gradual accumulation (27), complexifying our understanding of the events that govern TR-T REG accumulation.Notably, fate-mapping systems (Foxp3 eGFPCreERT2 x ROSA26 STOP- eYFP ) in neonate mice reveal that T REG cells seed non-lymphoid organs like the lungs and liver in the first 8 days of life, persisting for up to 12 weeks with little renewal (25).Critically, exposure to an inflammatory event prior to day 8, but not after, significantly reduces TCR diversity of liver and lung TR-T REG and causes long-lasting alterations to their transcriptional program (25), revealing how critical the neonatal period is to the establishment of tissue homeostasis.Here, the establishment of TR-T REG cells is heavily dependent on the acquisition of a core of transcriptional factors.Single-cell RNA-seq (19), bulk RNA-seq (17), microarray and ATAC-seq (16, 112) analysis of T REG from visceral adipose tissue (VAT), lung, skin or colon reveal the epigenetic and transcriptional landscape of these cells is primarily determined by the organ, with only a small set of core genes shared between them.In various non-lymphoid tissues, TR-T REG cells express a shared a set of core genes, including Il1rl1 (ST2), Gata3, Tnfrsf4, Rora, Il10 and Gzmb (16, 19).On the other hand, there is a significant difference in gene expression between the transcriptional signature and DNA methylation profile of colonic and skin-isolated T REG , including increased Dgat2, a gene involved in lipid synthesis (16,19), in skin TR-T REG cells, revealing these cells acquire tissue-specific abilities that allow them to persist in these microenvironments.

Tissue-specific migratory properties of TR-T REG
Following TCR engagement and clonal expansion, the development of TR-T REG involves the adoption of migratory properties through the acquisition and loss of chemokine receptors and other adhesion molecules.Indeed, as they undergo deep transcriptional changes and rapid clonal expansion, they also begin to express chemokine receptors that lead them to egress from the lymph node and migrate to a selected tissue.As with other T cells, activated T REG cells downregulate the surface expression of the Lselectin CD62L and upregulate the expression of the glycoprotein type I CD44 (132).Similarly, T REG cells from human tumors (140) and skin (141), as well as murine T REG cells isolated from multiple nonlymphoid organs (15), display low levels of CCR7, preventing their recirculation in lymphoid organs (142).However, the combination of chemokine receptors TR-T REG cells possess is specific to the type of tissue these cells travelled to.In adult mice, RNA sequencing of two distinct populations of T REG cells isolated from barrier tissues reveals that CCR7 -T REG possess an organ-specific chemokine receptor signature, regardless of their expression of the IL-33 receptor ST2 (17), suggesting that the migration of all TR-T REG cells is determined by a shared group of chemokine receptors.This combination of chemokine receptors can also be appreciated in the seminal work by Miragaia and colleagues, as they observed skin-localised T REG cells preferentially expressed Ccr6, while colonic T REG cells displayed higher levels of Ccr1 and Ccr5; yet, both subsets showed similar levels of Ccr4, Ccr8 and Ccxr4 (19).Unfortunately, we have yet to determine which combination of chemokine receptors is part of their migratory program and which are locally upregulated to provide further movement inside the tissues.

Core transcription factors of TR-T REG cells
Interestingly, while these experiments highlight the transcriptional diversity of TR-T REG, so did they help identify a core identity that govern their residency program (19).Some members of this list include transcriptional regulators that have been clearly associated to tissue The acquisition of a tissue-resident program requires a series of epigenetic and transcriptional changes that involve modulation of Foxp3 expression or activity.After thymic egress into the periphery, T REG cells are TCR-activated by self or non-self-antigens, and undergo a series of epigenetic and transcriptional changes that guide their maturation into TR-T REG cells.While not entirely understood, this process seems to happen in a step-wise manner.First, TCR-engaged T REG cells upregulate key transcriptional programs in part driven by the transcription factor BATF, which, in conjunction with Foxp3, promotes the accessibility of Foxp3 and expression of BATF-driven genes including Ctla4, Icos, Gata3, Irf4.Key to the stability of their epigenetic landscape, T REG cells require Dnmt1 and its partner Uhrf1 to promote the methylation of CpG-rich regions and control the accessibility to inflammatory genes, including Ifng, Il6, Il12, Il17a, Il22, Ccr1, Cxcr6, Runx2 and Stat3.Finally, Foxp3 partners with Lef1 to promote the expression of genes involved in its core program, including Foxp3, Il2ra and Tgfb1, and also with TCF1 to suppress the expression of genes associated with inflammation like Il6ra, Ifngr2 and Stat3.Importantly, BATF and IRF4 can, in turn, suppress Foxp3 expression, a process that, while not fully understood, may enable the temporal accessibility of genes normally repressed by FoxP3.Once in the tissue, BATF enables the continued suppression of genes like Rorc (RORgT), Il6ra and Stat3.GATA3 promotes the transcription of Foxp3, but may be further involved in the expression of other GATA3-associated genes, like Il1rl1(ST2).IRF4 is also required for the expression of core TR-T REG genes, including Icos, Il1rl1 and Il10.Moreover, there is evidence that Lef1 and Tcf7 (TCF1) mRNA expression are significantly decreased in TR-T REG cells, suggesting they are no longer required.Finally, BLIMP-1 expression is increased, and can actively inhibit the action of Dnmt3a, promoting the accessibility of key genes in T REG cells such as Foxp3.Consistently, murine models with Foxp3-conditional deletion of BATF, GATA3, IRF4, TCF1 and BLIMP-1 reveal how critical these regulators are for the function of TR-T REG cells.
residency in other T cell subsets, like tissue-resident T RM CD8 + cells (143,144), including Runx3 and Blimp1 (145).In addition, murine and human TR-T REG also possess unique key markers including transcription factors Ikzf2, Gata3, and Rora.

Helios
An important transcription factor associated with TR-T REG cells is Helios.While the majority of T REG cells in circulation readily express Helios, siRNA-mediated silencing of Helios expression in human and murine T REG cells does not impede their survival and suppressive capacity in vitro (146,147).On the other hand, the conditional deletion of Helios in murine T REG cells (Foxp3 CRE Ikzf2 fl/fl ) leads to the development of a progressive, rather than a scurfy-like, lymphoproliferative disease in adult mice (147), revealing it is not required for the development of T REG cells, but rather for the preservation of T REG cell fitness at barrier tissues.Importantly, Helios potentiates the suppressive function of T REG by directly interacting with Foxp3 and promoting histone deacetylation (148), providing further evidence Helios plays a supportive role to the program provided by Foxp3.
However, not all lymphoid and tissue-resident T REG cells express Helios.Originally thought to be solely expressed by tT REG cells (30), it is now well-appreciated that Helios expression in both murine and human Helios -T REG cells is inducible (31,149) in vivo and in vitro, respectively.Some of the key features that differentiate splenic Helios + from Helios -T REG is the little overlap they share between their respective TCR repertoire, and the expression of genes involved in the differentiation of specialized T H 17 cells, including Rorc, Il6ra and Il23r (31), suggesting a division of labor between two T REG subsets that may have long-reaching consequences in the tissue adaptation of TR-T REG cells.For example, Cruz-Morales et al. showed that colonic Helios + Gata3 + T REG differ greatly from Helios -RORgT + T REG cells by their requirement of CD28, but not MHC-II, to proliferate locally (20), providing a potential point of distinction between colonic Helios + TR-T REG and RORgT + emT REG .Nonetheless, further investigation into the role of Helios in the differentiation and maintenance of TR-T REG cells is required.

Gata3
Gata3 is the transcription factor 3 of the Gata-binding family that comprises six known members.In T cells, it has been shown to govern T cell development, proliferation and maintenance (150) and is particularly important to promote the transcriptional signature of helper type 2 T cells (T H 2) (151).Skin, gastrointestinal, visceral adipose tissue, and pulmonary TR-T REG cell were all shown to express Gata3 (22, 152), albeit with different intensities.This observation could be explained by the different states of activity of these T REG cells, as Gata3 expression is significantly increased in both murine and human T REG cells upon TCR engagement (22).Interestingly, the signaling pathway that leads T REG to express this TF does not require IL-4a cytokine associated with Gata3 expression in conventional T cells ( 153)and depends largely on exogenous IL-2 (22).Deletion of Gata3 in murine T REG cells does not lead to the development of spontaneous autoimmunity before 6 months of age ( 22), after which the mice develop intestinal pathology and dermatitis (104).This is because Gata3-deprived TR-T REG are not hindered in their development, but rather fail to respond to an inflammatory threat, displaying decreased tissue migration, proliferation, transcriptional stability, and suppressive capacity (22,23,104).
While not necessary for the maintenance of peripheral tolerance, Gata3 contributes to the functional adaptation of TR-T REG cells.Gata3 recognizes the CNS2 region of Foxp3 (23), promoting Foxp3 activity and stabilising the transcriptional program of T REG cells to avoid their conversion to proinflammatory T cells under stress (22).In addition, Gata3 partners with Foxp3 to form a complex that contributes to the regulation of a wide array of T REG -associated genes (104).Gastrointestinal, skin, pulmonary, and VAT TR-T REG cells express the IL-33 receptor ST2 (17,24,154), a known target of Gata3 in T cells (155).Unfortunately, while Gata3 is known to remodel the Il10 locus in CD4 + T cells (156), the link between Gata3 and IL-10 has yet to be established in TR-T REG cells.As such, there are many indicators that Gata3 is an important contributor to the tissue adaptation of T REG cells, and future investigation into the epigenetic, transcriptional, and post-transcriptional impact of this TF is warranted.

RORa
Another gene that is consistently found in RNA-seq data from TR-T REG cells is Rora.This gene codes for the retinoic acid receptor-related orphan receptor alpha (RORa), a transcription factor which has been found to be expressed in differentiated T cells, including T H 1, T H 2 and T H 17 (157) cells.Unfortunately, we know very little on the role of RORa in TR-T REG .In T cells, Rora is expressed upon TCR activation, and is closely associated with the expression of their lineage defining T H 1, T H 2 or T H 17 signature (158).Similarly, RORa plays a supporting role in the transcriptional signature of TR-T REG cells.For example, a Foxp3 conditional deletion of RORa does not alter the accumulation of skin localised TR-T REG cells but enables the evasion of immune responses during skin treatment with MC903, a chemical inducer of atopic dermatitis (18).Thus, as with Gata3, RORa is not required during the transcriptional transformation of tissue-migrating eT REG cells, but rather for their function once in the tissue.

Blimp 1
The B lymphocyte-induced maturation protein-1 (Blimp 1) is a transcriptional regulator that is particularly expressed by T REG cells located in secondary lymphoid organs or non-lymphoid tissues (159).A conditional knock out of Prdm1 (Blimp-1) in murine T REG (Foxp3 Cre Prdm1 fl/fl ) generates an increase in the accumulation of T REG cells, accompanied by small increase in T CONV cell abundance that is insufficient to induce autoimmunity (159), confirming Blimp-1 is not essential to the generation, migration or even function of eT REG .Rather, Blimp-1 prevents the methylation of multiple genes, including CNS2 in the Foxp3 locus, by inhibiting the action of the methyltransferase Dmnt3a downstream of IL-6 (160).In doing so, Blimp-1 prevents the full conversion of colonic T REG to non-suppressive RORgT + eT REG cells (161), suggesting that the role of Blimp-1 is to preserve the transcriptional program of TR-T REG cells.

Tissue-specific survival mechanisms of TR-T REG cells
TR-T REG cells have shown a remarkable capacity to communicate with their immediate environment, adopting cytokine receptors, sensing molecular changes in its environment, and providing direct cell-to-cell contact with immune and nonimmune cells (162).TR-T REG achieve this by adopting unique phenotypic characteristics, such as the ability to sense local danger signals and compete in microenvironments with limited IL-2 availability, allowing them to maintain their identity in nonlymphoid organs.

IL-33
IL-33 is a cytokine of the IL-1 family of alarmins constitutively expressed by endothelial and epithelial cells (163) and by activated macrophages and dendritic cells (164).The IL-33 receptor ST2 is transcriptionally upregulated and detected on the surface of TR-T REG (17,120).This is consistent with the fact that the expression of Il1rl1 (ST2) is closely associated to the expression of BATF and is part of the transcriptional signature elicited after DNA methylation in TR-T REG cells (16, 120).However, not all tissue isolated T REG express ST2 in mice at the steady state, nor do skin, lung, gut, or VAT-isolated T REG cells express ST2 with the same intensity (17).As such, while suggested as a marker of TR-T REG cells (17), there is currently no clear evidence that ST2 expression is exclusive to TR-T REG cells, and further investigation into this receptor is warranted.Moreover, the importance of ST2 in the differentiation and function of TR-T REG cells remains ill-defined.For example, while IL-33 can directly promote the homeostatic expansion of T REG cells (24,165), a Foxp3-specific conditional knock-down of ST2 (Foxp3 CRE Il1rl1 fl/fl ) does not impair T REG accumulation in the lungs (166).Rather, IL-33 orchestrates T REG -mediated suppression of local gd T (166), T H 1, and T H 17 cells during tissue injury (24,167).To complicate things, it is unclear if these mechanisms depend entirely on the expression of ST2 by T REG cells (168).Indeed, innate immune cells can readily respond to IL-33 and provide proliferative signals to promote TR-T REG expansion and survival (169).As such, rather than providing a survival signal, ST2 may act as a sensing mechanism for local TR-T REG to rapidly reactivate and produce suppressing cytokines.

Icos
While not exclusive to TR-T REG , the inducible co-stimulator Icos plays a crucial role in both TR-T REG and emT REG cells to maintain their identity and survival within non-lymphoid organs (21).In mice, a Foxp3 conditional knock out of Icos (Foxp3 YFP-CRE Icos fl/fl ) does not generate autoimmunity, but rather prevents tissuelocalised T REG cells from suppressing oxalone-induced dermatitis (170), suggesting Icos is particularly required for T REG cells to control tissue injury.Specifically, Icos coordinates with mTORC1 signaling to support T REG proliferation and the expression of suppressive molecules (171), and is particularly critical for TR-T REG and emT REG cells to persist in the absence of IL-2 signaling by providing anti-apoptotic signals (15).Together, Icos and CD28 act as potent activators of the PI3K/Akt signaling pathway that triggers the phosphorylation of the transcription factor Foxo1 (171,172).In turn, this sequesters Foxo1 in the cytoplasm and leads to downregulation of genes like Klf2 and Ccr7 (173).In the absence of IL-2, T REG cells become susceptible to apoptosis, highlighting the critical role of sustained Icos-IcosL signaling in their survival as they migrate to the tissue (15).On the other hand, abrogating the PI3K-activating capacity of Icos by removing a YMFM motif in its cytoplasmic tail increases VAT TR-T REG accumulation and function (174), suggesting that Icos may have tissue-specific roles for T REG cells.Thus, while there is abundant evidence that Icos promotes the activation and survival of TR-T REG cells, tissuespecific differences are likely at play and must be considered when investigating TR-T REG cell sub-populations.

The metabolic adaptation of TR-T REG cells
Genes involved in fatty acid b-oxidation (FAO) can be readily detected in antigen-experienced T REG cells isolated from nonlymphoid tissues, including in visceral adipose tissue (VAT), the skin, the colon, and the lungs, suggesting TR-T REG default to FAO in non-inflamed tissues (19, 120).However, these transcriptional approaches have not formally demonstrated that TR-T REG cells require FAO to persist in all tissues.Most of the current evidence comes from VAT-isolated TR-T REG , which express the peroxisome proliferator-activated receptor gamma (PPARg), a ligand-activated transcription factor.Functionally, PPARg provides a complex signal to engage FAO in VAT T REG cells (175), providing them with a competitive advantage over T CONV cells to survive, accumulate, and function (176).This crucial metabolic strategy enables VAT T REG cells to catabolize long-chain fatty acids (LCFAs) from the environment, turning to FAO to sustain their demand for energy (177,178).While this process is shared between T REG and T CONV cells, T REG cells utilise fatty acids differently as they do not build endogenous fatty acids from acetyl-CoA, but rely on the acquisition of exogenous fatty acids to meet their metabolic needs (179).Concomitantly, efficient lipid storage by VAT TR-T REG cells is essential to protect them against lipo-toxicity and to provide the metabolic precursors needed for energy generation.These include scavenger proteins such as CD36 and enzymes involved in triglyceride production, such as DGAT1 and DGAT2.Skin and VAT-isolated PPARg + T REG cells readily express CD36, providing them with the ability to capture and secure LCFAs (175,180).DGAT are a family of enzymes involved in triglyceride production and lipid droplet (LD) formation that are preferentially expressed in activated T REG cells (181).Foxp3 itself is a strong repressor of Glut1 (182), the glucose transporter, and favors the expression of FAO genes (178).Yet, this mechanism acts in a feed-back loop, with DGAT1 promoting Foxp3 expression by diminishing protein kinase C (PKC) activity downstream of the TCR (181,183).Interestingly, by tracing the tissue distribution of splenic T REG cells with shared TCR sequences, Li et al. demonstrated that PPARg-expressing eT REG cells localise to other non-lymphoid sites, including the skin and the liver (184), providing new translational evidence that FAO proteins are expressed by other TR-T REG cells.Nonetheless, while these observations highlight the importance of FAO for VAT TR-T REG cells to sustain their bioenergetic demands, it remains to be determined if this metabolic strategy is required to sustain other TR-T REG cells.

The inflammatory adaptation of TR-T REG
One of the most recent and exciting discoveries has been the observation that activated eT REG can further specialize to adopt T H 1, T H 2, T H 17, and even T FH -like features.Importantly, they can express master transcription factors that are part of a transcriptional program typically expressed by helper T cells, including T-bet (T H 1), RORgT(T H 17), Gata3(T H 2), and BCL6 (T FH ).The differentiation, migration, and tissue accumulation of functionally-specialized T REG cells in tissues is a dynamic process that can occur in microbiota-rich barrier sites (10) or during tissue injury (185, 186).Indeed, contrary to the core genes necessary for the generation and maintenance of TR-T REG cells, the role of these "master" transcription factors is not associated with a residency program; rather, these TFs promote a set of specialized functions that allow T REG cells to suppress or orchestrate local immune responses (Figure 3).For example, single-cell analysis performed at distinct times during an Influenza A infection in mice portrays how Gata3 + T REG cells are progressively replaced by antigenspecific T-bet + CXCR3 + T REG cells in the course of disease, suggesting that, contrary to the permanent presence of TR-T REG , T H 1-specialized T REG cells are generated concurrently with the antiviral T H 1 response and follow the pattern of accumulation of these cells (185,187).
Interestingly, some of these specialized T REG cells (RORgT + T REG ) are present at the steady-state in mucosal tissues such as the colon, blurring attempts at defining what constitutes the bona fide TR-T REG phenotype in these tissues.Indeed, key events leading to the generation of specialized T REG cells include the requirement for TCR signaling and aerobic glycolysis to facilitate clonal expansion and differentiation (188).Moreover, Irf4 ( 27) is a necessary stepping-stone for the differentiation of specialized T REG cells (9,28,29).A typical example of these specialized T REG cells is observed in the colon, where resident T REG cells displaying two distinct TCR repertoires can be segregated based on their transcriptional program.Indeed, both RORgT + T REG and Gata3 + T REG are readily detected in the colon; however, absence of a local microflora only hinders the specific generation of RORgT + T REG (189,190) since their TCR repertoire is largely biased towards bacterial antigens (72,191,192).Since specific signals are required for T REG cells to acquire these programs, it is possible to dissect the required pathways that lead T REG cells to acquire these specialized programs.

The effects of polarizing signals on the fate of T REG cells
Some of the better described signals that promote the generation of specialized T REG cells include cytokines that drive the phosphorylation and nuclear translocation of STAT and SMAD proteins (193).In turn, these signals promote the expression of genes that define T cell fate, including the acquisition of master transcription factors T-bet, Gata3, or RORgT.What is particularly interesting, however, is that the pathways that lead T REG to adopt these TFs can also undermine their Foxp3-dependent transcriptional program, either through the loss of Foxp3 expression, the expression of pro-inflammatory genes, or the engagement of apoptosis.As such, at the time when activated T REG cells undergo important epigenetic and transcriptional changes, certain inflammatory signals can promote the loss of Foxp3 expression and their conversion into inflammatory "exT REG " cells.Several key transcription factors have been described to be involved in this inflammatory adaptation process of T REG cells.

T-bet + eT REG
T-bet is a T-box transcription factor expressed in a wide variety of immune cells, and mostly recognized for its role in defining the transcriptional landscape of T H 1 cells (194).Using a unique murine model that enables the tracking of murine T-bet-expressing T REG (Foxp3 Thy1.1 Tbx21 tdTomato-T2A-CreERT2 R26YFP -fl-stop-fl ), Levine and colleagues showed that the conditional deletion of T-bet in Foxp3 + T REG cells does not lead to autoimmunity in adult mice, although it does generate a mild increase in T H 1 activity (195), suggesting T-bet has little to no impact on the way T REG preserve tissue function at the steady-state.Notably, T-bet is a critical regulator for the expression of CXCR3 (196), a chemokine receptor that orchestrates eT REG migration to sites of T H 1-driven inflammation (196,197).Highlighting the role of TCR engagement, T-bet + eT REG cells that progressively accumulate in the lungs of mice infected with acute Influenza A infection recognize viral proteins (185,198).Thus, as with T H 1 cell polarization, the generation of T-bet + eT REG occurs progressively during inflammation and is closely associated to the clonal expansion of antigen-specific CD4 + T H 1 cells.
The signals that promote the generation of T H 1 cells include IFNg (STAT1) and IL-12 (STAT4).Interestingly, an IFNg-STAT1 signal drives the initial expression of T-bet during TCR engagement, while a subsequent IL-12-STAT4 signal is required for their definitive differentiation (199,200).This initial T-bet expression can, in turn, promote the expression of the IL-12 receptor (IL-12Rb2) (201)(202)(203).However, contrary to T H 1 cells, eT REG cells seem to depend exclusively on the presence of IFNg for the acquisition of T-bet (196,204).By activating murine CD4 + Foxp3 + cells in vitro, Koch and colleagues demonstrated that T REG cells acquire T-bet expression and its associated target, CXCR3, only if they possess the receptor IFNgR1 (205), suggesting that IFNg-producing T H 1 cells are responsible for the polarization of T H 1-like eT REG cells.
The control of IL-12 signalling by T REG cells is critical, as excessive pSTAT4 can lead T REG cells to lose Foxp3 expression (206) by, notably, limiting chromatin accessibility of STAT5 to the Foxp3 locus (207).Yet, STAT4 is a major regulator of Ifng in CD4 + T cells (208), and both human and murine T REG exposed to IL-12 produce low levels of IFNg (187,205,206,(209)(210)(211)(212), revealing excessive IL-12 can still be perceived by T H 1-like eT REG cells.However, contrary to STAT1, STAT4 signaling is associated with less suppressive T REG cells and can even lead to the complete loss of Foxp3 expression (187,205,206,(209)(210)(211), suggesting T-bet + eT REG are in a constant struggle to avoid the loss of genes involved in their suppressive functions.In this regard, T-bet + T REG cells possess mechanisms to avoid overt STAT4 signaling.For example, IFNginduced T-bet + eT REG cells suppress IL-12Rb2 surface expression, preventing excessive phosphorylation of STAT4 and further T H 1like commitment (205).Moreover, non-labelled proteomics on circulating human T REG cell populations revealed that, compared to memory or naïve T REG , eT REG maintain low cytosolic levels of STAT4 (213).
There is growing evidence for the role of IL-18 on the function of tissue-resident T-bet + eT REG cells.While the origin of IL-18R1 + eT REG cell remain to be fully understood, T H 1 polarizing conditions, and particularly IL-12, allow T REG cells to adopt the expression of both T-bet and IL-18R1 (187), suggesting that, like for T conv cells, eT REG require a STAT4-dependent chromatin remodeling to express IL-18R1 (214,215).In vitro, IL-18 promotes the expansion and suppressive capacity of IL-12-generated T-bet + T REG cells (187), suggesting this signal can counter the destabilising effects of IL-12.In vivo, T-bet + eT REG cells express IL-18R1 when they accumulate in the lungs during an Influenza A infection (187).Here, IL-18 enhances the production of amphiregulin in local T REG cells, facilitating tissue restoration after pulmonary Influenza A infection (216).In addition, a Foxp3 conditional knock-out of Il18r1 (Foxp3 ERT2-CRE Il18r1 fl/fl ) allowed us to demonstrate that IL-18 is specifically required for eT REG cells to suppress IL-17A responses in the lungs after an Influenza A infection (187).Similarly, IL-18R1 deficiency in T REG cells fails to control the onset of a T cell-mediated colitis (217) as well as inflammation in an experimental model of ovalbumin-induced asthma (218), confirming IL-18 is an important contributor to eT REG function.However, these observations do not necessarily mean that IL-18R1 expression is restricted to T-bet + T REG , as we have observed RORgT expression among a subset of IL-18R1 + T REG cells (187) and IL-18R1 expression has been described in T H 17 cells (217).Collectively, these observations illustrate how the T H 1 adaptation of eT REG cells allows for the suppression of tissue inflammation.

Gata3 + eT REG
The transcription factor Gata3, which is an important component of the transcriptional program of TR-T REG , is best described for its role in driving T H 2 cell differentiation (219).In both human and murine CD4 + T cells, Gata3 promotes T H 2associated genes, allowing for the expression of genes associated to their function, such as IL-4, IL-5, and IL-13 (151, 219).There are numerous accounts of tissue-homing T REG cells expressing high levels of Gata3 during acute T H 2-driven immunity, such as what is observed during asthma (220) or helminth infections (221,222).
The signals driving GATA-3 expression in T REG cells are not fully understood.Two signals have been described to be sufficient to induce Gata3 expression during T H 2 differentiation, namely an IL2/ STAT5-dependent and an IL-4/STAT6-dependent signal (223)(224)(225).In homeostatic conditions, IL-2 (STAT5) is sufficient to promote the expression of Gata3 during TCR engagement (22).However, in T H 2-driven responses, T REG cells require IL-4R to acquire GATA-3 expression and their T H 2-like characteristics (226).This distinction between STAT5 and STAT6-dependent induction of Gata3 may pave the way towards understanding how T H 2-like eT REG cells differ from TR-T REG cells.For example, mice with a Foxp3-specific conditional knock-down of Il4ra (Foxp3 CRE Il4ra fl/fl ) fail to prevent exacerbated asthma-like symptoms when challenged with house-dust-mite (HDM) (226) and helminthdriven inflammation, despite the presence of T REG cells in situ (221).
While IL-4 can favor T REG cell-mediated functions, sustained IL-4 can also force T REG cells to lose Foxp3 expression and their suppressive capacity both in vitro (227) and in vivo (221,222,227).STAT6 can promote the activity of the histone deacetylase HDAC9, which decreases chromatin accessibility to the Foxp3 locus (228).To prevent this, eT REG cells require strategies to avoid excessive IL-4 signaling.First, by maintaining high levels of CD25 expression, eT REG cells remain sensitive to IL-2, whose STAT5 signal competes with STAT6 activity (229).Second, tissue-localised T REG cells prevent further commitment into the T H 2 lineage by producing the E3 ubiquitin ligase Itch (230,231).Finally, murine in vitroinduced T REG cells exposed to IL-4 express higher levels of the JAK/ STAT inhibitor SOCS2 to prevent further STAT6 phosphorylation and the expression of pro-inflammatory cytokines (232).Thus, while it remains to be fully confirmed in tissue-resident T REG cells, there is cumulating evidence that IL-4 is important for the commitment of Gata3 + eT REG cells, and responsible for their transcriptional destabilisation and conversion into T H 2-like ex-T REG cells.
Finally, IL-33, which contributes to the proliferation of TR-T REG cells (165), can also govern the function of Gata3 + eT REG cells during inflammation.In this regard, IL-33-responding activated T REG cells where shown to produce high amounts of IL-10 and TGF-b (233), playing a key role in maintaining intestinal homeostasis (24).Similarly, ST2 + T REG cells promote the suppression of anti-tumor immune responses (234)(235)(236).However, IL-33 can also drive the production of the T H 2 associated cytokines IL-5 and IL-13 in pulmonary eT REG cells (233,237,238) and interfere with their capacity to supress T H 2 responses (238).Thus, the role of IL-33 on Gata3 + T REG cells is specific to the inflammatory context and may depend on whether it targets TR-T REG cells or eT REG cells accompanying T H 2 responses.

RORgT + eT REG
While complex and not entirely defined, the signaling events that lead T REG cells to adopt a T H 17-like phenotype include some of the same polarizing JAK-STAT and SMAD signals that are required for the generation of T H 17 cells.Indeed, the promoter functions of both Stat3 (239) and RORgT (240) are required to establish a T H 17 cell transcriptional program (241), and T REG cells have been shown to share part of this transcriptional program through the acquisition of these TFs (239).In the gut, RORgT + T REG cells play an essential part in maintaining gut homeostasis and contribute to maintain local homeostasis by, notably, suppressing T H 17-driven responses (242).Transcriptionally, RORgT + T REG cells from the mouse colon at steady-state express higher levels of Il23r, Il1r1, Maf, Irf4, and Ikzf3 than their RORgT -counterparts (191), revealing they possess a unique landscape that encompasses some key T H 17-associated genes.Moreover, RORgT is required for IL-10 production by colonic T REG cells and prevention of T cell-mediated colitis (191).Similarly, RORgT is required for T REG cells to control T H 17mediated autoimmune arthritis and EAE (192,243), suggesting that RORgT expression allows emT REG cells to target and suppress T H 17-driven responses.However, the role of RORgT and its impact on the transcriptional landscape of emT REG cells remains ill-defined and is likely driven by the inflammatory microenvironments these cells are exposed to.
While many cytokines can promote the nuclear translocation of Stat3 in T H 17 cells, the simultaneous signals provided by TGF-b (SMAD2/3) and IL-6 (Stat3) are sufficient, in vitro, to induce RORgT expression in T REG cells (162,192).Interestingly, a delicate balance is achieved between the signal provided by TGFb and IL-6.For example, TGF-b and IL-6 synergistically promote the proteasome-dependent degradation pathway of Foxp3 ( 244), contributing to a partial loss of Foxp3 function.Interestingly, once colonic RORgT + T REG cells are generated, they display a significantly stable phenotype, with maintained demethylation of T REG -specific genes like Foxp3, Ikzf2, Ctla4, Gitr and Ikzf4 (Eos) (191).In fact, these cells possess intrinsic mechanisms to avoid their full conversion towards T H 17 cells.As with IL-12 and T H 1 cells, subsequent signals provided by IL-23(Stat3) can further destabilise the transcriptional program of RORgT + T REG cells and even engage an apoptotic cascade in these cells (245).Indeed, Il23r is amongst the genes upregulated by Stat3 and RORgT (246), making RORgT + eT REG particularly sensitive to IL-23 (245).In a recent report, Jacobse and colleagues demonstrated IL-23R expression is restricted to RORgT + T REG under homeostatic conditions in the colon, and colonic T REG cells maintain a competitive advantage over WT T REG cells to survive in these conditions (245).Concomitantly, the authors demonstrate that T REG cells isolated from the lamina propria of patients with active IBD express high levels of Il23r and pro-apoptotic genes (126), suggesting an evolutionary conserved mechanism that orchestrates RORgT + eT REG survival and function.
In addition to IL-23, IL-1b was found to promote the differentiation of human CD4 + CD25 high CD127 low Foxp3 + T REG cells into IL-17-producing cells (247,248), suggesting IL-1 may promote a pro-inflammatory phenotype in T REG cells.However, the role of IL-1 in RORgT + eT REG cells remains ill-defined.Through a T-cell mediated colitis model in mice, we demonstrated that a knock-out of IL-1R1 in T REG cells favors an accumulation of Gata3 + T REG cells over RORgT + T REG cells in the colon, as IL-1 directly promotes RORgT + T REG expansion (167).Despite this effect, a lack of IL-1 signaling in T REG cells results in more abundant accumulation in the colon compared to WT T REG (167), suggesting IL-1 is a negative signal for the maintenance of colonic T REG cells.Interestingly, there are specific situations where this effect is beneficial.For example, IL-1R1 -/-mice infected with Cryptococcus neoformans are particularly sensitive to the infection, as they cannot mount an effective T cell response (249).In their lungs, these mice lack RORgT + T REG cells and have increased ST2 + T REG cells in the lungs compared to WT mice (167), suggesting sustained immunosuppression.To counter this, activated T REG cells express high levels of the decoy receptor IL-1R2, which allows them to neutralize IL-1 signalling (250-252).

Conclusion
In this review, we aimed to detail some of the major elements that govern the trajectory of a precursor T REG P cell to a highly specialized TR-T REG cell.It is particularly interesting that the trajectory of a T REG cells is, in most regards, highly like that of the conventional T cell as it undergoes further polarization prior to reaching peripheral tissues.Importantly, the epigenetic malleability of T REG cells is central to their ability to perform outside of the thymus, as these transformations allow them to sense tissue-derived signals that, in turn, modulate their suppressive functions.However, while we have accumulated a lot of information in recent years, much remains to be understood on how these tissue and inflammation-specific adaptations govern the function of TR-T REG cells.For example, the notion that T REG cells can adopt a specific differentiation path and revert to their previous state, labelled "plasticity" (253,254), remains to be proven experimentally.
Finally, recent reviews have addressed how Foxp3 gene editing, IL-2 therapy, and the use of T REG cells as cellular therapies represent key strategies to engage human T REG cells (255).However, most of our current knowledge on TR-T REG cells has not been specifically exploited by T REG -targeting therapeutical approaches.There is, however, some evidence these strategies may facilitate the expression of a tissue residency program.For example, the development of muteins or low-dose therapies (256) aimed at promoting IL-2 signaling in T REG cells can promote the expression of genes associated with TR-T REG cell function, such as Il1rl1(ST2), as well as migratory and other tissue resident genes (257).Thus, it is of interest to understand how T REG targeting strategies can influence both the developmental trajectory and the function of tissue resident T REG cells.In addition, understanding the migratory cues that enable TR-T REG cells to recognize specific tissues can have long reaching therapeutical benefits.Chimeric antigen receptor (CAR) T REG cells have been proposed as a new avenue to circumvent the constraints of low T REG cells numbers and the unknown TCR repertoire of T REG in autoimmune or graftversus-host (GvHD) diseases (258).However, this approach is still very novel, and, in the absence of additional modifications, is expected to suffer from the same limitations of CAR-T cells (258, 259), including failing to adopt metabolic strategies to survive, preventing exhaustion, and maintaining their function in tissues.Thus, it is by establishing a solid understanding of the entire pathway leading T REG cells to adapt to non-lymphoid organs that we provide the basis for the development of better T REG cellbased therapies.

FIGURE 3
FIGURE 3 Specific inflammatory signals alter the trajectory of T REG cells in non-lymphoid sites by engaging specialized programs prior and during their migration to inflamed tissues.During active inflammation, the presence of cytokines such as IFNg, IL-2, IL-6, and TGF-b can divert the differentiation of T REG cells to adopt helper-like phenotypes, allowing them to migrate to specific sites of inflammation alongside conventional T cells.Importantly, by acquiring these master transcription factors, effector T REG cells (eT REG ) become responsive to signals provided by IL-12, IL-4 or IL-23.While these cytokines further promote the transcriptional program engaged by these specialized T REG cells, they can ultimately diminish their suppressive functions and allow them them to contribute to inflammation as exT REG cells.Importantly, it remains to be determined if the resulting population of emT REG cells in the tissue after inflammation acquire a residency program that lead them to form part of the TR-T REG cell population.