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The transcription factor Foxp3 is essential for the function of regulatory T cells. Rudensky and colleagues show Foxp3 participates in large protein complexes that regulate gene expression of many of these components in self-reinforcing networks. The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T reg cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400–800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T reg cell biology.

Antigen-specific regulatory T cells (Tregs) suppress pathogenic autoreactivity and are potential therapeutic candidates for autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis is associated with autoreactivity to the Smith (Sm) autoantigen and the human leucocyte antigen (HLA)-DR15 haplotype; hence, we investigated the potential of Sm-specific Tregs (Sm-Tregs) to suppress disease. Here we identify a HLA-DR15 restricted immunodominant Sm T cell epitope using biophysical affinity binding assays, then identify high-affinity Sm-specific T cell receptors (TCRs) using high-throughput single-cell sequencing. Using lentiviral vectors, we transduce our lead Sm-specific TCR into Tregs derived from patients with SLE who are anti-Sm and HLA-DR15 positive. Compared with polyclonal mock-transduced Tregs, Sm-Tregs potently suppress Sm-specific pro-inflammatory responses in vitro and suppress disease progression in a humanized mouse model of lupus nephritis. These results show that Sm-Tregs are a promising therapy for SLE. Antigen specific regulatory T cells (Treg) play key roles in the peripheral tolerance to suppress autoreactive immune cells and represent potential avenue for therapeutic intervention. Here the authors identify Smith specific Treg and engineer Treg based cell therapy showing suppression of inflammation in a murine model of lupus nephritis.

Conventional type 1 dendritic cells (DC1) contribute to the development of pathogenic T helper type 1 (Th1) cells in part via the production of the proinflammatory cytokine interleukin-12. Thus, depletion of DC1 has the potential to dampen autoimmune responses. Here, we developed X-C motif chemokine receptor 1 (XCR1)-specific chimeric antigen receptor (CAR)-T cells and CAR-Tregs that specifically targeted DC1. XCR1 CAR-T cells were successfully generated as CD4 + and CD8 + T cells, expressed XCR1 CAR efficiently, and induced XCR1-dependent activation, cytokine production and proliferation. XCR1 CAR-T cells selectively depleted DC1 when transferred into RAG2 −/− mice with a compensatory increase in conventional type 2 DC (DC2) and plasmacytoid DC (pDC). XCR1 CAR-T cell-mediated depletion of DC1 modestly suppressed the onset of Th1-driven experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Diphtheria toxin-mediated DC1 depletion in XCR1-diphtheria toxin receptor mice also suppressed EAE, suggesting that DC1 depletion was responsible for EAE suppression. XCR1 CAR-Tregs were successfully generated and suppressed effector T cells in the presence of XCR1 + cells. Therapeutic treatment with XCR1 CAR-Tregs suppressed Th1-driven EAE. Therefore, we conclude that depletion of DC1 with XCR1 CAR-T cells or immune suppression with XCR1 CAR-Tregs can modestly suppress Th1-driven EAE.

Among all T and NK cell subsets, regulatory T (Treg) cells typically respond to the lowest concentrations of IL-2 due to elevated surface expression of the IL-2R alpha chain (IL2RA; CD25) and the high affinity IL-2 receptor (IL-2R) complex. This enhanced sensitivity forms the basis for low-dose (LD) IL-2 therapy for the treatment of inflammatory diseases, where efficacy correlates with increased Treg cell number and expression of functional markers. Despite strong preclinical support for this approach, moderate and variable clinical efficacy has raised concerns that adequate Treg selectivity still cannot be achieved with LD IL-2, and/or that doses are too low to stimulate effective Treg-mediated suppression within tissues. This has prompted development of IL-2 variants with greater Treg selectivity, achieved through attenuated affinity for the signaling chains of the IL-2R complex (IL2RB or CD122 and IL2RG or CD132) and, consequently, greater reliance on high CD25 levels for full receptor binding and signaling. While certain IL-2 variants have advanced to the clinic, it remains unknown if the full range of IL-2R signaling potency and Treg-selectivity observed with low concentrations of wildtype IL-2 can be sufficiently recapitulated with attenuated IL-2 muteins at high concentrations. Using a panel of engineered IL-2 muteins, we investigated how a range of IL-2R signaling intensity, benchmarked by the degree of STAT5 phosphorylation, relates to biologically relevant Treg cell responses such as proliferation, lineage and phenotypic marker expression, and suppressor function. Our results demonstrate that a surprisingly wide dynamic range of IL-2R signaling intensity leads to productive biological responses in Treg cells, with negligible STAT5 phosphorylation associating with nearly complete downstream effects such as Treg proliferation and suppressor activity. Furthermore, we show with both and humanized mouse systems that different biological responses in Treg cells require different minimal IL-2R signaling thresholds. Our findings suggest that more than minimal IL-2R signaling, beyond that capable of driving Treg cell proliferation, may be required to fully enhance Treg cell stability and suppressor function .

Mucosal-associated invariant T (MAIT) cells are an abundant class of innate T cells restricted by the MHC I-related molecule MR1. MAIT cells can recognize bacterially-derived metabolic intermediates from the riboflavin pathway presented by MR1 and are postulated to play a role in innate antibacterial immunity through production of cytokines and direct bacterial killing. MR1 tetramers, typically stabilized by the adduct of 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal (MeG), are important tools for the study of MAIT cells. A long-standing problem with 5-A-RU is that it is unstable upon storage. Herein we report an efficient synthetic approach to the HCl salt of this ligand, which has improved stability during storage. We also show that synthetic 5-A-RU•HCl produced by this method may be used in protocols for the stimulation of human MAIT cells and production of both human and mouse MR1 tetramers for MAIT cell identification.

Interleukin-2 (IL-2), along with T-cell receptor (TCR) signaling, are required to control regulatory T cell (Treg) homeostasis and function in vivo . Due to the heightened sensitivity to IL-2, Tregs retain the ability to respond to low-dose or attenuated forms of IL-2, as currently being developed for clinical use to treat inflammatory diseases. While attenuated IL-2 increases Treg selectivity, the question remains as to whether a weakened IL-2 signal sufficiently enhances Treg suppressive function(s) toward disease modification. To understand this question, we characterized the in vivo activity and transcriptomic profiles of two different attenuated IL-2 muteins in comparison with wildtype (WT) IL-2. Our study showed that, in addition to favoring Tregs, the attenuated muteins induced disproportionately robust effects on Treg activation and conversion to effector Treg (eTreg) phenotype. Our data furthermore suggested that Tregs activated by attenuated IL-2 muteins showed reduced dependence on TCR signal, at least in part due to the enhanced ability of IL-2 muteins to amplify the TCR signal in vivo . These results point to a new paradigm wherein IL-2 influences Tregs’ sensitivity to antigenic signal, and that the combination effect may be leveraged for therapeutic use of attenuated IL-2 muteins.

Tregs have been implicated in control of homeostasis in the immune system and beyond. These cells restrain inflammatory responses to self antigens, commensal microorganisms, allergens, and pathogens and adapt their homeostatic and functional capabilities to a particular environment. In this review, we discuss a general model of integration of environmental cues by Tregs in which specialized Treg homeostatic, migratory, and suppression programs are established in dynamically changing inflammatory environments by maintaining an optimal threshold of activation of transcription factors involved in regulation of the corresponding type of effector immune responses.

Foxp3 gene and T-cell fate Regulatory T (T reg ) cells act to suppress immune system activation, so tight control over their numbers and activity is an important part of immune homeostasis. Here it is shown that particular conserved non-coding sequence elements at the Foxp3 locus play a role in controlling the size and composition of the T reg -cell population. Immune homeostasis relies on tight control over the size of a population of regulatory T cells (T reg ) that can suppress over-exuberant immune responses. Cells commit to the T reg lineage by upregulating the transcription factor Foxp3. Conserved non-coding DNA sequence elements at the Foxp3 locus are now shown to control the composition, size and maintenance of the T reg cell population. Immune homeostasis is dependent on tight control over the size of a population of regulatory T (T reg ) cells capable of suppressing over-exuberant immune responses. The T reg cell subset is comprised of cells that commit to the T reg lineage by upregulating the transcription factor Foxp3 either in the thymus (tT reg ) or in the periphery (iT reg ) 1 , 2 . Considering a central role for Foxp3 in T reg cell differentiation and function 3 , 4 , we proposed that conserved non-coding DNA sequence (CNS) elements at the Foxp3 locus encode information defining the size, composition and stability of the T reg cell population. Here we describe the function of three Foxp3 CNS elements (CNS1–3) in T reg cell fate determination in mice. The pioneer element CNS3, which acts to potently increase the frequency of T reg cells generated in the thymus and the periphery, binds c-Rel in in vitro assays. In contrast, CNS1, which contains a TGF-β–NFAT response element, is superfluous for tT reg cell differentiation, but has a prominent role in iT reg cell generation in gut-associated lymphoid tissues. CNS2, although dispensable for Foxp3 induction, is required for Foxp3 expression in the progeny of dividing T reg cells. Foxp3 binds to CNS2 in a Cbf-β–Runx1 and CpG DNA demethylation-dependent manner, suggesting that Foxp3 recruitment to this ‘cellular memory module’ facilitates the heritable maintenance of the active state of the Foxp3 locus and, therefore, T reg lineage stability. Together, our studies demonstrate that the composition, size and maintenance of the T reg cell population are controlled by Foxp3 CNS elements engaged in response to distinct cell-extrinsic or -intrinsic cues.

Regulatory T cells expressing the transcription factor T-bet selectively suppress T H 1 and CD8 T cells, but not T H 2 or T H 17 activation and associated autoimmunity. Adaptive immune responses are tailored to different types of pathogens through differentiation of naive CD4 T cells into functionally distinct subsets of effector T cells (T helper 1 (T H 1), T H 2, and T H 17) defined by expression of the key transcription factors T-bet, GATA3, and RORγt, respectively 1 . Regulatory T (T reg ) cells comprise a distinct anti-inflammatory lineage specified by the X-linked transcription factor Foxp3 (refs 2 , 3 ). Paradoxically, some activated T reg cells express the aforementioned effector CD4 T cell transcription factors, which have been suggested to provide T reg cells with enhanced suppressive capacity 4 , 5 , 6 . Whether expression of these factors in T reg cells—as in effector T cells—is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here we demonstrate that expression of the T H 1-associated transcription factor T-bet in mouse T reg cells, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss of function or elimination of T-bet-expressing T reg cells—but not of T-bet expression in T reg cells—resulted in severe T H 1 autoimmunity. Conversely, following depletion of T-bet − T reg cells, the remaining T-bet + cells specifically inhibited T H 1 and CD8 T cell activation consistent with their co-localization with T-bet + effector T cells. These results suggest that T-bet + T reg cells have an essential immunosuppressive function and indicate that T reg cell functional heterogeneity is a critical feature of immunological tolerance.

Distinct classes of protective immunity are guided by activation of STAT transcription factor family members in response to environmental cues. CD4⁺ regulatory T cells (Tregs) suppress excessive immune responses, and their deficiency results in a lethal, multi-organ autoimmune syndrome characterized by T helper 1 (TH1) and T helper 2 (TH2) CD4⁺ T cell-dominated lesions. Here we show that pathogenic TH17 responses in mice are also restrained by Tregs. This suppression was lost upon Treg-specific ablation of Stat3, a transcription factor critical for TH17 differentiation, and resulted in the development of a fatal intestinal inflammation. These findings suggest that Tregs adapt to their environment by engaging distinct effector response-specific suppression modalities upon activation of STAT proteins that direct the corresponding class of the immune response.