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.

The gut microbiome-prostate cancer crosstalk is modulated by dietary polyunsaturated long-chain fatty acids.

The gut microbiota modulates response to hormonal treatments in prostate cancer (PCa) patients, but whether it influences PCa progression remains unknown. Here, we show a reduction in fecal microbiota alpha-diversity correlating with increase tumour burden in two distinct groups of hormonotherapy naïve PCa patients and three murine PCa models. Fecal microbiota transplantation (FMT) from patients with high PCa volume is sufficient to stimulate the growth of mouse PCa revealing the existence of a gut microbiome-cancer crosstalk. Analysis of gut microbial-related pathways in mice with aggressive PCa identifies three enzymes responsible for the metabolism of long-chain fatty acids (LCFA). Supplementation with LCFA omega-3 MAG-EPA is sufficient to reduce PCa growth in mice and cancer up-grading in pre-prostatectomy PCa patients correlating with a reduction of gut Ruminococcaceae in both and fecal butyrate levels in PCa patients. This suggests that the beneficial effect of omega-3 rich diet is mediated in part by modulating the crosstalk between gut microbes and their metabolites in men with PCa.

The effect of dose-interval on antibody response to mRNA COVID-19 vaccines: a prospective cohort study.

Background: Vaccination against COVID-19 is highly effective in preventing severe disease and hospitalization, but primary COVID mRNA vaccination schedules often differed from those recommended by the manufacturers due to supply chain issues. We investigated the impact of delaying the second dose on antibody responses to COVID mRNA-vaccines in a prospective cohort of health-care workers in Quebec. Methods: We recruited participants from the McGill University Health Centre who provided serum or participant-collected dried blood samples (DBS) at 28-days, 3 months, and 6 months post-second dose and at 28-days after a third dose. IgG antibodies to SARS-CoV2 spike (S), the receptor-binding domain (RBD), nucleocapsid (N) and neutralizing antibodies to the ancestral strain were assessed by enzyme-linked immunosorbent assay (ELISA). We examined associations between long (≤89 days) versus short (<89 days) between-dose intervals and antibody response through multivariable mixed-effects models adjusted for age, sex, prior covid infection status, time since vaccine dose, and assay batch. Findings: The cohort included 328 participants who received up to three vaccine doses (>80% Pfizer-BioNTech). Weighted averages of the serum (n=744) and DBS (n=216) cohort results from the multivariable models showed that IgG anti-S was 31% higher (95% CI: 12% to 53%) and IgG anti-RBD was 37% higher (95% CI: 14% to 65%) in the long vs. short interval participants, across all time points. Interpretation: Our study indicates that extending the covid primary series between-dose interval beyond 89 days (approximately 3 months) provides stronger antibody responses than intervals less than 89 days. Our demonstration of a more robust antibody response with a longer between dose interval is reassuring as logistical and supply challenges are navigated in low-resource settings.

The impact of Foxp3+ regulatory T-cells on CD8+ T-cell dysfunction in tumour microenvironments and responses to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have been a breakthrough in cancer therapy, inducing durable remissions in responding patients. However, they are associated with variable outcomes, spanning from disease hyperprogression to complete responses with the onset of immune-related adverse events. The consequences of checkpoint inhibition on Foxp3+ regulatory T (Treg ) cells remain unclear but could provide key insights into these variable outcomes. In this review, we first cover the mechanisms that underlie the development of hot and cold tumour microenvironments, which determine the efficacy of immunotherapy. We then outline how differences in tumour-intrinsic immunogenicity, T-cell trafficking, local metabolic environments and inhibitory checkpoint signalling differentially impair CD8+ T-cell function in tumour microenvironments, all the while promoting Treg -cell suppressive activity. Finally, we focus on the mechanisms that enable the induction of polyfunctional CD8+ T-cells upon checkpoint blockade and discuss the role of ICI-induced Treg -cell reactivation in acquired resistance to treatment.

Comparison of Omicron breakthrough infection versus monovalent SARS-CoV-2 intramuscular booster reveals differences in mucosal and systemic humoral immunity.

Our understanding of the quality of cellular and humoral immunity conferred by COVID-19 vaccination alone versus vaccination plus SARS-CoV-2 breakthrough (BT) infection remains incomplete. While the current (2023) SARS-CoV-2 immune landscape of Canadians is complex, in late 2021 most Canadians had either just received a third dose of COVID-19 vaccine, or had received their two-dose primary series and then experienced an Omicron BT. Herein we took advantage of this coincident timing to contrast cellular and humoral immunity conferred by three doses of vaccine versus two doses plus BT. Our results show thatBT infection induces cell-mediated immune responses to variants comparable to an intramuscular vaccine booster dose. In contrast, BT subjects had higher salivary immunoglobulin (Ig)G and IgA levels against the Omicron spike and enhanced reactivity to the ancestral spike for the IgA isotype, which also reacted with SARS-CoV-1. Serumneutralizing antibody levels against the ancestral strain and the variants were also higher after BT infection. Our results support the need for the development of intranasal vaccines that could emulate the enhanced mucosal and humoral immunity induced by Omicron BT without exposing individuals to the risks associated with SARS-CoV-2 infection.

The extrafollicular B cell response is a hallmark of childhood idiopathic nephrotic syndrome.

The efficacy of the B cell-targeting drug rituximab (RTX) in childhood idiopathic nephrotic syndrome (INS) suggests that B cells may be implicated in disease pathogenesis. However, B cell characterization in children with INS remains limited. Here, using single-cell RNA sequencing, we demonstrate that a B cell transcriptional program poised for effector functions represents the major immune perturbation in blood samples from children with active INS. This transcriptional profile was associated with an extrafollicular B cell response marked by the expansion of atypical B cells (atBCs), marginal zone-like B cells, and antibody-secreting cells (ASCs). Flow cytometry of blood from 13 children with active INS and 24 healthy donors confirmed the presence of an extrafollicular B cell response denoted by the expansion of proliferating RTX-sensitive extrafollicular (CXCR5-) CD21low T-bet+ CD11c+ atBCs and short-lived T-bet+ ASCs in INS. Together, our study provides evidence for an extrafollicular origin for humoral immunity in active INS.

BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine.

Recent publications have explored intranasal (i.n.) adenovirus-based (Ad) vaccines as an effective strategy for SARS-CoV-2 in pre-clinical models. However, the effects of prior immunizations and infections have yet to be considered. Here, we investigate the immunomodulatory effects of Mycobacterium bovis BCG pre-immunization followed by vaccination with an S-protein-expressing i.n. Ad, termed Ad(Spike). While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish did not affect Ad(Spike)-generated humoral immunity, it promoted the generation of cytotoxic/Th1 responses over suppressive FoxP3+ TREG cells in the lungs of infected mice. Thus, this vaccination strategy may prove useful in limiting future pandemics by potentiating the long-term efficacy of mucosal vaccines within the context of the widely distributed BCG vaccine.

Selection for immune evasion in SARS-CoV-2 revealed by high-resolution epitope mapping and sequence analysis.

Here, we exploit a deep serological profiling strategy coupled with an integrated, computational framework for the analysis of SARS-CoV-2 humoral immune responses. Applying a high-density peptide array (HDPA) spanning the entire proteomes of SARS-CoV-2 and endemic human coronaviruses allowed identification of B cell epitopes and relate them to their evolutionary and structural properties. We identify hotspots of pre-existing immunity and identify cross-reactive epitopes that contribute to increasing the overall humoral immune response to SARS-CoV-2. Using a public dataset of over 38,000 viral genomes from the early phase of the pandemic, capturing both inter- and within-host genetic viral diversity, we determined the evolutionary profile of epitopes and the differences across proteins, waves, and SARS-CoV-2 variants. Lastly, we show that mutations in spike and nucleocapsid epitopes are under stronger selection between than within patients, suggesting that most of the selective pressure for immune evasion occurs upon transmission between hosts.

The functional adaptation of effector Foxp3+ regulatory T cells to pulmonary inflammation.

During infections, the timings of effector differentiation of pulmonary immune responses are of paramount importance, as pathogen persistence and unsuppressed inflammation can rapidly lead to a loss of function, increased frailty, and death. Thus, both an efficient clearance of the danger and a rapid resolution of inflammation are critical to host survival. We now know that tissue-localized FoxP3+ regulatory T cells, a subset of CD4+ T cells, are highly attuned to the type of immune response, acquiring unique phenotypic characteristics that allow them to adapt their suppressive functions with the nature of inflammatory cells. To achieve this, activated effector TREG cells acquire specialized TH 1, TH 2, and TH 17-like characteristics that allow them to migrate, survive, and time their function(s) through refined mechanisms. Herein, we describe how this process requires a unique developmental path that includes the acquisition of master transcription factors and the expression of receptors adapted to sense local danger signals that are found during pulmonary inflammation. In turn, we offer an overview of how these characteristics promote the capacity of local effector TREG cells to proliferate, survive, and display suppressive strategies to resolve lung injury.

IL-18 is required for the TH1-adaptation of TREG cells and the selective suppression of TH17 responses in acute and chronic infections.

Interleukin (IL)-18, a member of the IL-1 family of alarmins, is abundantly released in the lungs following influenza A (IAV) infections yet its role in orchestrating the local adaptive immune response remains ill defined. Through genetic disruption of the IL-18 receptor, we demonstrate that IL-18 not only promotes pulmonary TH1 responses but also influences regulatory T cells (TREG) function in the infected lungs. As the response unfolds, TREG cells accumulating in the lungs express Helios, T-bet, CXCR3, and IL-18R1 and produce interferon γ in the presence of IL-12. During IAV, IL-18R1 is required for TREG cells to control TH17, but not TH1, responses and promote a return to lung homeostasis, revealing a novel mechanism of selective suppression. Moreover, this observation was not limited to the lungs, as skin-localized TREG cells require an IL-18 signal to specifically suppress IL-17A production by TH17 and γδ T cells in a model of chronic cutaneous Leishmania major infection. Overall, these results uncover how IL-18 orchestrates the tissue adaptation of TREG cells to selectively favor TH1 over TH17 responses during TH1-driven immune responses and provide a novel perspective into how IL-18 dictates the immune response during viral and parasitic infections.

The eIF4EBP-eIF4E axis regulates CD4+ T cell differentiation through modulation of T cell activation and metabolism.

CD4+ T cells are critical for adaptive immunity, differentiating into distinct effector and regulatory subsets. Although the transcriptional programs underlying their differentiation are known, recent research has highlighted the importance of mRNA translation in determining protein abundance. We previously conducted genome-wide analysis of translation in CD4+ T cells revealing distinct translational signatures distinguishing these subsets, identifying eIF4E as a central differentially translated transcript. As eIF4E is vital for eukaryotic translation, we examined how altered eIF4E activity affected T cell function using mice lacking eIF4E-binding proteins (BP-/-). BP-/- effector T cells showed elevated Th1 responses ex vivo and upon viral challenge with enhanced Th1 differentiation observed in vitro. This was accompanied by increased TCR activation and elevated glycolytic activity. This study highlights how regulating T cell-intrinsic eIF4E activity can influence T cell activation and differentiation, suggesting the eIF4EBP-eIF4E axis as a potential therapeutic target for controlling aberrant T cell responses.

NOD2 Agonism Counter-Regulates Human Type 2 T Cell Functions in Peripheral Blood Mononuclear Cell Cultures: Implications for Atopic Dermatitis.

Atopic dermatitis (AD) is known as a skin disease; however, T cell immunopathology found in blood is associated with its severity. Skin Staphylococcus aureus (S. aureus) and associated host-pathogen dynamics are important to chronic T helper 2 (Th2)-dominated inflammation in AD, yet they remain poorly understood. This study sought to investigate the effects of S. aureus-derived molecules and skin alarmins on human peripheral blood mononuclear cells, specifically testing Th2-type cells, cytokines, and chemokines known to be associated with AD. We first show that six significantly elevated Th2-related chemokine biomarkers distinguish blood from adult AD patients compared to healthy controls ex vivo; in addition, TARC/CCL17, LDH, and PDGF-AA/AB correlated significantly with disease severity. We then demonstrate that these robust AD-associated biomarkers, as well as associated type 2 T cell functions, are readily reproduced from healthy blood mononuclear cells exposed to the alarmin TSLP and the S. aureus superantigen SEB in a human in vitro model, including IL-13, IL-5, and TARC secretion as well as OX-40-expressing activated memory T cells. We further show that the agonism of nucleotide-binding oligomerization domain-containing protein (NOD)2 inhibits this IL-13 secretion and memory Th2 and Tc2 cell functional activation while inducing significantly increased pSTAT3 and IL-6, both critical for Th17 cell responses. These findings identify NOD2 as a potential regulator of type 2 immune responses in humans and highlight its role as an endogenous inhibitor of pathogenic IL-13 that may open avenues for its therapeutic targeting in AD.

ICOS-Deficient Regulatory T Cells Can Prevent Spontaneous Autoimmunity but Are Impaired in Controlling Acute Inflammation.

ICOS is induced in activated T cells and its main role is to boost differentiation and function of effector T cells. ICOS is also constitutively expressed in a subpopulation of Foxp3+ regulatory T cells under steady-state condition. Studies using ICOS germline knockout mice or ICOS-blocking reagents suggested that ICOS has supportive roles in regulatory T (Treg) cell homeostasis, migration, and function. To avoid any compounding effects that may arise from ICOS-deficient non-Treg cells, we generated a conditional knockout system in which ICOS expression is selectively abrogated in Foxp3-expressing cells (ICOS FC mice). Compared to Foxp3-Cre control mice, ICOS FC mice showed a minor numerical deficit of steady-state Treg cells but did not show any signs of spontaneous autoimmunity, indicating that tissue-protective Treg populations do not heavily rely on ICOS costimulation. However, ICOS FC mice showed more severe inflammation in oxazolone-induced contact hypersensitivity, a model of atopic dermatitis. This correlated with elevated numbers of inflammatory T cells expressing IFN-γ and/or TNF-α in ICOS FC mice compared with the control group. In contrast, elimination of ICOS in all T cell compartments negated the differences, confirming that ICOS has a dual positive role in effector and Treg cells. Single-cell transcriptome analysis suggested that ICOS-deficient Treg cells fail to mature into T-bet+CXCR3+ "Th1-Treg" cells in the draining lymph node. Our results suggest that regimens that preferentially stimulate ICOS pathways in Treg cells might be beneficial for the treatment of Th1-driven inflammation.

A Hemagglutinin 1 Carrying Plant-Based Virus-like Particle Vaccine Generates an Efficacious Cellular Response by Exploiting IL-1 Signaling in Both Adult and Aged Mice.

Inactivated influenza vaccines have struggled to provide consistent protection in older individuals. Circumventing immune senescence, an aging of the immune response characterized by weak humoral responses to vaccines, and unchecked inflammation during infection require novel immunization strategies. Plant-based virus-like particles (VLPs) bearing recombinant hemagglutinin proteins have been shown to provide protection in older animals in preclinical challenge studies, despite eliciting relatively low or absent humoral responses. The nature of the cellular response induced by these vaccines and its evolution during infection have not yet been fully characterized, however. Using a murine model that recapitulates features of human immune senescence, we assessed T cell responses to vaccination with a VLP bearing the hemagglutinin of H1N1/California 07/2009 (H1-VLP) before and after challenge in young and aged BALB/c mice (2 and 18 mo old, respectively). We report that two i.m. doses of H1-VLP (3 µg) vaccine 21 d apart generated H1-specific Th1 and Th2 cells associated with the prevention of prolonged pulmonary inflammation and mortality in both adult and aged mice. While investigating the regulation of cellular immunity, we identified a unique IL-1R1+ tissue-adapted regulatory T cell population in the lungs of both H1-VLP-vaccinated adult and aged mice, suggesting a novel regulatory T cell population associated with vaccine-mediated protection. Collectively, this study provides preclinical evidence that the plant-based H1-VLP vaccine may act, in part, by preventing exacerbated immune responses against influenza A.

Co-Targeting Luminal B Breast Cancer with S-Adenosylmethionine and Immune Checkpoint Inhibitor Reduces Primary Tumor Growth and Progression, and Metastasis to Lungs and Bone.

Breast cancer (BCa) is the most prevalent cancer in females and has a high rate of mortality, especially due to increased metastasis to skeletal and non-skeletal sites. Despite the marked clinical accomplishment of immune checkpoint inhibitor (CPI) therapy in patients with several cancers, it has had limited success in luminal subtypes of BCa. Accordingly, recent efforts have focused on combination therapy with CPI, including epigenetic modulators, to increase response rates of CPI in luminal BCa. We have previously shown that S-adenosylmethionine (SAM), the ubiquitous methyl donor, has strong anti-cancer effects in various cancers, including all subtypes of BCa. In the current study, we took a novel approach and examined the effect of CPI alone and in combination with SAM on tumor growth and metastasis in a syngeneic mouse model of luminal B BCa. We showed that SAM decreases cell proliferation, colony-formation (survival), and invasion of luminal B BCa cell lines (Eo771, R221A) in vitro. In in vivo studies, in Eo771 tumor-bearing mice, either SAM or anti-PD-1 antibody treatment alone significantly reduced tumor growth and progression, while the SAM+anti-PD-1 combination treatment had the highest anti-cancer efficacy of all groups. The SAM+anti-PD-1 combination reduced the percentage of animals with lung metastasis, as well as total metastatic lesion area, compared to control. Additionally, the SAM+anti-PD-1 combination significantly reduced the skeletal lesion area and protected tibial integrity to a greater extent than the monotherapies in an Eo771 bone metastasis model. Transcriptome analysis of Eo771 primary tumors revealed significant downregulation of pro-metastatic genes, including Matrix metalloproteinases (MMPs) and related pathways. On the other hand, CD8+ T cell infiltration, CD8+ T cell cytotoxicity (elevated granzymes), and immunostimulatory genes and pathways were significantly upregulated by the combination treatment. The results presented point to a combination of SAM with CPI as a possible treatment for luminal B BCa that should be tested in clinical studies.

Age- and sex-mediated differences in T lymphocyte populations of kidney transplant recipients.

BACKGROUND: Graft failure rates increase through childhood and adolescence, decline in adulthood, and are higher in female than male kidney transplant recipients (KTR) until middle age. We aimed to describe age- and sex-related differences in T-cell subsets among KTR to determine which differences may help to explain the differences in kidney graft failure rates. METHODS: Effector T (Teff)-cell and regulatory T (Treg)-cell phenotypes in PBMCs from healthy controls and KTR, who were at least 1 year post-transplant with stable graft function under immunosuppression, were analyzed by flow cytometry. The effects of age, sex, and status (KTR or control) were analyzed using linear regressions. RESULTS: We enrolled 20 male and 21 female KTR and 20 male and 20 female controls between 3 and 29 years of age. CD3+ T-cell frequencies were not associated with age or sex but were higher in KTR than controls. There were no differences in CD4+ and CD8+ frequencies. Th1 (IFNγ+ IL-4- IL-17A-) and Th17 (IL-17A+) frequencies within the CD4+ T-cell population were higher at older ages. The frequencies of FOXP3 + Helios + Treg cells in CD4+ CD25+ CD127- T cells were lower in females than males and in KTR than controls. CONCLUSIONS: Increasing frequencies of Th1 and Th17 cells with increasing age mirrors the increasing graft failure rates from childhood to young adulthood. Importantly, sex differences in frequencies of circulating Treg cells may suggest a role in the sex differences in graft failure rates.

Successful Milk Oral Immunotherapy Promotes Generation of Casein-Specific CD137+ FOXP3+ Regulatory T Cells Detectable in Peripheral Blood.

Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (TREG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific TREG in a cohort of subjects undergoing OIT. Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4+ T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4+ T cell phenotypes were quantified by flow cytometry. Results: Our culture system induced activated casein-specific FOXP3+Helios+ TREG cells and FOXP3- TEFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific TREG cells increased significantly with escalating doses of milk during OIT while casein-specific TEFF cell frequencies remained constant. Moreover, expanded casein-specific TREG cells expressed higher levels of FOXP3 compared to polyclonal TREG cells, suggesting a more robust TREG phenotype. The induction of casein-specific TREG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific TREG cells negatively correlated with the time required to reach the maintenance phase of desensitization. Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3+ TREG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro TREG response to casein may correlate with the time to reach maintenance in CMP-OIT.

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.

FOXP3 and Tip60 Structural Interactions Relevant to IPEX Development Lead to Potential Therapeutics to Increase FOXP3 Dependent Suppressor T Cell Functions.

Regulatory T (Treg) cells play a role in the maintenance of immune homeostasis and are critical mediators of immune tolerance. The Forkhead box P3 (FOXP3) protein acts as a regulator for Treg development and function. Mutations in the FOXP3 gene can lead to autoimmune diseases such as Immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome in humans, often resulting in death within the first 2 years of life and a scurfy like phenotype in Foxp3 mutant mice. We discuss biochemical features of the FOXP3 ensemble including its regulation at various levels (epigenetic, transcriptional, and post-translational modifications) and molecular functions. The studies also highlight the interactions of FOXP3 and Tat-interacting protein 60 (Tip60), a principal histone acetylase enzyme that acetylates FOXP3 and functions as an essential subunit of the FOXP3 repression ensemble complex. Lastly, we have emphasized the role of allosteric modifiers that help stabilize FOXP3:Tip60 interactions and discuss targeting this interaction for the therapeutic manipulation of Treg activity.