T-regulatory cells require Sin3a for stable expression of Foxp3.

Histone deacetylases 1 and 2 play a major role in the transcriptional regulation of T-regulatory (Treg) cells via interactions with a myriad of coregulatory factors. Sin3a has been well established as a Hdac1/2 cofactor, while its role within Tregs has not been established. In this study, the effects of conditional deletion of Sin3a within Foxp3+ Tregs were evaluated.

Obesity-related IL-18 Impairs T-Regulatory Cell Function and Promotes Lung Ischemia-Reperfusion Injury.

Primary graft dysfunction (PGD) is a severe form of acute lung injury, leading to increased early morbidity and mortality after lung transplant. Obesity is a major health problem, and recipient obesity is one of the most significant risk factors for developing PGD. We hypothesized that T-regulatory cells (Tregs) are able to dampen early ischemia-reperfusion events and thereby decrease the risk of PGD, whereas that action is impaired in obese recipients.

A Biological Circuit Involving Mef2c, Mef2d, and Hdac9 Controls the Immunosuppressive Functions of CD4+Foxp3+ T-Regulatory Cells.

The Mads/Mef2 (Mef2a/b/c/d) family of transcription factors (TFs) regulates differentiation of muscle cells, neurons and hematopoietic cells. By functioning in physiological feedback loops, Mef2 TFs promote the transcription of their repressor, Hdac9, thereby providing temporal control of Mef2-driven differentiation. Disruption of this feedback is associated with the development of various pathologic states, including cancer.

MEF2D sustains activation of effector Foxp3+ Tregs during transplant survival and anticancer immunity.

The transcription factor MEF2D is important in the regulation of differentiation and adaptive responses in many cell types. We found that among T cells, MEF2D gained new functions in Foxp3+ T regulatory (Treg) cells due to its interactions with the transcription factor Foxp3 and its release from canonical partners, like histone/protein deacetylases. Though not necessary for the generation and maintenance of Tregs, MEF2D was required for the expression of IL-10, CTLA4, and Icos, and for the acquisition of an effector Treg phenotype.

Limited efficacy of rapamycin monotherapy in vascularized composite allotransplantation.

Background: Vascularized composite allotransplantation (VCA) is a novel and life-enhancing procedure to restore a patient's function and/or appearance. Current immunosuppression in VCA recipients is based on calcineurin inhibitor (CNI) therapy that can lead to severe complications, such that inducing immune tolerance is a major goal of VCA research. In contrast to CNI, rapamycin (RPM) is thought to be beneficial to the development of immune tolerance by suppressing T-effector cells (Teffs) and expanding T-regulatory (Treg) cells.

Donor bone-marrow CXCR4+ Foxp3+ T-regulatory cells are essential for costimulation blockade-induced long-term survival of murine limb transplants.

Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM.

HDAC10 deletion promotes Foxp3 T-regulatory cell function.

Foxp3 T-regulatory (Treg) cells are capable of suppressing immune responses. Lysine acetylation is a key mechanism of post-translational control of various transcription factors, and when acetylated, Foxp3 is stabilized and transcriptionally active. Therefore, understanding the roles of various histone/protein deacetylases (HDAC) are key to promoting Treg-based immunotherapy.

Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity.

Foxp3+ Tregs are key to immune homeostasis, but the contributions of various large, multiprotein complexes that regulate gene expression remain unexplored. We analyzed the role in Tregs of the evolutionarily conserved CoREST complex, consisting of a scaffolding protein, Rcor1 or Rcor2, plus Hdac1 or Hdac2 and Lsd1 enzymes. Rcor1, Rcor2, and Lsd1 were physically associated with Foxp3, and mice with conditional deletion of Rcor1 in Foxp3+ Tregs had decreased proportions of Tregs in peripheral lymphoid tissues and increased Treg expression of IL-2 and IFN-γ compared with what was found in WT cells.

Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells.

Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival.

Utility of IL-2 Complexes in Promoting the Survival of Murine Orthotopic Forelimb Vascularized Composite Allografts.

Background: Vascularized composite allografts (VCA) are novel, life-enhancing forms of transplantation (Tx). However, host immune responses to the various VCA components, especially those involving skin, are complex and make selection of appropriate therapy challenging. Although the interplay between Foxp3+ T regulatory (Treg) cells and CD4 and CD8 effector T cells is of central importance in determining the acceptance or rejection of solid organ allografts, there is little information available concerning the contribution of Treg cells to VCA survival.

Histone/protein deacetylase 11 targeting promotes Foxp3+ Treg function.

Current interest in Foxp3+ T-regulatory (Treg) cells as therapeutic targets in transplantation is largely focused on their harvesting pre-transplant, expansion and infusion post-transplantation. An alternate strategy of pharmacologic modulation of Treg function using histone/protein deacetylase inhibitors (HDACi) may allow more titratable and longer-term dosing. However, the effects of broadly acting HDACi vary, such that HDAC isoform-selective targeting is likely required.

Foxp3 Reprograms T Cell Metabolism to Function in Low-Glucose, High-Lactate Environments.

Immune cells function in diverse metabolic environments. Tissues with low glucose and high lactate concentrations, such as the intestinal tract or ischemic tissues, frequently require immune responses to be more pro-tolerant, avoiding unwanted reactions against self-antigens or commensal bacteria. T-regulatory cells (Tregs) maintain peripheral tolerance, but how Tregs function in low-glucose, lactate-rich environments is unknown.

Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3+ T-regulatory Cell Function and Promotes Antitumor Immunity.

Foxp3+ T-regulatory (Treg) cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or "secondary" modulation, e.g. using anti-CTLA-4 monoclonal antibody.

Proximity Ligation Assay to Quantify Foxp3 Acetylation in Regulatory T Cells.

Determining protein acetylation by immunoprecipitation and immunoblotting can be challenging, especially if the tissue of interest is low in quantity, and when good quality acetylation site-specific antibodies are not available. Proximity ligation assays allow a sensitive and quantitative method to assess Foxp3 acetylation in regulatory T cells, with as little as 1.5 × 10 cells within two days turnaround time.

HDAC5 controls the functions of Foxp3(+) T-regulatory and CD8(+) T cells.

Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy.

Essential role of mitochondrial energy metabolism in Foxp3⁺ T-regulatory cell function and allograft survival.

Conventional T (Tcon) cells and Foxp3(+) T-regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)-induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild-type cells but disproportionally affected Treg cells.

FOXP3+ regulatory T cell development and function require histone/protein deacetylase 3.

Treg dysfunction is associated with a variety of inflammatory diseases. Treg populations are defined by expression of the oligomeric transcription factor FOXP3 and inability to produce IL-2, a cytokine required for T cell maintenance and survival. FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs).

Two histone/protein acetyltransferases, CBP and p300, are indispensable for Foxp3+ T-regulatory cell development and function.

T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability.

Mbd2 promotes foxp3 demethylation and T-regulatory-cell function.

Use of Foxp3-positive (Foxp3(+)) T-regulatory (Treg) cells as potential cellular therapy in patients with autoimmunity, or post-stem cell or -organ transplantation, requires a sound understanding of the transcriptional regulation of Foxp3. Conserved CpG dinucleotides in the Treg-specific demethylation region (TSDR) upstream of Foxp3 are demethylated only in stable, thymus-derived Foxp3(+) Treg cells. Since methyl-binding domain (Mbd) proteins recruit histone-modifying and chromatin-remodeling complexes to methylated sites, we tested whether targeting of Mbd2 might promote demethylation of Foxp3 and thereby promote Treg numbers or function.

Inhibition of p300 impairs Foxp3⁺ T regulatory cell function and promotes antitumor immunity.

Forkhead box P3 (Foxp3)(+) T regulatory (T(reg)) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors. Foxp3(+) T(reg) cells are therefore considered promising targets to enhance antitumor immunity, and approaches for their therapeutic modulation are being developed. However, although studies showing that experimentally depleting Foxp3(+) T(reg) cells can enhance antitumor responses provide proof of principle, these studies lack clear translational potential and have various shortcomings.

Foxp3+ T-regulatory cells require DNA methyltransferase 1 expression to prevent development of lethal autoimmunity.

Protocols to use Foxp3+ T-regulatory (Treg) cells for cellular therapy, especially postallogeneic stem cell transplantation, are currently being developed and tested by various groups. Inhibitors of DNA methyltransferase (Dnmt) enzymes have been advocated as a means to promote and stabilize Foxp3 expression in Tregs undergoing expansion in vitro before their injection in vivo. We investigated the effects of conditionally deleting two Dnmt enzymes that co-immunoprecipitated with Foxp3 in Treg isolates.

Histone deacetylases 6 and 9 and sirtuin-1 control Foxp3+ regulatory T cell function through shared and isoform-specific mechanisms.

Therapeutic inhibition of the histone deacetylases HDAC6, HDAC9, or sirtuin-1 (Sirt1) augments the suppressive functions of regulatory T cells (T(regs)) that contain the transcription factor Foxp3 (Forkhead box P3) and is useful in organ transplant patients or patients with autoimmune diseases. However, it is unclear whether distinct mechanisms are involved for each HDAC or whether combined inhibition of HDACs would be more effective. We compared the suppressive functions of T(regs) from wild-type C57BL/6 mice with those from mice with either complete or cell-specific deletion of various HDACs, as well as with those of T(regs) treated with isoform-selective HDAC inhibitors.

Two lysines in the forkhead domain of foxp3 are key to T regulatory cell function.

Background: The forkhead box transcription factor, Foxp3, is master regulator of the development and function of CD4+CD25+ T regulatory (Treg) cells that limit autoimmunity and maintain immune homeostasis. The carboxyl-terminal forkhead (FKH) domain is required for the nuclear localization and DNA binding of Foxp3. We assessed how individual FKH lysines contribute to the functions of Foxp3 in Treg cells.

Sirtuin-1 targeting promotes Foxp3+ T-regulatory cell function and prolongs allograft survival.

Sirtuin 1 (Sirt1), a class III histone/protein deacetylase, is central to cellular metabolism, stress responses, and aging, but its contributions to various host immune functions have been little investigated. To study the role of Sirt1 in T cell functions, we undertook targeted deletions by mating mice with a floxed Sirt1 gene to mice expressing CD4-cre or Foxp3-cre recombinase, respectively. We found that Sirt1 deletion left conventional T-effector cell activation, proliferation, and cytokine production largely unaltered.