Ulf H. Beier

Helios Expression Is a Marker of T Cell Activation and Proliferation

Foxp3+ T-regulatory cells (Tregs) normally serve to attenuate immune responses and are key to maintenance of immune homeostasis. Over the past decade, Treg cells have become a major focus of research for many groups, and various functional subsets have been characterized. Recently, the Ikaros family member, Helios, was reported as a marker to discriminate naturally occurring, thymic-derived Tregs from those peripherally induced from naïve CD4+ T cells. We investigated Helios expression in murine and human T cells under resting or activating conditions, using well-characterized molecules of naïve/effector/memory phenotypes, as well as a set of Treg-associated markers. We found that Helios-negative T cells are enriched for naïve T cell phenotypes and vice versa. Moreover, Helios can be induced during T cell activation and proliferation, but regresses in the same cells under resting conditions. We demonstrated comparable findings using human and murine CD4+Foxp3+ Tregs, as well as in CD4+ and CD8+ T cells. Since Helios expression is associated with T cell activation and cellular division, regardless of the cell subset involved, it does not appear suitable as a marker to distinguish natural and induced Treg cells.

Histone Deacetylase 6 and Heat Shock Protein 90 Control the Functions of Foxp3+ T-Regulatory Cells

ABSTRACT Foxp3+ T-regulatory cells (Tregs) are key to immune homeostasis such that their diminished numbers or function can cause autoimmunity and allograft rejection. Foxp3+ Tregs express multiple histone/protein deacetylases (HDACs) that regulate chromatin remodeling, gene expression, and protein function. Pan-HDAC inhibitors developed for oncologic applications enhance Treg production and Treg suppression function but have limited nononcologic utility given their broad actions and various side effects. We show, using HDAC6-deficient mice and wild-type (WT) mice treated with HDAC6-specific inhibitors, that HDAC6 inhibition promotes Treg suppressive activity in models of inflammation and autoimmunity, including multiple forms of experimental colitis and fully major histocompatibility complex (MHC)-incompatible cardiac allograft rejection. Many of the beneficial effects of HDAC6 targeting are also achieved by inhibition of the HDAC6-regulated protein heat shock protein 90 (HSP90). Hence, selective targeting of a single HDAC isoform, HDAC6, or its downstream target, HSP90, can promote Treg-dependent suppression of autoimmunity and transplant rejection.

Sirtuin-1 Targeting Promotes Foxp3+ T-Regulatory Cell Function and Prolongs Allograft Survival

ABSTRACT 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. However, Sirt1 targeting promoted the expression of Foxp3, a key transcription factor in T-regulatory (Treg) cells, and increased Treg suppressive functions in vitro and in vivo. Consistent with these data, mice with targeted deletions of Sirt1 in either CD4+ T cells or Foxp3+ Treg cells exhibited prolonged survival of major histocompatibility complex (MHC)-mismatched cardiac allografts. Allografts in Sirt1-targeted recipients showed long-term preservation of myocardial histology and infiltration by Foxp3+ Treg cells. Comparable results were seen in wild-type allograft recipients treated with Sirt1 inhibitors, such as EX-527 and splitomicin. Hence, Sirt1 may inhibit Treg functions, and its targeting may have therapeutic value in autoimmunity and transplantation.

Histone Deacetylases 6 and 9 and Sirtuin-1 Control Foxp3+ Regulatory T Cell Function Through Shared and Isoform-Specific Mechanisms

Combined inhibition of distinct histone deacetylases enhances the suppressive effects of regulatory T cells. Enhancing Regulatory T Cell Function Some therapies designed to help patients suffering from autoimmune diseases or recovering from organ transplant involve the removal of the patient’s regulatory T cells (Tregs), which suppress the undesirable proinflammatory actions of effector T cells, so that they can be expanded in number ex vivo before being put back into the patient. One problem associated with this approach is that the phenotype of the administered Tregs is unstable, and so their suppressive function is lost over time. Previous studies have shown that inhibition of certain members of the family of histone deacetylases (HDACs) enhances the function of Tregs. Through experiments with mice deficient in distinct HDACs and treated with HDAC-specific inhibitors, Beier et al. have demonstrated the mechanisms by which the simultaneous inhibition of combinations of distinct HDACs improved the suppressive function of Tregs and enhanced their stability, which suggests that such an approach may be of benefit to patients reliant on such therapies. Therapeutic inhibition of the histone deacetylases HDAC6, HDAC9, or sirtuin-1 (Sirt1) augments the suppressive functions of regulatory T cells (Tregs) 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 Tregs 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 Tregs treated with isoform-selective HDAC inhibitors. The improvement of Treg suppressive function mediated by inhibition of HDAC6, but not Sirt1, required an intact heat shock response. Although HDAC6, HDAC9, and Sirt1 all deacetylated Foxp3, each protein had different effects on transcription factors that control expression of the gene encoding Foxp3. For example, loss of HDAC9, but not other HDACs, was associated with stabilization of the acetylated form of signal transducer and activator of transcription 5 (STAT5) and promoted its transcriptional activity. Thus, targeting different HDACs increased Treg function through multiple and additive mechanisms, which suggests the therapeutic potential for using combinations of HDAC inhibitors in the management of autoimmunity and organ transplantation.

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

Forkhead box P3 (Foxp3)+ T regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors. Foxp3+ Treg 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+ Treg cells can enhance antitumor responses provide proof of principle, these studies lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and nonhistone proteins. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (also known as Ep300 or KAT3B), in Foxp3+ Treg cells increased T cell receptor–induced apoptosis in Treg cells, impaired Treg cell suppressive function and peripheral Treg cell induction, and limited tumor growth in immunocompetent but not in immunodeficient mice. Our data thereby demonstrate that p300 is important for Foxp3+ Treg cell function and homeostasis in vivo and in vitro, and identify mechanisms by which appropriate small-molecule inhibitors can diminish Treg cell function without overtly impairing T effector cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.

Histone/protein deacetylases and T-cell immune responses

Clinical and experimental studies show that inhibition of histone/protein deacetylases (HDAC) can have important anti-neoplastic effects through cytotoxic and proapoptotic mechanisms. There are also increasing data from nononcologic settings that HDAC inhibitors (HDACi) can exhibit useful anti-inflammatory effects in vitro and in vivo, unrelated to cytotoxicity or apoptosis. These effects can be cell-, tissue-, or context-dependent and can involve modulation of specific inflammatory signaling pathways as well as epigenetic mechanisms. We review recent advances in the understanding of how HDACi alter immune and inflammatory processes, with a particular focus on the effects of HDACi on T-cell biology, including the activation and functions of conventional T cells and the unique T-cell subset, composed of Foxp3(+) T-regulatory cells. Although studies are still needed to tease out details of the various biologic roles of individual HDAC isoforms and their corresponding selective inhibitors, the anti-inflammatory effects of HDACi are already promising and may lead to new therapeutic avenues in transplantation and autoimmune diseases.

Histone/protein deacetylases control Foxp3 expression and the heat shock response of T-regulatory cells

Lysine ɛ-acetylation is a post-translational modification that alters the biochemical properties of many proteins. The reaction is catalyzed by histone/protein acetyltransferases (HATs), and is reversed by histone/protein deacetylases (HDACs). As a result, HATs and HDACs constitute an important, though little recognized, set of proteins that control the functions of T-regulatory (Treg) cells. Targeting certain HDACs, especially HDAC6, HDAC9, and Sirtuin-1 (Sirt1), can augment Treg suppressive potency by several distinct and potentially additive mechanisms. These involve promoting Forkhead box p3 (Foxp3) gene expression and preserving Foxp3 lysine ɛ-acetylation, which infers resistance to ubiquitination and proteasomal degradation, and increases DNA binding. Moreover, depleting certain HDAC can enhance the heat shock response, which increases the tenacity of Treg to survive under stress, and helps preserve a suppressive phenotype. As a result, HDAC inhibitor therapy can be used to enhance Treg functions in vivo and have beneficial effects on allograft survival and autoimmune diseases.

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. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg‐dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase‐9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9‐/‐ Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild‐type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg‐dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo.—Beier, U. H., Angelin, A., Akimova, T., Wang, L., Liu, Y., Xiao, H., Koike, M. A., Hancock, S. A., Bhatti, T. R., Han, R., Jiao, J., Veasey, S. C., Sims, C. A., Baur, J. A., Wallace, D. C., Hancock, W. W. Essential role of mitochondrial energy metabolism in Foxp3+ T‐regulatory cell function and allograft survival. FASEB J. 29, 2315‐2326 (2015). www.fasebj.org

HDAC inhibitor therapy in autoimmunity and transplantation

Pharmacological inhibitors of histone/protein deacetylases (HDACi) have considerable therapeutic potential as anti-inflammatory and immunosuppressive drugs. The utility of HDACi as anti-inflammatory agents is dependent upon their proving safe and effective in experimental models. Current pan-HDACi compounds are ill-suited to this role, given the broad distribution of target HDACs and their complex and multifaceted mechanisms of action. By contrast, the development of isoform-selective HDACi may provide important new tools for treatment in autoimmunity and transplantation. This review discusses which HDACs are worthwhile targets in inflammation, and the progress made towards their therapeutic inhibition, including the use of HDAC subclass and isoform-selective HDACi to promote the functions of Foxp3+ T-regulatory cells.

Mbd2 Promotes Foxp3 Demethylation and T-Regulatory-Cell Function

ABSTRACT 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. Surprisingly, while chromatin immunoprecipitation (ChIP) analysis showed Mbd2 binding to the Foxp3-associated TSDR site in Treg cells, Mbd2 targeting by homologous recombination, or small interfering RNA (siRNA), decreased Treg numbers and impaired Treg-suppressive function in vitro and in vivo. Moreover, we found complete TSDR demethylation in wild-type (WT) Treg cells but >75% methylation in Mbd2−/− Treg cells, whereas reintroduction of Mbd2 into Mbd2-null Treg cells restored TSDR demethylation, Foxp3 gene expression, and Treg-suppressive function. Lastly, thymic Treg cells from Mbd2−/− mice had normal TSDR demethylation, but compared to WT Treg cells, peripheral Mbd2−/− Treg cells had a marked impairment of binding of Tet2, the DNA demethylase enzyme, at the TSDR site. These data show that Mbd2 has a key role in promoting TSDR demethylation, Foxp3 expression, and Treg-suppressive function.