Wayne Hancock - Academia.edu (original) (raw)

Papers by Wayne Hancock

Research paper thumbnail of A Novel Role for Histone Deacetylase 6 in the Regulation of the Tolerogenic STAT3/IL-10 Pathway in APCs

Journal of Immunology, Aug 8, 2014

Antigen-presenting cells (APCs) are critical in T-cell activation and in the induction of T-cell ... more Antigen-presenting cells (APCs) are critical in T-cell activation and in the induction of T-cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them, histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. Here we show for the first time, that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells resulted in diminished production of the immunosuppressive cytokine IL-10, and induction of inflammatory APCs that effectively activate antigen-specific naïve T-cells and restore the responsiveness of anergic CD4 + T-cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising aminoacids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are

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

Frontiers in Immunology

The Mads/Mef2 (Mef2a/b/c/d) family of transcription factors (TFs) regulates differentiation of mu... more 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. Beside their direct involvement in oncogenesis, Mef2 TFs indirectly control tumor progression by regulating antitumor immunity. We recently reported that in CD4+CD25+Foxp3+ T-regulatory (Treg) cells, Mef2d is required for the acquisition of an effector Treg (eTreg) phenotype and for the activation of an epigenetic program that suppresses the anti-tumor immune responses of conventional T and B cells. We now report that as with Mef2d, the deletion of Mef2c in Tregs switches off the expression of Il10 and Icos and leads to enhanced antitumor immunity in syngeneic ...

Research paper thumbnail of Nuclear Coregulatory Complexes in Tregs as Targets to Promote Anticancer Immune Responses

Frontiers in Immunology

T-regulatory (Treg) cells display considerable heterogeneity in their responses to various cancer... more T-regulatory (Treg) cells display considerable heterogeneity in their responses to various cancers. The functional differences among this cell type are heavily influenced by multiprotein nuclear complexes that control their gene expression. Many such complexes act mechanistically by altering epigenetic profiles of genes important to Treg function, including the forkhead P3 (Foxp3) transcription factor. Complexes that form with certain members of the histone/protein deacetylase (HDAC) class of enzymes, like HDACs 1, 2, and 3, along with histone methyltransferase complexes, are important in the induction and stabilization of Foxp3 and Treg identity. The functional behavior of both circulating and intratumoral Tregs greatly impacts the antitumor immune response and can be predictive of patient outcome. Thus, targeting these regulatory complexes within Tregs may have therapeutic potential, especially in personalized immunotherapies.

Research paper thumbnail of Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells

Cancers, 2019

Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransfer... more 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. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acu...

Research paper thumbnail of Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity

Journal of Clinical Investigation, 2020

Research paper thumbnail of HDAC10 deletion promotes Foxp3+ T-regulatory cell function

Scientific Reports, 2020

Foxp3+ T-regulatory (Treg) cells are capable of suppressing immune responses. Lysine acetylation ... more 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. Several of the 11 classical HDAC enzymes are necessary for optimal Treg function while others are dispensable. We investigated the effect of HDAC10 in murine Tregs. HDAC10 deletion had no adverse effect on the health of mice, which retained normal CD4+ and CD8+ T cell function. However, HDAC10−/− Treg exhibited increased suppressive function in vitro and in vivo. C57BL/6 Rag1−/− mice adoptively transferred with HDAC10−/− but not wild Treg, were protected from developing colitis. HDAC10−/− but not wild-type mice receiving fully MHC-mismatched cardiac transplants became tolerant and showed long-term allograft su...

Research paper thumbnail of How little is known about the role of human Foxp3+ Tregs in tumors

Expert opinion on therapeutic targets, Jan 10, 2018

Research paper thumbnail of Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60

PloS one, 2017

Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with... more Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-...

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

Transplantation, 2017

Background-Vascularized composite allografts (VCA) are novel, life-enhancing forms of transplanta... more 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+ Tregulatory (Treg) cells and CD4 and CD8 effector Tcells 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. In addition, the effects of therapeutic expansion in vivo of host Treg cell populations on VCA survival are unknown. Methods-We established a fully major histocompatibility complex-disparate (BALB/c-> C57BL/6) murine orthotopic forelimb Tx model to explore the benefits of pre-and post-Tx IL-2/ anti-IL-2 monoclonal antibody complex (IL-2C) administration to expand the host Treg cell population and thereby attempt to promote Treg cell-dependent VCA survival.

Research paper thumbnail of Human lung tumor FOXP3+ Tregs upregulate four "Treg-locking" transcription factors

JCI insight, Jan 17, 2017

Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses... more Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses, but the properties of human intratumoral Tregs are still largely unknown, in part due to significant methodologic problems. We studied the phenotypic, functional, epigenetic, and transcriptional features of Tregs in 92 patients with non-small-cell lung cancer, comparing the features of Tregs within tumors versus corresponding blood, lung, and lymph node samples. Intratumoral Treg numbers and suppressive function were significantly increased compared with all other sites but did not display a distinctive phenotype by flow cytometry. However, by undertaking simultaneous evaluation of mRNA and protein expression at the single-cell level, we demonstrated that tumor Tregs have a phenotype characterized by upregulated expression of FOXP3 mRNA and protein as well as significantly increased expression of EOS, IRF4, SATB1, and GATA1 transcription factor mRNAs. Expression of these "Treg-loc...

Research paper thumbnail of T-cells lacking HDAC11 have increased effector functions and mediate enhanced alloreactivity in a murine model

Blood, Jul 26, 2017

Histone acetylation and the families of enzymes responsible for controlling these epigenetic mark... more Histone acetylation and the families of enzymes responsible for controlling these epigenetic marks have been implicated in regulating T-cell maturation and phenotype. Here, we demonstrate a previously undefined role of histone deacetylase 11 (HDAC11) in regulating T-cell effector functions. Using EGFP-HDAC11 transgenic reporter mice, we found that HDAC11 expression was lower in effector relative to naïve and central memory T-cell populations, and activation of resting T-cells resulted in its decreased expression. Experiments using HDAC11 knockout (KO) mice revealed that T-cells from these mice displayed enhanced proliferation, pro-inflammatory cytokine production and effector molecule expression. Additionally, HDAC11KO T-cells had increased expression of Eomesodermin (Eomes) and TBX21 (Tbet), transcription factors previously shown to regulate inflammatory cytokine and effector molecule production. Conversely, overexpression of HDAC11 resulted in decreased expression of these genes. ...

Research paper thumbnail of Proximity Ligation Assay to Quantify Foxp3 Acetylation in Regulatory T Cells

Methods in molecular biology (Clifton, N.J.), 2017

Determining protein acetylation by immunoprecipitation and immunoblotting can be challenging, esp... more 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(5) cells within two days turnaround time. This method is of potential use in other similar scenarios, when post-translational modifications of a protein of interest need to be determined with only a small amount of sample and in the absence of specific antibodies that can assess the post-translational modification in the protein of interest.

Research paper thumbnail of Isoform-Selective HDAC Inhibitor Therapy for Transplantation

Transplantation, 2016

The 18 known mammalian histone/protein deacetylases (HDACs) are divided into 4 groups, termed cla... more The 18 known mammalian histone/protein deacetylases (HDACs) are divided into 4 groups, termed class I (HDAC1, HDAC2, HDAC3, HDAC8), class IIa (HDAC4, HDAC5, HDAC7, HDAC9), class IIb (HDAC6, HDAC10), class III (SIRT1-7) and class IV (HDAC11) enzymes 1. While these enzymes were initially defined by their ability to deacetylate histones and dampen histone-DNA and histone-protein interactions, they are now recognized as regulating the functions of thousands of nonhistone proteins 2. A large number of broadly active pharmacologic HDAC inhibitors (pan-HDACi) have been developed and are in clinical trials as anti-cancer agents due to their abilities to promote tumor cell-cycle arrest, differentiation and apoptosis 3. There is also interest in the potential use of HDACi therapy for autoimmunity and transplantation, but there are concerns that the various pan-HDACi compounds may be too broadly acting and/or toxic for clinical use beyond oncology.

Research paper thumbnail of Targeting Sirtuin-1 prolongs murine renal allograft survival and function

Kidney International, 2016

Current immunosuppressive medications used after transplantation have significant toxicities. Fox... more Current immunosuppressive medications used after transplantation have significant toxicities. Foxp3 + T-regulatory (Treg) cells can prevent allograft rejection without compromising protective host immunity. Interestingly, inhibiting the class III histone/protein deacetylase Sirtuin-1 can augment Foxp3 + Treg suppressive function through increasing Foxp3 acetylation. Here we determined whether Sirtuin-1 targeting can stabilize biological allograft function. BALB/c kidney allografts were transplanted into C57BL/6 recipients with a CD4-conditional deletion of Sirtuin-1 (Sirt1 fl/fl CD4 cre) or mice treated with a Sirtuin-1 specific inhibitor (EX-527), and the native kidneys removed. Blood chemistries and hematocrit were followed weekly. Sirt1 fl/fl CD4 cre recipients showed markedly longer survival and improved kidney function. Sirt1 fl/fl CD4 cre recipients exhibited donor specific tolerance, accepted BALB/c, but rejected third-party C3H cardiac allografts. C57BL/6 recipients of BALB/c renal allografts that were treated with EX-527 showed improved survival and renal function at 1, but not 10 mg/kg/day. Pharmacologic inhibition of Sirtuin-1 also improved renal allograft survival and function with dosing effects having relevance to outcome. Thus, inhibiting Sirtuin-1 can be a useful asset in controlling T-cell mediated rejection. However, effects on non-T cells that could adversely affect allograft survival and function merit consideration.

Research paper thumbnail of Hdac Inhibitors and Therapeutic Methods Using the Same

Research paper thumbnail of Thiol-Based Potent and Selective HDAC6 Inhibitors Promote Tubulin Acetylation and T-Regulatory Cell Suppressive Function

ACS medicinal chemistry letters, Jan 12, 2015

Several new mercaptoacetamides were synthesized and studied as HDAC6 inhibitors. One compound, 2b... more Several new mercaptoacetamides were synthesized and studied as HDAC6 inhibitors. One compound, 2b, bearing an aminoquinoline cap group, was found to show 1.3 nM potency at HDAC6, with >3000-fold selectivity over HDAC1. 2b also showed excellent efficacy at increasing tubulin acetylation in rat primary cortical cultures, inducing a 10-fold increase in acetylated tubulin at 1 μM. To assess possible therapeutic effects, compounds were assayed for their ability to increase T-regulatory (Treg) suppressive function. Some but not all of the compounds increased Treg function, and thereby decreased conventional T cell activation and proliferation in vitro.

Research paper thumbnail of Pim-2 kinase influences Tregulatory cell function and stability by mediating Foxp3 N-terminal phosphorylation

The Journal of biological chemistry, Jan 18, 2015

Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limi... more Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4+CD25+Foxp3+ regulatory T cells (Treg) play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg development and function. Foxp3 function itself, is directly regulated by multiple post-translational modifications (PTMs) that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macro-molecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here, we have identified a mechanism regulating Foxp3 level and activity that operate through discreet phosphorylation. The Pim-2 kinase can phosphorylate Foxp3 leading to decreased suppressive functions of Treg cells. The amino terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to ...

Research paper thumbnail of Class-Specific Histone/Protein Deacetylase Inhibition Protects Against Renal Ischemia Reperfusion Injury and Fibrosis Formation

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, Jan 23, 2015

Renal ischemia-reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure.... more Renal ischemia-reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure. Histone/protein deacetylases (HDACs) regulate gene accessibility and higher order protein structures and may alter cellular responses to a variety of stresses. We investigated whether use of pan- and class-specific HDAC inhibitors (HDACi) could improve IRI tolerance in the kidney. Using a model of unilateral renal IRI, we investigated early renal function after IRI, and calculated fibrosis after IRI using an automated scoring system. We found that pan-HDAC inhibition using trichostatin (TSA) yielded significant renal functional benefit at 24-96 hours (p < 0.001). Treated mice developed significantly less fibrosis at 30 days (p < 0.0004). Class I HDAC inhibition with MS-275 yielded similar effects. Protection from fibrosis formation was also noted in a cold ischemia transplant model (p < 0.008) with a trend toward improved cold ischemic survival in TSA-treated mice. These effect...

Research paper thumbnail of A Novel Role for Histone Deacetylase 6 in the Regulation of the Tolerogenic STAT3/IL-10 Pathway in APCs

The Journal of Immunology, 2014

APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modif... more APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4+ T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain compri...

Research paper thumbnail of Structural and biological features of FOXP3 dimerization relevant to regulatory T cell function

Cell reports, Jan 28, 2012

FOXP3 is a key transcription factor for regulatory T cell function. We report the crystal structu... more FOXP3 is a key transcription factor for regulatory T cell function. We report the crystal structure of the FOXP3 coiled-coil domain, through which a loose or transient dimeric association is formed and modulated, accounting for the activity variations introduced by disease-causing mutations or posttranslational modifications. Structure-guided mutagenesis revealed that FOXP3 coiled-coil-mediated homodimerization is essential for Treg function in vitro and in vivo. In particular, we identified human FOXP3 K250 and K252 as key residues for the conformational change and stability of the FOXP3 dimer, which can be regulated by protein posttranslational modifications such as reversible lysine acetylation. These studies provide structural and mechanistic explanations for certain disease-causing mutations in the coiled-coil domain of FOXP3 that are commonly found in IPEX syndrome. Overall, the regulatory machinery involving homooligomerization, acetylation, and heteroassociation has been dis...

Research paper thumbnail of A Novel Role for Histone Deacetylase 6 in the Regulation of the Tolerogenic STAT3/IL-10 Pathway in APCs

Journal of Immunology, Aug 8, 2014

Antigen-presenting cells (APCs) are critical in T-cell activation and in the induction of T-cell ... more Antigen-presenting cells (APCs) are critical in T-cell activation and in the induction of T-cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them, histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. Here we show for the first time, that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells resulted in diminished production of the immunosuppressive cytokine IL-10, and induction of inflammatory APCs that effectively activate antigen-specific naïve T-cells and restore the responsiveness of anergic CD4 + T-cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising aminoacids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are

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

Frontiers in Immunology

The Mads/Mef2 (Mef2a/b/c/d) family of transcription factors (TFs) regulates differentiation of mu... more 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. Beside their direct involvement in oncogenesis, Mef2 TFs indirectly control tumor progression by regulating antitumor immunity. We recently reported that in CD4+CD25+Foxp3+ T-regulatory (Treg) cells, Mef2d is required for the acquisition of an effector Treg (eTreg) phenotype and for the activation of an epigenetic program that suppresses the anti-tumor immune responses of conventional T and B cells. We now report that as with Mef2d, the deletion of Mef2c in Tregs switches off the expression of Il10 and Icos and leads to enhanced antitumor immunity in syngeneic ...

Research paper thumbnail of Nuclear Coregulatory Complexes in Tregs as Targets to Promote Anticancer Immune Responses

Frontiers in Immunology

T-regulatory (Treg) cells display considerable heterogeneity in their responses to various cancer... more T-regulatory (Treg) cells display considerable heterogeneity in their responses to various cancers. The functional differences among this cell type are heavily influenced by multiprotein nuclear complexes that control their gene expression. Many such complexes act mechanistically by altering epigenetic profiles of genes important to Treg function, including the forkhead P3 (Foxp3) transcription factor. Complexes that form with certain members of the histone/protein deacetylase (HDAC) class of enzymes, like HDACs 1, 2, and 3, along with histone methyltransferase complexes, are important in the induction and stabilization of Foxp3 and Treg identity. The functional behavior of both circulating and intratumoral Tregs greatly impacts the antitumor immune response and can be predictive of patient outcome. Thus, targeting these regulatory complexes within Tregs may have therapeutic potential, especially in personalized immunotherapies.

Research paper thumbnail of Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells

Cancers, 2019

Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransfer... more 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. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acu...

Research paper thumbnail of Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity

Journal of Clinical Investigation, 2020

Research paper thumbnail of HDAC10 deletion promotes Foxp3+ T-regulatory cell function

Scientific Reports, 2020

Foxp3+ T-regulatory (Treg) cells are capable of suppressing immune responses. Lysine acetylation ... more 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. Several of the 11 classical HDAC enzymes are necessary for optimal Treg function while others are dispensable. We investigated the effect of HDAC10 in murine Tregs. HDAC10 deletion had no adverse effect on the health of mice, which retained normal CD4+ and CD8+ T cell function. However, HDAC10−/− Treg exhibited increased suppressive function in vitro and in vivo. C57BL/6 Rag1−/− mice adoptively transferred with HDAC10−/− but not wild Treg, were protected from developing colitis. HDAC10−/− but not wild-type mice receiving fully MHC-mismatched cardiac transplants became tolerant and showed long-term allograft su...

Research paper thumbnail of How little is known about the role of human Foxp3+ Tregs in tumors

Expert opinion on therapeutic targets, Jan 10, 2018

Research paper thumbnail of Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60

PloS one, 2017

Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with... more Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-...

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

Transplantation, 2017

Background-Vascularized composite allografts (VCA) are novel, life-enhancing forms of transplanta... more 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+ Tregulatory (Treg) cells and CD4 and CD8 effector Tcells 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. In addition, the effects of therapeutic expansion in vivo of host Treg cell populations on VCA survival are unknown. Methods-We established a fully major histocompatibility complex-disparate (BALB/c-> C57BL/6) murine orthotopic forelimb Tx model to explore the benefits of pre-and post-Tx IL-2/ anti-IL-2 monoclonal antibody complex (IL-2C) administration to expand the host Treg cell population and thereby attempt to promote Treg cell-dependent VCA survival.

Research paper thumbnail of Human lung tumor FOXP3+ Tregs upregulate four "Treg-locking" transcription factors

JCI insight, Jan 17, 2017

Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses... more Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses, but the properties of human intratumoral Tregs are still largely unknown, in part due to significant methodologic problems. We studied the phenotypic, functional, epigenetic, and transcriptional features of Tregs in 92 patients with non-small-cell lung cancer, comparing the features of Tregs within tumors versus corresponding blood, lung, and lymph node samples. Intratumoral Treg numbers and suppressive function were significantly increased compared with all other sites but did not display a distinctive phenotype by flow cytometry. However, by undertaking simultaneous evaluation of mRNA and protein expression at the single-cell level, we demonstrated that tumor Tregs have a phenotype characterized by upregulated expression of FOXP3 mRNA and protein as well as significantly increased expression of EOS, IRF4, SATB1, and GATA1 transcription factor mRNAs. Expression of these "Treg-loc...

Research paper thumbnail of T-cells lacking HDAC11 have increased effector functions and mediate enhanced alloreactivity in a murine model

Blood, Jul 26, 2017

Histone acetylation and the families of enzymes responsible for controlling these epigenetic mark... more Histone acetylation and the families of enzymes responsible for controlling these epigenetic marks have been implicated in regulating T-cell maturation and phenotype. Here, we demonstrate a previously undefined role of histone deacetylase 11 (HDAC11) in regulating T-cell effector functions. Using EGFP-HDAC11 transgenic reporter mice, we found that HDAC11 expression was lower in effector relative to naïve and central memory T-cell populations, and activation of resting T-cells resulted in its decreased expression. Experiments using HDAC11 knockout (KO) mice revealed that T-cells from these mice displayed enhanced proliferation, pro-inflammatory cytokine production and effector molecule expression. Additionally, HDAC11KO T-cells had increased expression of Eomesodermin (Eomes) and TBX21 (Tbet), transcription factors previously shown to regulate inflammatory cytokine and effector molecule production. Conversely, overexpression of HDAC11 resulted in decreased expression of these genes. ...

Research paper thumbnail of Proximity Ligation Assay to Quantify Foxp3 Acetylation in Regulatory T Cells

Methods in molecular biology (Clifton, N.J.), 2017

Determining protein acetylation by immunoprecipitation and immunoblotting can be challenging, esp... more 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(5) cells within two days turnaround time. This method is of potential use in other similar scenarios, when post-translational modifications of a protein of interest need to be determined with only a small amount of sample and in the absence of specific antibodies that can assess the post-translational modification in the protein of interest.

Research paper thumbnail of Isoform-Selective HDAC Inhibitor Therapy for Transplantation

Transplantation, 2016

The 18 known mammalian histone/protein deacetylases (HDACs) are divided into 4 groups, termed cla... more The 18 known mammalian histone/protein deacetylases (HDACs) are divided into 4 groups, termed class I (HDAC1, HDAC2, HDAC3, HDAC8), class IIa (HDAC4, HDAC5, HDAC7, HDAC9), class IIb (HDAC6, HDAC10), class III (SIRT1-7) and class IV (HDAC11) enzymes 1. While these enzymes were initially defined by their ability to deacetylate histones and dampen histone-DNA and histone-protein interactions, they are now recognized as regulating the functions of thousands of nonhistone proteins 2. A large number of broadly active pharmacologic HDAC inhibitors (pan-HDACi) have been developed and are in clinical trials as anti-cancer agents due to their abilities to promote tumor cell-cycle arrest, differentiation and apoptosis 3. There is also interest in the potential use of HDACi therapy for autoimmunity and transplantation, but there are concerns that the various pan-HDACi compounds may be too broadly acting and/or toxic for clinical use beyond oncology.

Research paper thumbnail of Targeting Sirtuin-1 prolongs murine renal allograft survival and function

Kidney International, 2016

Current immunosuppressive medications used after transplantation have significant toxicities. Fox... more Current immunosuppressive medications used after transplantation have significant toxicities. Foxp3 + T-regulatory (Treg) cells can prevent allograft rejection without compromising protective host immunity. Interestingly, inhibiting the class III histone/protein deacetylase Sirtuin-1 can augment Foxp3 + Treg suppressive function through increasing Foxp3 acetylation. Here we determined whether Sirtuin-1 targeting can stabilize biological allograft function. BALB/c kidney allografts were transplanted into C57BL/6 recipients with a CD4-conditional deletion of Sirtuin-1 (Sirt1 fl/fl CD4 cre) or mice treated with a Sirtuin-1 specific inhibitor (EX-527), and the native kidneys removed. Blood chemistries and hematocrit were followed weekly. Sirt1 fl/fl CD4 cre recipients showed markedly longer survival and improved kidney function. Sirt1 fl/fl CD4 cre recipients exhibited donor specific tolerance, accepted BALB/c, but rejected third-party C3H cardiac allografts. C57BL/6 recipients of BALB/c renal allografts that were treated with EX-527 showed improved survival and renal function at 1, but not 10 mg/kg/day. Pharmacologic inhibition of Sirtuin-1 also improved renal allograft survival and function with dosing effects having relevance to outcome. Thus, inhibiting Sirtuin-1 can be a useful asset in controlling T-cell mediated rejection. However, effects on non-T cells that could adversely affect allograft survival and function merit consideration.

Research paper thumbnail of Hdac Inhibitors and Therapeutic Methods Using the Same

Research paper thumbnail of Thiol-Based Potent and Selective HDAC6 Inhibitors Promote Tubulin Acetylation and T-Regulatory Cell Suppressive Function

ACS medicinal chemistry letters, Jan 12, 2015

Several new mercaptoacetamides were synthesized and studied as HDAC6 inhibitors. One compound, 2b... more Several new mercaptoacetamides were synthesized and studied as HDAC6 inhibitors. One compound, 2b, bearing an aminoquinoline cap group, was found to show 1.3 nM potency at HDAC6, with >3000-fold selectivity over HDAC1. 2b also showed excellent efficacy at increasing tubulin acetylation in rat primary cortical cultures, inducing a 10-fold increase in acetylated tubulin at 1 μM. To assess possible therapeutic effects, compounds were assayed for their ability to increase T-regulatory (Treg) suppressive function. Some but not all of the compounds increased Treg function, and thereby decreased conventional T cell activation and proliferation in vitro.

Research paper thumbnail of Pim-2 kinase influences Tregulatory cell function and stability by mediating Foxp3 N-terminal phosphorylation

The Journal of biological chemistry, Jan 18, 2015

Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limi... more Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4+CD25+Foxp3+ regulatory T cells (Treg) play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg development and function. Foxp3 function itself, is directly regulated by multiple post-translational modifications (PTMs) that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macro-molecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here, we have identified a mechanism regulating Foxp3 level and activity that operate through discreet phosphorylation. The Pim-2 kinase can phosphorylate Foxp3 leading to decreased suppressive functions of Treg cells. The amino terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to ...

Research paper thumbnail of Class-Specific Histone/Protein Deacetylase Inhibition Protects Against Renal Ischemia Reperfusion Injury and Fibrosis Formation

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, Jan 23, 2015

Renal ischemia-reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure.... more Renal ischemia-reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure. Histone/protein deacetylases (HDACs) regulate gene accessibility and higher order protein structures and may alter cellular responses to a variety of stresses. We investigated whether use of pan- and class-specific HDAC inhibitors (HDACi) could improve IRI tolerance in the kidney. Using a model of unilateral renal IRI, we investigated early renal function after IRI, and calculated fibrosis after IRI using an automated scoring system. We found that pan-HDAC inhibition using trichostatin (TSA) yielded significant renal functional benefit at 24-96 hours (p < 0.001). Treated mice developed significantly less fibrosis at 30 days (p < 0.0004). Class I HDAC inhibition with MS-275 yielded similar effects. Protection from fibrosis formation was also noted in a cold ischemia transplant model (p < 0.008) with a trend toward improved cold ischemic survival in TSA-treated mice. These effect...

Research paper thumbnail of A Novel Role for Histone Deacetylase 6 in the Regulation of the Tolerogenic STAT3/IL-10 Pathway in APCs

The Journal of Immunology, 2014

APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modif... more APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4+ T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain compri...

Research paper thumbnail of Structural and biological features of FOXP3 dimerization relevant to regulatory T cell function

Cell reports, Jan 28, 2012

FOXP3 is a key transcription factor for regulatory T cell function. We report the crystal structu... more FOXP3 is a key transcription factor for regulatory T cell function. We report the crystal structure of the FOXP3 coiled-coil domain, through which a loose or transient dimeric association is formed and modulated, accounting for the activity variations introduced by disease-causing mutations or posttranslational modifications. Structure-guided mutagenesis revealed that FOXP3 coiled-coil-mediated homodimerization is essential for Treg function in vitro and in vivo. In particular, we identified human FOXP3 K250 and K252 as key residues for the conformational change and stability of the FOXP3 dimer, which can be regulated by protein posttranslational modifications such as reversible lysine acetylation. These studies provide structural and mechanistic explanations for certain disease-causing mutations in the coiled-coil domain of FOXP3 that are commonly found in IPEX syndrome. Overall, the regulatory machinery involving homooligomerization, acetylation, and heteroassociation has been dis...