Search tips
Search criteria

Results 1-25 (52)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
more »
Document Types
1.  FOXP3+ regulatory T cell development and function require histone/protein deacetylase 3 
The Journal of Clinical Investigation  2015;125(3):1111-1123.
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). Here, we determined that HDAC3 mediates both the development and function of the two main Treg subsets, thymus-derived Tregs and induced Tregs (iTregs). We determined that HDAC3 and FOXP3 physically interact and that HDAC3 expression markedly reduces Il2 promoter activity. In murine models, conditional deletion of Hdac3 during thymic Treg development restored Treg production of IL-2 and blocked the suppressive function of Tregs. HDAC3-deficient mice died from autoimmunity by 4–6 weeks of age; however, injection of WT FOXP3+ Tregs prolonged survival. Adoptive transfer of Hdac3-deficient Tregs, unlike WT Tregs, did not control T cell proliferation in naive mice and did not prevent allograft rejection or colitis. HDAC3 also regulated the development of iTregs, as HDAC3-deficient conventional T cells were not converted into iTregs under polarizing conditions and produced large amounts of IL-2, IL-6, and IL-17. We conclude that HDAC3 is essential for the normal development and suppressive functions of thymic and peripheral FOXP3+ Tregs.
PMCID: PMC4362235  PMID: 25642770
2.  Two Histone/Protein Acetyltransferases, CBP and p300, Are Indispensable for Foxp3+ T-Regulatory Cell Development and Function 
Molecular and Cellular Biology  2014;34(21):3993-4007.
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. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells.
PMCID: PMC4386456  PMID: 25154413
3.  Heat Shock Protein-70 is Required for Optimal Liver Regeneration After Partial Hepatectomy in Mice 
Liver regeneration is a complex process that restores functional tissue following resection or injury, and is accompanied by transient ATP depletion and metabolic stress in hepatic parenchymal cells. Heat shock protein-70 (Hsp70) functions as a chaperone during periods of cellular stress, and induces expression of several inflammatory cytokines identified as key players during early liver regeneration. We therefore hypothesized that Hsp70 would be required in the initiation of regeneration. Investigations were carried out in a 70% partial-hepatectomy (PHx) mouse model with mice lacking inducible Hsp70 (Hsp70−/−). Liver regeneration was assessed post-operatively using the ratio of liver weight to body weight (LW/BW), and sera and tissues were collected for analysis. In addition, gene expression of Hsp related genes were assessed in a cohort of 23 human living-donor liver transplantation donors. In mice, the absence of Hsp70 was associated with reduced postoperative LW/BW, Ki-67 staining, and TNF-α expression compared to wild-type mice. TNF-α expression was also reduced in livers from Hsp70−/− mice following induction with LPS (1 mg/kg). Clinically, the transcription of multiple Hsp genes was upregulated following donor hepatectomy, especially Hsp70 family members. Together, these results suggest that the early phase of successful liver regeneration requires the presence of Hsp70 to induce TNF-α. Further studies are required to determine whether Hsp70 contributes to liver regeneration as a chaperone, by stabilizing specific interactions required for growth signalling, or as a paracrine inflammatory signal, as can occur in models of shock.
PMCID: PMC3947447  PMID: 24357103
heat shock response; living-donor liver transplantation; TNF-alpha
4.  Mbd2 Promotes Foxp3 Demethylation and T-Regulatory-Cell Function 
Molecular and Cellular Biology  2013;33(20):4106-4115.
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.
PMCID: PMC3811679  PMID: 23979593
5.  Inhibition of p300 impairs Foxp3+ T-regulatory cell function and promotes anti-tumor immunity 
Nature medicine  2013;19(9):1173-1177.
Foxp3+ T-regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity, but can also curtail host immune responses to various types of tumors1,2. Foxp3+ Tregs are therefore considered promising targets to enhance anti-tumor immunity, and efforts are underway to develop approaches for their therapeutic modulation. However, while studies showing that Foxp3+ Treg depletion experimentally can enhance anti-tumor responses provide proof-of-principle, they 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 non-histone proteins3,4. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (Ep300, KAT3B), in Foxp3+ Tregs, increased TCR-induced apoptosis in Tregs, impaired Treg suppressive function and peripheral Treg induction, and limited tumor growth in immunocompetent, but not in immunodeficient, hosts. Our data thereby demonstrate that p300 is important for Foxp3+ Treg function and homeostasis in vivo and in vitro, and identify novel mechanisms by which appropriate small molecule inhibitors can diminish Treg function without overtly impairing T-effector (Teff) cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.
PMCID: PMC3793393  PMID: 23955711
6.  Differing Effects of Rapamycin or Calcineurin Inhibitor on T-Regulatory Cells in Pediatric Liver and Kidney Transplant Recipients 
In a cross-sectional study, we assessed effects of calcineurin inhibitor (CNI) or rapamycin on T-regulatory (Treg) cells from children with stable liver (n=53) or kidney (n=9) allografts several years post-transplant. We analyzed Treg number, phenotype, suppressive function, and methylation at the Treg-specific demethylation region (TSDR) using Tregs and peripheral blood mononuclear cells. 48 patients received CNI (39 as monotherapy) and 12 patients received rapamycin (9 as monotherapy). Treg numbers diminished over time on either regimen, but reached significance only with CNI (r=−0.424, p=0.017). CNI levels inversely correlated with Treg number (r=−0.371, p=0.026), and positively correlated with CD127+ expression by Tregs (r=0.437, p=0.023). Patients with CNI levels >3.6 ng/ml had weaker Treg function than those with levels <3.6 ng/ml, whereas rapamycin therapy positively correlated with Treg numbers (r=0.628, p=0.029) and their expression of CTLA4 (r=0.726, p=0.041). Overall, CTLA4 expression, TSDR demethylation and an absence of CD127 were important for Treg suppressive function. We conclude that rapamycin has beneficial effects on Treg biology, whereas long-term and high dose CNI use may impair Treg number, function and phenotype, potentially acting as a barrier to attaining host hyporesponsiveness to an allograft.
PMCID: PMC3513508  PMID: 22994804
Immunosuppression; immunoregulation; clinical transplantation
7.  Inhibitory Effects of Pirfenidone on Dendritic Cells and Lung Allograft Rejection 
Transplantation  2012;94(2):114-122.
Pirfenidone (PFD) is an anti-fibrotic agent with beneficial effects upon proinflammatory disorders. In this study, we further investigated PFD and long acting form, “deuterated (d)PFD” immune modulating properties by evaluating their effects on mouse dendritic cells (DCs).
The effects of PFD upon DCs were examined in vivo using an orthotopic mouse lung transplant model and in vitro utilizing isolated bone marrow derived DCs in response to lipopolysaccharide and allogeneic stimulation.
In mouse lung transplants, PFD and dPFD treatment improved allograft lung function based on peak airway pressure, less infiltrates/consolidation on microCT scan imaging, and reduced lung rejection/injury. DC activation from lung allografts was suppressed with PFD and there appeared to be a greater effect of PFD upon CD11c+CD11b−CD103+ lung DCs. In addition, PFD reduced the expression of a number of proinflammatory cytokines/chemokines from lung allografts. In vitro, DCs treated with PFD showed decreased expression of MHC class II and co-stimulatory molecules and impaired DC’s capacity to stimulate T cell activation while antigen uptake was preserved. PFD directly inhibited the release of inflammatory cytokines from isolated DCs, and was associated with a reduction of stress protein kinases and attenuated LPS-dependent MAPKp38 phosphorylation.
PFD has lung allograft protective properties and in addition to its known effects on T cell biology, PFD’s immune modulating activities encompass inhibitory effects upon DC activation and function.
PMCID: PMC3402635  PMID: 22743547
pirfenidone; lung transplantation; dendritic cell; acute rejection; mouse
8.  Histone/protein deacetylases control Foxp3 expression and the heat shock response of T-regulatory cells 
Current opinion in immunology  2011;23(5):670-678.
Lysine ε-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 ε-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.
PMCID: PMC3190028  PMID: 21798734
9.  Combination of isoform-selective histone/protein deacetylase inhibitors improves Foxp3+ T-regulatory cell function 
Cell Cycle  2012;11(18):3351-3352.
PMCID: PMC3466538  PMID: 22918251
transplantation; immunotherapy; HDAC6; HDAC9; Sirt1
10.  Inhibition of HDAC9 Increases T Regulatory Cell Function and Prevents Colitis in Mice 
Gastroenterology  2009;138(2):583-594.
Foxp3+ T regulatory cells (Tregs) help prevent autoimmunity, and increases in their numbers of functions could decrease the development of inflammatory bowel disease. Like other cells, Foxp3+ Tregs express histone/protein deacetylases (HDACs), which regulate chromatin remodeling and gene expression. We investigated whether disruption of a specific class IIa HDAC, HDAC9, activity in Tregs affects the pathogenesis of colitis in mice.
We tested the effects of various HDAC inhibitors (HDACi) in models of colitis using wild-type mice. We also transferred Tregs and non-Treg cells from HDAC9−/− or wild-type mice to immunodeficient mice. HDAC9 contributions to the functions of Tregs were determined during development and progression of colitis.
Pan-HDACi, but not class I-specific HDACi, increased the functions of Foxp3+ Tregs, prevented colitis, and reduced established colitis in mice, indicating the role of class II HDACs in controlling Treg function. The abilities of pan-HDACi to prevent/reduce colitis were associated with increased numbers of Foxp3+ Tregs and their suppressive functions. Colitis was associated with increased local expression of HDAC9; HDAC9−/− mice resistant to development of colitis. HDAC9−/− Tregs expressed increased levels of the heat shock protein (HSP) 70, compared with controls. Immunoprecipitation experiments indicated an interaction between HSP70 and Foxp3. Inhibition of HSP70 reduced the suppressive functions of HDAC9−/− Tregs; Tregs that overexpressed HSP70 had increased suppressive functions.
Strategies to decrease HDAC9 expression or function in Tregs or to increase expression of HSP70 might be used to treat colitis and other autoimmune disorders.
PMCID: PMC3369426  PMID: 19879272
11.  Mesenchymal Stromal Cell-Derived Factors Promote Tissue Repair in a Small-for-Size Ischemic Liver Model but Do Not Protect against Early Effects of Ischemia and Reperfusion Injury 
Journal of Immunology Research  2015;2015:202975.
Loss of liver mass and ischemia/reperfusion injury (IRI) are major contributors to postresectional liver failure and small-for-size syndrome. Mesenchymal stromal cell- (MSC-) secreted factors are described to stimulate regeneration after partial hepatectomy. This study investigates if liver-derived MSC-secreted factors also promote liver regeneration after resection in the presence of IRI. C57BL/6 mice underwent IRI of 70% of their liver mass, alone or combined with 50% partial hepatectomy (PH). Mice were treated with MSC-conditioned medium (MSC-CM) or unconditioned medium (UM) and sacrificed after 6 or 24 hours (IRI group) or after 48 hours (IRI + PH group). Blood and liver tissue were analyzed for tissue injury, hepatocyte proliferation, and gene expression. In the IRI alone model, serum ALT and AST levels, hepatic tissue damage, and inflammatory cytokine gene expression showed no significant differences between both treatment groups. In the IRI + PH model, significant reduction in hepatic tissue damage as well as a significant increase in hepatocyte proliferation was observed after MSC-CM treatment. Conclusion. Mesenchymal stromal cell-derived factors promote tissue regeneration of small-for-size livers exposed to ischemic conditions but do not protect against early ischemia and reperfusion injury itself. MSC-derived factors therefore represent a promising treatment strategy for small-for-size syndrome and postresectional liver failure.
PMCID: PMC4561317  PMID: 26380314
12.  Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs1 
Clinical immunology (Orlando, Fla.)  2010;136(3):348-363.
Histone/protein deacetylases (HDACs) decrease histone and protein acetylation, typically leading to suppression of gene transcription and modulation of various protein functions. We found significant differences in expression of HDAC before and after stimulation of human T regulatory (Treg) and T effector cells, suggesting the potential for future selective targeting of Tregs with HDAC inhibitors (HDACi). Use of various HDACi small molecules enhanced, by up to 4.5-fold (average 2-fold), the suppressive functions of both freshly isolated and expanded human Tregs, consistent with our previous murine data. HDACi use increased Treg expression of CTLA-4, a key negative regulator of immune response, and we found a direct and significant correlation between CTLA-4 expression and Treg suppression. Hence, HDACi compounds are promising pharmacologic tools to increase Treg suppressive functions, and this action may potentially be of use in patients with autoimmunity or post-transplantation.
PMCID: PMC2917523  PMID: 20478744
13.  Pirfenidone Inhibits T Cell Activation, Proliferation, Cytokine and Chemokine Production, and Host Alloresponses 
Transplantation  2009;88(3):330-338.
We previously showed that pirfenidone, an anti-fibrotic agent, reduces lung allograft injury/rejection. In this study, we tested the hypothesis that pirfenidone has immune modulating activities and evaluated its effects on the function of T cell subsets, which play important roles in allograft rejection.
We first evaluated whether pirfenidone alters T cell proliferation and cytokine release in response to T cell receptor (TCR) activation, and whether pirfenidone alters regulatory T cells (CD4+CD25+) suppressive effects using an in vitro assay. Additionally, pirfenidone effects on alloantigen-induced T cell proliferation in vivo were assessed by adoptive transfer of CFSE-labeled T cells across a parent->F1 MHC mismatch, as well as using a murine heterotopic cardiac allograft model (BALB/c->C57BL/6).
Pirfenidone was found to inhibit the responder frequency of TCR-stimulated CD4+ cell total proliferation in vitro and in vivo, whereas both CD4 and CD8 proliferation index were reduced by pirfenidone. Additionally, pirfenidone inhibited TCR-induced production of multiple pro-inflammatory cytokines and chemokines. Interestingly, there was no change on TGF-β production by purified T cells, and pirfenidone had no effect on the suppressive properties of naturally occurring regulatory T cells. Pirfenidone alone showed a small but significant (p < 0.05) effect on the in vivo allogeneic response while the combination of pirfenidone and low dose rapamycin had more remarkable effect in reducing the alloantigen response with prolonged graft survival.
Pirfenidone may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses.
PMCID: PMC2726969  PMID: 19667934
rodent; T cells; transplantation
14.  Dynamic Interactions Between TIP60 and p300 Regulate FOXP3 Function Through a Structural Switch Defined by a Single Lysine on TIP60 
Cell reports  2014;7(5):1471-1480.
The human FOXP3 molecule is an oligomeric transcriptional factor able to mediate activities that characterize T regulatory cells, a class of lymphocytes central to the regulation of immune responses. The activity of FOXP3 is regulated at the post-translational level, in part by two histone acetyltransferases (HAT), TIP60 and p300. TIP60 and p300 work cooperatively to regulate FOXP3 activity. Initially p300 and TIP60 interactions lead to the activation of TIP60 and facilitate acetylation of K327 of TIP60, which functions as a molecular switch to allow TIP60 to change binding partners. Subsequently, p300 is released from this complex and TIP60 interacts with and acetylates FOXP3. Maximal induction of FOXP3 activities is observed when both p300 and TIP60 are able to undergo cooperative interactions. Conditional knockout of TIP60 in Treg cells significantly decreases the Treg population in the peripheral immune organs, leading to a scurfy-like fatal autoimmune disease.
PMCID: PMC4064594  PMID: 24835996
15.  Histone deacetylase inhibitors and transplantation 
Current opinion in immunology  2007;19(5):589-595.
Summary of recent advances
Simply detecting the presence or absence of Foxp3, a transcription factor characteristic of naturally occurring CD4+ CD25+ regulatory T cells (Tregs), now appears of minimal value in predicting the outcome of immunologic responses, since dividing human CD4+ effector T cells can induce Foxp3 without attaining repressive functions, and additional molecular interactions, as well epigenetic events, affect Foxp3-dependent Treg functions in humans and mice. Experimentally, in vivo and in vitro studies show histone deacetylase inhibitors (HDACi) can enhance the numbers and suppressive function of regulatory T cells (Tregs), by promoting Foxp3+ cell production, enhancing chromatin remodeling within Tregs, and inducing acetylation of Foxp3 protein itself. Human studies consistent with a role for HDACi in controlling Fox3-dependent Treg functions are also available. We review these molecular interactions and how they may be exploited therapeutically to enhance Treg-dependent functions, including post-transplantation.
PMCID: PMC2693068  PMID: 17719760
16.  Recruitment of Foxp3+ T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor 
The Journal of Experimental Medicine  2005;201(7):1037-1044.
Although certain chemokines and their receptors guide homeostatic recirculation of T cells and others promote recruitment of activated T cells to inflammatory sites, little is known of the mechanisms underlying a third function, migration of Foxp3+ regulatory T (T reg) cells to sites where they maintain unresponsiveness. We studied how T reg cells are recruited to cardiac allografts in recipients tolerized with CD154 monoclonal antibody (mAb) plus donor-specific transfusion (DST). Real-time polymerase chain reaction showed that intragraft Foxp3 levels in tolerized recipients were ∼100-fold higher than rejecting allografts or allografts associated with other therapies inducing prolonged survival but not tolerance. Foxp3+ cells were essential for tolerance because pretransplant thymectomy or peritransplant depletion of CD25+ cells prevented long-term survival, as did CD25 mAb therapy in well-functioning allografts after CD154/DST therapy. Analysis of multiple chemokine pathways showed that tolerance was accompanied by intragraft up-regulation of CCR4 and one of its ligands, macrophage-derived chemokine (CCL22), and that tolerance induction could not be achieved in CCR4−/− recipients. We conclude that Foxp3 expression is specifically up-regulated within allografts of mice displaying donor-specific tolerance, that recruitment of Foxp3-expressing T reg cells to an allograft tissue is dependent on the chemokine receptor, CCR4, and that, in the absence of such recruitment, tolerizing strategies such as CD154 mAb therapy are ineffectual.
PMCID: PMC2213137  PMID: 15809349
17.  Genetic Variation in the Prostaglandin E2 Pathway Is Associated with Primary Graft Dysfunction 
Rationale: Biologic pathways with significant genetic conservation across human populations have been implicated in the pathogenesis of primary graft dysfunction (PGD). The evaluation of the role of recipient genetic variation in PGD has thus far been limited to single, candidate gene analyses.
Objectives: We sought to identify genetic variants in lung transplant recipients that are responsible for increased risk of PGD using a two-phase large-scale genotyping approach.
Methods: Phase 1 was a large-scale candidate gene association study of the multicenter, prospective Lung Transplant Outcomes Group cohort. Phase 2 included functional evaluation of selected variants and a bioinformatics screening of variants identified in phase 1.
Measurements and Main Results: After genetic data quality control, 680 lung transplant recipients were included in the analysis. In phase 1, a total of 17 variants were significantly associated with PGD, four of which were in the prostaglandin E2 family of genes. Among these were a coding variant in the gene encoding prostaglandin E2 synthase (PTGES2; P = 9.3 × 10−5) resulting in an arginine to histidine substitution at amino acid position 298, and three variants in a block containing the 5′ promoter and first intron of the PTGER4 gene (encoding prostaglandin E2 receptor subtype 4; all P < 5 × 10−5). Functional evaluation in regulatory T cells identified that rs4434423A in the PTGER4 gene was associated with differential suppressive function of regulatory T cells.
Conclusions: Further research aimed at replication and additional functional insight into the role played by genetic variation in prostaglandin E2 synthetic and signaling pathways in PGD is warranted.
PMCID: PMC3977709  PMID: 24467603
lung transplantation; genetics; prostaglandin; regulatory T cells
18.  Targeting Sirtuin-1 Alleviates Experimental Autoimmune Colitis by Induction of Foxp3+ T-Regulatory Cells 
Mucosal immunology  2014;7(5):1209-1220.
Induced Foxp3+ T-regulatory cells (iTreg) are essential to gastrointestinal immune homeostasis and loss of the ability to develop iTregs may lead to autoimmune colitis. We previously showed a role for Sirtuin-1 (Sirt1) in control of Treg function and hypothesized that targeting of Sirt1 might enhance iTreg development and thereby represent a potential therapy for inflammatory bowel disease (IBD). We adoptively transferred CD4+CD25−Foxp3− T effector (TE) cells from wild-type (C57BL/6) or fl-Sirt1/CD4cre mice into B6/Rag1−/− mice and monitored the mice until they lost 10-15% of their weight. Adoptive transfer of TE cells lacking Sirt1 to B6/Rag1−/− mice resulted in a 2.8-fold increase in iTreg formation compared to mice receiving wild-type TE cells and correlated with attenuated colitis and reduced weight loss (1.04±1.4% vs. 13.97±2.2%, respectively, p<0.001). In a second model of IBD, we used pharmacologic Sirt1 targeting of mice receiving multiple cycles of dextran sodium sulfate (DSS) in their drinking water, alternated with fresh water. Likewise, wild-type mice receiving cyclic DSS and a Sirt1 inhibitor, EX-527, had reduced weight loss (5.8±5.9% vs. 13.2±6.9%, p=0.03) and increased iTreg formation compared to controls. Sirt1 appears a promising target for pharmacologic therapy of IBD as a result of promoting iTreg development.
PMCID: PMC4138288  PMID: 24549276
Inflammatory bowel disease; Th1-mediated colitis; DSS colitis; iTreg
19.  Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer 
The Journal of Clinical Investigation  2014;124(12):5466-5480.
Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%–25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62LloCD54hi) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.
PMCID: PMC4348966  PMID: 25384214
20.  Modulation of LIGHT-HVEM Costimulation Prolongs Cardiac Allograft Survival 
LIGHT (TNFSF14), a tumor necrosis factor superfamily member expressed by activated T cells, binds to herpes virus entry mediator (HVEM) which is constitutively expressed by T cells and costimulates T cell activation in a CD28-independent manner. Given interest in regulating the effector functions of T cells in vivo, we examined the role of LIGHT-HVEM costimulation in a murine cardiac allograft rejection model. Normal hearts lacked LIGHT or HVEM mRNA expression, but allografts showed strong expression of both genes from day 3 after transplant, and in situ hybridization and immunohistology-localized LIGHT and HVEM to infiltrating leukocytes. To test the importance of LIGHT expression on allograft survival, we generated LIGHT−/− mice by homologous recombination. The mean survival of fully major histocompatibility complex–mismatched vascularized cardiac allografts in LIGHT−/− mice (10 days, P < 0.05) or cyclosporine A (CsA)-treated LIGHT+/+ mice (10 days, P < 0.05) was only slightly prolonged compared with LIGHT+/+ mice (7 days). However, mean allograft survival in CsA-treated LIGHT−/− allograft recipients (30 days) was considerably enhanced (P < 0.001) compared with the 10 days of mean survival in either untreated LIGHT−/− mice or CsA-treated LIGHT+/+ controls. Molecular analyzes showed that the beneficial effects of targeting of LIGHT in CsA-treated recipients were accompanied by decreased intragraft expression of interferon (IFN)-γ, plus IFN-γ–induced chemokine, inducible protein-10, and its receptor, CXCR3. Treatment of LIGHT+/+ allograft recipients with HVEM-Ig plus CsA also enhanced mean allograft survival (21 days) versus wild-type controls receiving HVEM-Ig (mean of 7 days) or CsA alone (P < 0.001). Our data suggest that T cell to T cell–mediated LIGHT/HVEM-dependent costimulation is a significant component of the host response leading to cardiac allograft rejection.
PMCID: PMC2193745  PMID: 11901205
transplantation; allograft rejection; T cell activation; costimulation; TNF superfamily
21.  Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection 
Although mononuclear cell infiltration is a hallmark of cellular rejection of a vascularized allograft, efforts to inhibit rejection by blocking leukocyte-endothelial cell adhesion have proved largely unsuccessful, perhaps in part because of persistent generation of chemokines within rejecting grafts. We now provide, to our knowledge, the first evidence that in vivo blockade of specific chemokine receptors is of therapeutic significance in organ transplantation. Inbred mice with a targeted deletion of the chemokine receptor CCR1 showed significant prolongation of allograft survival in 4 models. First, cardiac allografts across a class II mismatch were rejected by CCR1+/+ recipients but were accepted permanently by CCR1–/– recipients. Second, CCR1–/– mice rejected completely class I– and class II–mismatched BALB/c cardiac allografts more slowly than control mice. Third, levels of cyclosporin A that had marginal effects in CCR1+/+ mice resulted in permanent allograft acceptance in CCR1–/– recipients. These latter allografts showed no sign of chronic rejection 50–200 days after transplantation, and transfer of CD4+ splenic T cells from these mice to naive allograft recipients significantly prolonged allograft survival, whereas cells from CCR1+/+ mice conferred no such benefit. Finally, both CCR1+/+ and CCR1–/– allograft recipients, when treated with a mAb to CD4, showed permanent engraftment, but these allografts showed florid chronic rejection in the former strain and were normal in CCR1–/– mice. We conclude that therapies to block CCR1/ligand interactions may prove useful in preventing acute and chronic rejection clinically.
PMCID: PMC382589  PMID: 10619859
22.  Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis 
Journal of Clinical Investigation  1999;104(11):1549-1557.
During the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1α), CCR2 (MCP-1), CCR5 (RANTES, MIP-1α, MIP-1β), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1–/– mice. We found that neutrophil accumulation in CCR1–/– mice was comparable to that in wild-type animals but that renal recruitment of CD4+ and CD8+ T cells and macrophages increased significantly. Moreover, CCR1–/– mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1–/– mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-γ and TNF-α. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1α, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.
J. Clin. Invest. 104:1549–1557 (1999).
PMCID: PMC409862  PMID: 10587518
23.  Regulation of T Cell Differentiation and Alloimmunity by the Cyclin-Dependent Kinase Inhibitor p18ink4c 
PLoS ONE  2014;9(3):e91587.
Cellular proliferation in response to mitogenic stimuli is negatively regulated by the Cip/Kip and the Ink4 families of cyclin-dependent kinase (CDK) inhibitors. Several of these proteins are elevated in anergic T cells, suggesting a potential role in the induction or maintenance of tolerance. Our previous studies showed that p27kip1 is required for the induction of T cell anergy and transplantation tolerance by costimulatory blockade, but a role for Ink4 proteins in these processes has not been established. Here we show that CD4+ T cells from mice genetically deficient for p18ink4c divide more rapidly than wild-type cells in response to antigenic, costimulatory and growth factor signals. However, this gain of proliferative function was accompanied by a moderate increase in the rate of cell death, and was accompanied by an overall defect in the generation of alloreactive IFNγ-producing effector cells. Consistent with this, p18ink4c-deficient T cells were unable to induce graft-vs-host disease in vivo, and p18ink4c deficiency cooperated with costimulatory blockade to significantly increase the survival of fully mismatched allografts in a cardiac transplantation model. While both p18ink4c and p27kip1 act to restrict T cell proliferation, p18ink4c exerts an opposite effect from p27kip1 on alloimmunity and organ transplant rejection, most likely by sustaining T cell survival and the development of effector function. Our studies point to additional important links between the cell cycle machinery and the processes of T cell differentiation, survival and tolerance.
PMCID: PMC3948896  PMID: 24614758
24.  Cyclin-dependent kinase 2 controls peripheral immune tolerance1 
Adaptive immunity requires signals from both the T cell antigen receptor and the costimulatory molecule CD28. These receptors activate multiple signaling pathways, including the cyclin-dependent kinase (CDK) cascade, and antigenic signals in the absence of costimulation results in a tolerant state that is enforced by the CDK inhibitory protein p27kip1. We find that CDK2, the major target of p27kip1, is highly active in T cells that infiltrate and reject cardiac allografts. To determine whether CDK2 is required for T cell alloimmunity, we utilized mice genetically deficient for CDK2. Blockade of CD28 costimulation alone was unable to inhibit the rejection of cardiac allografts by wild-type recipients, however, targeting this pathway in CDK2-deficient recipients led to long-term allograft survival. CDK2-deficient CD4+ T cells proliferated normally in response to stimulation in vitro and in vivo, however, genetic, shRNA, or small molecule-mediated antagonism of CDK2 resulted in decreased production of IL-2 and IFNγ. In addition, surviving grafts from CDK2-deficient recipients showed increased infiltration of Foxp3+ regulatory T cells (Treg), and Treg from CDK2-deficient mice exhibited increased suppressive activity in vitro and in an in vivo model of inflammatory bowel disease. These data suggest that that p27kip1 promotes peripheral tolerance through its ability to inhibit CDK2, which otherwise acts to promote conventional T cell differentiation and restrict Treg function.
PMCID: PMC3518671  PMID: 23136201
25.  Indoleamine 2,3-Dioxygenase and Metabolites Protect Murine Lung Allografts and Impair the Calcium Mobilization of T Cells 
The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan into kynurenine metabolites that suppress effector T-cell function. In this study, we investigated IDO and its metabolite, 3-hydroxyanthranilic acid (3HAA), in regulating lung allograft rejection, using a murine orthotopic lung transplant model with a major mismatch (BALB/c donor and C57BL6 recipient). IDO was overexpressed in murine donor lungs, using an established nonviral (polyethylenimine carrier)–based gene transfer approach, whereas 3HAA was delivered daily via intraperitoneal injection. Increased IDO expression or its metabolite, 3HAA, resulted in a remarkable therapeutic effect with near normal lung function and little acute rejection, approximately A1, compared with A3 in untreated allografts (grading based on International Society for Heart and Lung Transplantation guidelines). We found that a high IDO environment for 7 days in lung allografts resulted in impaired T-cell activation, the production of multiple effector cytokines (IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, IL-12, and IL-13), and the generation of effector memory T cells (CD62LloCD44hi phenotype). In isolated murine splenocytes, we observed that IDO/3HAA impaired T-cell receptor (TCR)–mediated T-cell activation, and more importantly, a decrease of intracellular calcium, phospholipase C-γ1 phosphorylation, and mitochondrial mass was evident. This work further illustrates the potential role of a high IDO environment in lung transplantation, and that the high IDO environment directly impairs TCR activation via the disruption of calcium signaling.
PMCID: PMC3488632  PMID: 22517796
3-hydroxyanthranilic acid; lung allograft rejection; nonviral gene transfer

Results 1-25 (52)