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.
A 60 years old chinese male scheduled for a removal of an intracardiac mass occupying majority of right ventricular space, right ventricular outflow tract and pulmonary artery. The giant cardiac mass was later diagnosed pathologically as metastatic liposarcoma. The patient had a history of surgical removal of myxoid liposarcoma from his left thigh many years ago. It is extremely rare for liposarcoma to metastatize to right ventricle and pulmonary artery. The anesthetic management of the surgical procedure to remove this kind of intracardiac mass poses significant challenges to anesthesia providers. Our patient developed refractory hypotension after induction of general anesthesia which necessitated urgent cardiopulmonary bypass. The surgical procedure was successful and the patient recovered from the surgery and was discharged home without significant complication. Accurate preoperative diagnosis and assessment of patient’s functional status, appropriate preoperative volume status, emergency cardiopulmonary bypass readiness, smooth and gentle induction of general anesthesia with less myocardial depressing agent, and closely monitoring patient’s vitals and hemodynamic parameters are imperative in managing this kind of patients.
Liposarcoma; Cardiac tumor; Hypotension; Cardiopulmonary bypass; Transesophageal echocardiography
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.
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.
GATA transcription factors and their Friend of Gata (FOG) cofactors control the development of diverse tissues. GATA4 and GATA6 are essential for the expansion of the embryonic liver bud, but their expression patterns and functions in the adult liver are unclear. We characterized the expression of GATA and FOG factors in whole mouse liver and purified hepatocytes. GATA4, GATA6, and FOG1 are the most prominently expressed family members in whole liver and hepatocytes. GATA4 chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) identified 4409 occupied sites, associated with genes enriched in ontologies related to liver function, including lipid and glucose metabolism. However, hepatocyte-specific excision of Gata4 had little impact on gross liver architecture and function, even under conditions of regenerative stress, and, despite the large number of GATA4 occupied genes, resulted in relatively few changes in gene expression. To address possible redundancy between GATA4 and GATA6, both factors were conditionally excised. Surprisingly, combined Gata4,6 loss did not exacerbate the phenotype resulting from Gata4 loss alone. This points to the presence of an unusually robust transcriptional network in adult hepatocytes that ensures the maintenance of liver function.
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.
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.
3-hydroxyanthranilic acid; lung allograft rejection; nonviral gene transfer
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.
pirfenidone; lung transplantation; dendritic cell; acute rejection; mouse
Changes in the density and species composition of planktonic rotifers as well as their relationship to several environmental variables were studied at Dadian Lake, a shallow subtropical lake, which was completely dredged and reconstructed. Samples were taken monthly (2006–2009) at five stations. The total rotifer abundance exponentially declined and reached a relatively stable stage in 2009. Polyarthra dolichoptera and Trichocerca pusilla dominated the rotifer community in most seasons. TN, TP, and CODMn went down at the beginning of the monitoring period, rebounded in the second winter, and then decreased and reached a stable state in 2009. CCA showed that the most significant variations were caused by fluctuations in temperature, CODMn, SRP, and NO2-N. The rotifer community experienced a two-stage succession and the difference of species between the stages was exhibited during warm seasons. GAMs indicated that the selected factors were responsible for 64.8% of the total rotifer abundance variance and 16.5~64.3% of the variances of individual species abundance. Most of the environmental parameters had effects on rotifer abundance that could only be described by complicated curves, characterised by unimodality and bimodality instead of linearity. Our study highlighted the temperature influence on rotifer species composition and total abundance in subtropical lakes.
Therapeutic targeting of histone/protein deacetylase 6 (HDAC6), HDAC9, or the sirtuin-1 (Sirt1) augments the suppressive functions of regulatory T cells (Tregs) that contain the transcription factor Foxp3. However, it is unclear whether distinct mechanisms are involved or whether combined inhibition of these targets would be more beneficial. We compared the suppressive functions of Tregs from wild-type C57BL/6 mice with those from mice with either global (HDAC6−/−, HDAC9−/−, and HDAC6−/−HDAC9−/−), or conditional (fl-Sirt1/CD4-Cre or fl-Sirt1/Foxp3-Cre) HDAC deletion, as well as treatment with isoform-selective HDAC inhibitors. We found that the heat shock response was important for the improvement of Treg suppressive function mediated by HDAC6 inhibition, but not Sirt1 inhibition. Furthermore, although HDAC6, HDAC9, and Sirt1 all deacetylated Foxp3, each protein had diverse effects on transcription factors controlling Foxp3 gene expression. For example, loss of HDAC9 was associated with stabilization of the acetylation of signal transducer and activator of transcription 5 (STAT5) and of its transcriptional activity. Hence, targeting different HDACs increased Treg function by multiple and additive mechanisms, which indicates the therapeutic potential for combinations of HDAC inhibitors in the management of autoimmunity and organ transplantation.
Early allograft dysfunction (EAD) occurring in the first week post-liver transplantation is associated with increased graft failure and mortality and is believed to be largely due to ischemia/reperfusion injury. We anticipated that the presence of EAD would be reflected by alterations in expression of serum proteins associated with an inflammatory response in the peri-operative period, and hypothesized that a specific pattern of expression might correlate with the development of EAD. The serum levels of 25 cytokines, chemokines, and immunoreceptors were measured by Luminex multiplex assays pre- and post-liver transplantation. Levels of each cytokine biomarker were compared in adult recipients with or without EAD at serial time points using samples collected pre-operatively and at 1, 7, 14, and 30 days post-transplant. EAD was defined according to standard criteria as maximum alanine transferase (ALT) or aspartate transferase (AST) levels on days 1–7 of >2000 U/ml, day 7 bilirubin level ≥10 mg/dl, or a day 7 international normalized ratio (INR) ≥1.7. Multivariable analyses showed that patients experiencing EAD had lower pre-operative IL-6 and higher IL-2R levels. Patients with EAD also showed higher MCP-1 (CCL2), IL-8 (CXCL8), and RANTES (CCL5) chemokine levels in the early post-operative period, suggesting up-regulation of the NF-κB pathway, in addition to higher levels of chemokines and cytokines associated with T cell immunity, including Mig (CXCL9), IP-10 (CXCL10) and IL-2R. These findings identify several possible biomarkers and pathways associated with EAD, that may guide future validation studies and investigation of specific cellular and molecular mechanisms of graft dysfunction. Furthermore, if validated, our findings may contribute to perioperative prediction of the occurrence of EAD and ultimately lead to identification of potential interventional therapies.
immune monitoring; multiplex analysis; chemokines; immunobiology
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.
transplantation; immunotherapy; HDAC6; HDAC9; Sirt1
The aim was to assess the early and mid-term clinical effects of transaortic extended septal myectomy (TAESM) on obstructive hypertrophic cardiomyopathy (HCM) in China.
Ninety-three consecutive patients [57 men; mean age 45.8 ± 13.4 (11–74) years] with obstructive HCM underwent TAESM in Fuwai hospital. Their clinical data were analysed retrospectively. All the patients had drug-refractory symptoms and left ventricular outflow tract (LVOT) obstruction with a resting or physically provoked gradient of ≥50 mmHg. Preoperative transthoracic, intra-operative transoesophageal and postoperative transthoracic echocardiography was performed to assess LVOT gradients, septal thickness, LVOT diameter, mitral valve function, etc. Systolic anterior motion (SAM) of the anterior mitral valve leaflet had been detected in all preoperatively.
All the surgical procedures of the 93 patients were technically successful. The average length of postoperative stay was 7.8 ± 3.7 days. The 30-day and in-hospital mortality was 0%. Initial postoperative transoesophageal echocardiography (TEE) demonstrated marked reduction in LVOT gradient (91.76 ± 25.08 to 14.34 ± 13.44 mmHg, P < 0.0005) and significant improvement in mitral regurgitation (MR; P < 0.0005). Concomitant surgical procedures were carried out in 37 (39.8%). Complete atrioventricular block occurred in 3, complete left bundle branch block in 44, intraventricular conduction delay in 18, complete right bundle branch block in 2, transient renal dysfunction in 2 and transient intra-aortic-balloon-pumping was needed in 2. No other complications were observed during hospital stay. During a follow-up of 10.72 ± 11.02 (1–24) months, there were no readmissions or deaths, and all patients subjectively reported an obvious decrease in limiting symptoms and a significant increase in physical ability. At the latest follow-up, the New York Heart Association functional class decreased from 3.09 ± 0.60 (2–4) preoperatively to 1.12 ± 0.32 (1–2) (P < 0.0005); the LVOT gradient remained low at 14.78 ± 14.01 mmHg; MR remained absent (51) or at mild-(41)-to-moderate-(1) levels and SAM resolved completely in 98.9% (92 of 93) patients.
TAESM provides excellent relief from LVOT obstruction in HCM patients, with a conspicuous clinical and echocardiographic outcome at early and mid-term follow-up. For obstructive HCM and cardiac comorbidities, concomitant cardiac procedures with TAESM can be performed with low risk and satisfactory results.
Hypertrophic cardiomyopathy; Surgical myectomy; Concomitant procedures
BACKGROUND & AIMS
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.
The forkhead box transcription factor, Foxp3, is master regulator of the development and function of CD4+CD25+ T regulatory (Treg) cells that limit autoimmunity and maintain immune homeostasis. The carboxyl-terminal forkhead (FKH) domain is required for the nuclear localization and DNA binding of Foxp3. We assessed how individual FKH lysines contribute to the functions of Foxp3 in Treg cells.
We found that mutation of FKH lysines at position 382 (K17) and at position 393 (K18) impaired Foxp3 DNA binding and inhibited Treg suppressive function in vivo and in vitro. These lysine mutations did not affect the level of expression of Foxp3 but inhibited IL-2 promoter remodeling and had important and differing effects on Treg-associated gene expression.
These data point to complex effects of post-translational modifications at individual lysines within the Foxp3 FKH domain that affect Treg function. Modulation of these events using small molecule inhibitors may allow regulation of Foxp3+ Treg function clinically.
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.
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.
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.
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.
Acute allograft rejection requires a multifaceted immune response involving trafficking of immune cells into the transplant and expression of effector cell functions leading to graft destruction. The chemokine receptor CXCR3 and its ligands, CXCL9, CXCL10 and CXCL11, constitute an important pathway for effector cell recruitment post-transplant. However, analysis of CXCR3 expression and function has been hampered by a general lack of availability of a neutralizing anti-CXCR3 monoclonal antibody (mAb) for use in experimental models.
We report the generation, characterization and use of CXCR3-173, a new hamster mAb specific for mouse CXCR3 that recognizes CXCR3 on cells from wild-type but not CXCR3-/- mice.
Using CXCR3-173 mAb, we demonstrate CXCR3 expression on primary memory phenotype CD4+ and CD8+ T cells, naturally occurring CD4+CD25+ Foxp3+ regulatory T cells, natural killer T (NKT) cells, and ~25% of NK cells. CXCR3-173 blocked chemotaxis in vitro in response to CXCL10 or CXCL11 but not CXCL9. When injected into mice, this mAb significantly prolonged both cardiac and islet allograft survival. When combined with a subtherapeutic regimen of rapamycin, CXCR3-173 mAb induced long-term (>100 d) survival of cardiac and islet allografts. The in vivo effects of CXCR3-173 mAb were not associated with effector lymphocyte depletion.
These data highlight the utility of CXCR3-173 mAb in developing immunotherapeutic approaches to inhibit transplant rejection and potentially other immune-mediated diseases in murine models.
transplant rejection; chemokine receptor; immunotherapy
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.
rodent; T cells; transplantation
Classical zinc-dependent histone deacetylases (HDACs) catalyse the removal of acetyl groups from histone tails and also from many non-histone proteins, including the transcription factor FOXP3, a key regulator of the development and function of regulatory T cells. Many HDAC inhibitors are in cancer clinical trials, but a subset of HDAC inhibitors has important anti-inflammatory or immunosuppressive effects that might be of therapeutic benefit in immuno-inflammatory disorders or post-transplantation. At least some of these effects result from the ability of HDAC inhibitors to enhance the production and suppressive functions of FOXP3+ regulatory T cells. Understanding which HDACs contribute to the regulation of the functions of regulatory T cells may further stimulate the development of new class- or subclass-specific HDAC inhibitors with applications beyond oncology.
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.
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.