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.
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
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.
Immunosuppression; immunoregulation; clinical transplantation
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 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
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.
Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane) (PDMS), a biocompatible silicone elastomer. We show that softer (Young’s Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4+ and CD8+ T cells compared with stiffer substrates (E >2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (non-significant) towards a greater proportion of CD62Lneg, effector-differentiated CD4+ and CD8+ T cells. Naïve CD4+ T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ producing TH1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation and TH differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.
T cells; Cell Activation; Cell Proliferation; Cytokines; Human
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
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.
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.
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
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.
transplantation; allograft rejection; T cell activation; costimulation; TNF superfamily
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 homo-dimerization 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 homo-oligomerization, acetylation, and hetero-association has been dissected, defining atomic insights into the biological and pathological characteristics of the FOXP3 complex.
FOXP3; IPEX Syndrome; Dimerization; Acetylation; Complex Assembly
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.
Erythropoiesis, the production of red blood cells, must be tightly controlled to ensure adequate oxygen delivery to tissues without causing thrombosis or stroke. Control of physiologic and pathologic erythropoiesis is dependent predominantly on erythropoietin (EPO), the expression of which is regulated by hypoxia-inducible factor (HIF) activity in response to low oxygen tension. Accumulating evidence indicates that oxygen-independent mediators, including inflammatory stimuli, cytokines, and growth factors, also upregulate HIF activity, but it is unclear whether these signals also result in EPO production and erythropoiesis in vivo. Here, we found that signaling through herpesvirus entry mediator (HVEM), a molecule of the TNF receptor superfamily, promoted HIF-1α activity in the kidney and subsequently facilitated renal Epo production and erythropoiesis in vivo under normoxic conditions. This Epo upregulation was mediated by increased production of NO by renal macrophages. Hvem-deficient mice displayed impaired Epo expression and aggravated anemia in response to erythropoietic stress. These data reveal that HVEM signaling functions to promote HIF-1α activity and Epo production, and thus to regulate erythropoiesis. Furthermore, our findings suggest that this molecular mechanism could represent a therapeutic target for Epo-responsive diseases, including anemia.
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.
Regulatory T cells (Tregs) are required for the maintenance of immune homeostasis as first clearly described by Herman Waldmann’s laboratory. Dysfunction of Treg cells also leads to fatal autoimmunity in humans and mice. Conversely, the activation of different classes of Tregs operative systemically and within the cancer microenvironment can suppress host anti-tumor immune responses and promote tumor progression. Therefore, the development of new therapeutic approaches to regulate the activity of Treg cells may have considerable clinical potential.
FOXP3 is the key transcriptional regulator of Treg development and function. The activity of FOXP3 is regulated by acetylation, a process catalyzed by distinct types of histone/protein acetyltransferases (HATs) that regulate the functions of many transcription factors, independently of FOXP3, as well as non-histone proteins, in addition to their effects on chromatin accessibility. Interactions between FOXP3 and these enzymes determine the suppressive function of FOXP3. Clearly, small molecules targeting these enzymes are candidates for the regulation of Treg function in vaccines and tumor therapies.
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
Collagen-induced arthritis (CIA) is an established mouse model of disease with hallmarks of clinical rheumatoid arthritis. Histone/protein deacetylase inhibitors (HDACi) are known to inhibit the pathogenesis of CIA and other models of autoimmune disease, although the mechanisms responsible are unclear. Regulatory T cell (Treg) function is defective in rheumatoid arthritis. FOXP3 proteins in Tregs are present in a dynamic protein complex containing histone acetyltransferase and HDAC enzymes, and FOXP3 itself is acetylated on lysine residues. We therefore investigated the effects of HDACi therapy on regulatory T cell function in the CIA model. Administration of an HDACi, valproic acid (VPA), significantly decreased disease incidence (p<0.005) and severity (p<0.03) in CIA. In addition, VPA treatment increased both the suppressive function of CD4+CD25+ Tregs (p<0.04) and the numbers of CD25+FOXP3+ Tregs in vivo. Hence, clinically approved HDACi such as VPA may limit autoimmune disease in vivo through effects on the production and function of FOXP3+ Treg cells.
T cell; suppression; autoimmunity; rheumatoid arthritis
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.
Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4+Foxp3+ T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4+Foxp3+ Tregs.