Histone/protein deacetylase inhibitor therapy for enhancement of Foxp3+ T-regulatory cell function posttransplantation.

T-regulatory (Treg) cells are like other cells present throughout the body in being subject to biochemical modifications in response to extracellular signals. An important component of these responses involves changes in posttranslational modifications (PTMs) of histones and many nonhistone proteins, including phosphorylation/dephosphorylation, ubiquitination/deubiquitination, and acetylation/deacetylation. Foxp3, the key transcription factor of Tregs, is constantly being rapidly turned over, and a number of these PTMs determine its level of expression and activity. Of interest in the transplant setting, modulation of the acetylation or deacetylation of key lysine residues in Foxp3 can promote the stability and function, leading to increased Treg production and increased Treg suppressive activity. This mini-review focuses on recent data concerning the roles that histone/protein deacetylases (HDACs) play in control of Treg function, and how small molecule HDAC inhibitors can be used to promote Treg-dependent allograft survival in experimental models. These data are discussed in the light of increasing interest in the identification and clinical evaluation of isoform-selective HDAC inhibitors, and their potential application as tools to modulate Foxp3+ Treg cell numbers and function in transplant recipients.

Class-specific histone/protein deacetylase inhibition protects against renal ischemia reperfusion injury and fibrosis formation.

Renal ischemia-reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure. Histone/protein deacetylases (HDACs) regulate gene accessibility and higher order protein structures and may alter cellular responses to a variety of stresses. We investigated whether use of pan- and class-specific HDAC inhibitors (HDACi) could improve IRI tolerance in the kidney. Using a model of unilateral renal IRI, we investigated early renal function after IRI, and calculated fibrosis after IRI using an automated scoring system. We found that pan-HDAC inhibition using trichostatin (TSA) yielded significant renal functional benefit at 24-96 hours (p < 0.001). Treated mice developed significantly less fibrosis at 30 days (p < 0.0004). Class I HDAC inhibition with MS-275 yielded similar effects. Protection from fibrosis formation was also noted in a cold ischemia transplant model (p < 0.008) with a trend toward improved cold ischemic survival in TSA-treated mice. These effects were not accompanied by induction of typical ischemic tolerance pathways or by priming of heat shock protein expression. In fact, heat shock protein 70 deletion or overexpression did not alter renal ischemia tolerance. Micro-RNA 21, known to be enhanced in vitro in renal tubular cells that survive stress, was enhanced by treatment with HDACi, pointing to possible mechanism.

Blunted expression of miR-199a-5p in regulatory T cells of patients with chronic obstructive pulmonary disease compared to unaffected smokers.

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal regulatory T cell (T(reg)) response and increases in T helper type 1 (Th1) and Th17 cell responses. It is unclear if dysregulation of microRNAs (miRNA) within T(reg) cells contributes to the abnormal inflammatory response in COPD. In this study, we aimed to compare the miRNA profile of COPD T(reg) cells with that of healthy controls and to explore the function of differentially expressed miRNAs. We first obtained T(reg) and T effector cells (Teff ) from peripheral blood of non-smokers, unaffected current smokers and COPD current smokers. Then, we assessed their miRNA expression by microarray analysis followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) validation of particular miRNAs. Six and 96 miRNAs were expressed differentially in COPD T(reg) cells versus T(reg) cells of healthy non-smokers and healthy smokers, whereas no differences were found in miRNA expression in T(eff) cells. We found that miR-199a-5p was repressed by approximately fourfold in T(reg) cells of COPD patients compared to healthy smokers (P < 0·05). In addition, miR-199a-5p was over-expressed in T(reg) cells compared to Teff cells (P < 0·001) and had significant over-representation of its target genes in the T(reg) transcriptome, being associated with the transforming growth factor (TGF)-ß activation pathway (P < 0·01). We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan® arrays of the human TGF-ß pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favour of a Th1 and Th17 response.

Anti-TCRß mAb induces long-term allograft survival by reducing antigen-reactive T cells and sparing regulatory T cells.

TCR specific antibodies may modulate the TCR engagement with antigen-MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRß (H57-597), TCRα or CD3 promptly reduced the number of CD4(+) and CD8(+) T cells in normal mice, but H57-597 mAb most potently increased the frequency of CD4(+) Foxp3(+) Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57-597 mAb, the expansion of SEB-reactive Vß8(+) T cells was completely abrogated while SEB-nonreactive Vß2(+) T cells remained unaffected. More importantly, transient H57-597 mAb treatment exerted long-lasting effect in preventing T cell responses to alloantigens, and produced long-term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor-specific long-term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57-597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin-sensitized recipients. Thus, transient modulation of the TCRß chain by H57-597 mAb exhibits potent, long-lasting therapeutic effects to control alloimmune responses.

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 posttransplant. We analyzed Treg number, phenotype, suppressive function, and methylation at the Treg-specific demethylation region (TSDR) using Tregs and peripheral blood mononuclear cells. Forty-eight 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.

Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes.

We investigated the pathogenesis of chronic allograft rejection in mouse cardiac allografts. Long-term survival occurred after administration of monoclonal antibody to CD4 or CD40-ligand (CD40L) plus donor cells. Both treatments induced permanent graft survival, but, in contrast to transplants in mice treated with CD4 monoclonal antibody, grafts in mice treated with CD40L monoclonal antibody lacked evidence of chronic rejection, including transplant arteriosclerosis. Freedom from chronic rejection in the group treated with CD40L monoclonal antibody correlated with vascular expression of the 'protective' genes heme oxygenase-1 (HO-1), Bcl-xL and A20. Moreover, arteriosclerosis was induced in allografts in immunoglobulin-deficient mice by antibody transfer only when the transfer was done before expression of protective genes. A direct role for protective gene expression in endothelial cells was demonstrated by in vitro experiments in which induction of HO-1 or Bcl-xL suppressed alloantibody-stimulated endothelial activation. Finally, induction of HO-1 in vivo protected allografts against chronic injury. These data show a role for protective genes in the prevention of chronic rejection, and indicate new approaches to protect grafts against development of transplant arteriosclerosis.

Accommodation of vascularized xenografts: expression of "protective genes" by donor endothelial cells in a host Th2 cytokine environment.

Organ xenografts under certain circumstances survive in the presence of anti-graft antibodies and complement, a situation referred to as "accommodation." We find that the endothelial cells (ECs) in hamster hearts that accommodate themselves in rats express genes, such as A20 and bcl-2, that in vitro protect ECs from apoptosis and prevent upregulation in those cells of proinflammatory genes such as cytokines, procoagulant and adhesion molecules. Hearts that are rejected do not express these genes. In addition, vessels of rejected hearts show florid transplant arteriosclerosis whereas those of accommodated hearts do not. Accommodated xenografts have an ongoing T helper cell type 2 (Th2) cytokine immune response, whereas the rejected grafts have a Th1 response. We propose a model for factors that contribute to the survival of xenografts and the avoidance of transplant arteriosclerosis.

Adenovirus-mediated gene transfer into cold-preserved liver allografts: survival pattern and unresponsiveness following transduction with CTLA4Ig.

The immune response of liver transplant recipients was modulated via adenovirus-mediated transduction of the cold-preserved liver with sequences encoding CTLA4Ig. Transplanted allografts demonstrated rapid transient local expression and recombinant protein production shortly after revascularization, resulting in intact liver function, indefinite survival of the recipient, and the development of donor-specific unresponsiveness. Lymphocytic infiltration of the graft was mainly of the T helper 2 (Th2) subset and was not associated with injury to primary cellular targets of the alloimmune response. These findings demonstrate a successful outcome of a feasible and potentially clinically relevant system of gene delivery of sequences encoding proteins capable of inhibiting the alloimmune response.

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance.

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.

Tumor necrosis factor production during human renal allograft rejection is associated with depression of plasma protein C and free protein S levels and decreased intragraft thrombomodulin expression.

Fibrin deposition is a common accompaniment of renal allograft rejection, indicating disruption of the normal physiologic balance between procoagulant and anticoagulant pathways. In vitro, tumor necrosis factor (TNF) induces endothelial expression of the procoagulant, tissue factor, and downregulation of thrombomodulin, a key component of the thrombomodulin/protein C (PC)/protein S (PS) pathway, which normally maintains an anticoagulant state by inactivating thrombin, preventing further thrombin formation by degrading factors Va and VIIIa, and decreasing plasminogen activator inhibitor activity. Raised levels of TNF were recently demonstrated within the blood of patients during episodes of renal allograft injection, and may be an early and discriminatory marker of rejection. This led us to investigate prospectively whether monitoring of serum TNF levels was of value clinically, and was associated with effects on circulating PC and PS levels, or alterations in intragraft thrombomodulin expression. Plasma samples (n = 454) were collected three times/week from all patients (n = 25) undergoing renal transplantation during a 9-month consecutive period, and assayed by ELISA and functional assays for TNF, PC, and free PS (FPS). Portions of renal biopsies, taken to evaluate episodes of acute deterioration of renal function, were evaluated by immunoperoxidase labeling for the presence and distribution of TNF, thrombomodulin, PC, PS, thrombin, fibrin, and factors V and VIII. Comparison of 78 plasma samples collected during 26 episodes of biopsy-proven acute cellular rejection with samples collected during periods of stable renal function (n = 349) showed that TNF levels rose significantly (390 +/- 242 pg/ml, p less than 0.01) above background levels 3 days before rising serum creatinine concentrations, and peaked (2,426 +/- 978 pg/ml) on the day of clinical rejection. PC-antigen (Ag) concentrations also decreased 3 days before rejection (68 +/- 13%, p less than 0.05), and were maximally depressed (49% +/- 16%, p less than 0.001) on the day of rejection. FPS levels were normal until the day before rejection (63% +/- 8%, p less than 0.01) and, like PC, were maximally depressed (43 +/- 10%) at rejection. Plasma TNF levels were significantly and inversely correlated with PC-Ag (p less than 0.001) and FPS (p less than 0.005) levels during rejection, regardless of whether such rejection episodes were steroid responsive or required OKT3 monoclonal antibody therapy. TNF, PC, and FPS levels were normal during episodes of cyclosporine toxicity and viral infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor beta, interleukin 4, and prostaglandin E expression in the brain.

Experimental autoimmune encephalomyelitis (EAE) in the Lewis rat is a self-limited inflammatory process localized to the central nervous system that is induced by the injection of myelin basic protein (MBP) in adjuvant. Oral administration of MBP suppresses EAE, and this suppression is mediated by CD8+ T cells that adoptively transfer protection and suppress both in vitro and in vivo by the release of transforming growth factor (TGF) beta after antigen-specific triggering. Furthermore, oral tolerance to MBP is enhanced by the concomitant oral administration of lipopolysaccharide (LPS). The present study was undertaken to determine whether the disease course in EAE and its suppression by oral tolerization to MBP is associated with distinct patterns of cytokine expression in the target organ. Detailed immunohistology of the brain was performed at the peak of clinical disease (day 14 after immunization) and after recovery (day 18) in control (ovalbumin [OVA]-fed), MBP-fed, and MBP plus LPS-fed animals. Brains from OVA-fed animals at the peak of disease showed perivascular infiltration with activated mononuclear cells which secreted the inflammatory cytokines interleukins (IL) 1, 2, 6, 8, TNF-alpha, and interferon gamma. The inhibitory cytokines TGF-beta and IL-4, and prostaglandin E2 (PGE2) were absent. In MBP orally tolerized animals there was a marked reduction of the perivascular infiltrate and downregulation of all inflammatory cytokines. In addition, there was upregulation of the inhibitory cytokine TGF-beta. In MBP plus LPS orally tolerized animals, in addition to upregulation of TGF-beta and reduction of inflammatory cytokines, there was enhanced expression of IL-4 and PGE2, presumably secondary to activation of an additional population of immunoregulatory cells. In OVA-fed animals that had recovered (day 18), staining for inflammatory cytokines diminished, and there was the appearance of TGF-beta and IL-4. These results suggest that suppression of EAE, either induced by oral tolerization or that which occurs during natural recovery is related to the secretion of inhibitory cytokines or factors that actively suppress the inflammatory process in the target organ.

P-selectin and platelet-activating factor mediate initial endotoxin-induced neutropenia.

Polymorphonuclear neutrophil (PMN) accumulation within damaged tissues, a hallmark of acute inflammation, is dependent upon initial adhesion to endothelial cells. In vitro studies suggest that P-selectin and platelet activating factor (PAF) are key molecules in this process by promoting the initial adhesion of PMN to endothelial cells. We report in vivo studies in which intravenous administration of lipopolysaccharide (LPS) to anesthetized rats caused a very rapid onset (< 5 min) of neutropenia, in association with induction of surface expression of P-selectin on microvascular endothelial cells in kidney, liver and lung; analogous induction of P-selectin expression by cultured endothelial cells was observed in response to LPS stimulation in vitro. In addition, treatment with an antibody (Ab) to P-selectin (or use of a PAF antagonist) blocked development of neutropenia in vivo for at least 15 min post-LPS injection, and Ab treatment was shown to block PMN accumulation in tissues. These studies document roles for P-selectin and PAF in the early adhesion of PMN to endothelial cells in vivo.

Donor-derived IP-10 initiates development of acute allograft rejection.

An allograft is often considered an immunologically inert playing field on which host leukocytes assemble and wreak havoc. However, we demonstrate that graft-specific physiologic responses to early injury initiate and promulgate destruction of vascularized grafts. Serial analysis of allografts showed that intragraft expression of the three chemokine ligands for the CXC chemo-kine receptor CXCR3 was induced in the order of interferon (IFN)-gamma-inducible protein of 10 kD (IP-10, or CXCL10), IFN-inducible T cell alpha-chemoattractant (I-TAC; CXCL11), and then monokine induced by IFN-gamma (Mig, CXCL9). Initial IP-10 production was localized to endothelial cells, and only IP-10 was induced by isografting. Anti-IP-10 monoclonal antibodies prolonged allograft survival, but surprisingly, IP-10-deficient (IP-10(-/-)) mice acutely rejected allografts. However, though allografts from IP-10(+/+) mice were rejected by day 7, hearts from IP-10(-/-) mice survived long term. Compared with IP-10(+/+) donors, use of IP-10(-/-) donors reduced intragraft expression of cytokines, chemokines and their receptors, and associated leukocyte infiltration and graft injury. Hence, tissue-specific generation of a single chemokine in response to initial ischemia/reperfusion can initiate progressive graft infiltration and amplification of multiple effector pathways, and targeting of this proximal chemokine can prevent acute rejection. These data emphasize the pivotal role of donor-derived IP-10 in initiating alloresponses, with implications for tissue engineering to decrease immunogenicity, and demonstrate that chemokine redundancy may not be operative in vivo.

Acquired tolerance to experimental autoimmune encephalomyelitis by intrathymic injection of myelin basic protein or its major encephalitogenic peptide.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP) in adjuvant, and serves as an experimental model for the study of multiple sclerosis. The role of the thymus in acquired tolerance in autoimmune models has not been thoroughly investigated. In this study, we examined the effects of intrathymic injection of MBP or its major encephalitogenic peptide on the course of EAE in Lewis rats. A single intrathymic injection of MBP 48 h pre- but not postimmunization protects animals from actively induced EAE. An intact MBP-primed thymus was required up to 10 d postimmunization, as thymectomy on days 1, 2, and 7 postimmunization abrogated the protective effect, whereas thymectomy on day 10 did not. The proliferative response of primed lymphocytes was significantly reduced in animals that were intrathymically injected with MBP. Protection against clinical EAE was induced by thymic injection of the major encephalitogenic region (residues 71-90) but not a nonencephalitogenic (21-40) MBP epitope. Immunohistologic examination of the brain from rats intrathymically injected with encephalitogenic peptide showed markedly reduced cellular infiltrate and virtual absence of activation and inflammatory cytokines as compared with rats intrathymically injected with the nonencephalitogenic peptide. These results indicate that the thymus may play an active role in acquired systemic immunologic tolerance in T cell-mediated experimental autoimmune diseases. This effect may be mediated by a process of clonal inactivation of autoreactive T cell clones circulating through the thymus.

CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2.

Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig-treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig-treated animals was associated with increased staining for the Th2-related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig-treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.

Loss of ATP diphosphohydrolase activity with endothelial cell activation.

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

Mechanisms of acquired thymic tolerance in experimental autoimmune encephalomyelitis: thymic dendritic-enriched cells induce specific peripheral T cell unresponsiveness in vivo.

Experimental autoimmune encephalomyelitis (EAE), an experimental model for the study of multiple sclerosis, is an autoimmune disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP). MBP-reactive CD4+ T cells, predominantly expressing the V beta 8.2 T cell receptor (TCR), migrate from the peripheral lymphoid organs and initiate the inflammatory response in the brain. We have previously shown that a single intrathymic injection of MBP or its major encephalitogenic peptide (p71-90), but not a nonencephalitogenic peptide (p21-40), induces antigen-specific systemic tolerance and inhibits the induction of EAE in Lewis rats. In this study, we investigated the mechanisms of induction and maintenance of acquired thymic tolerance in this model. First, we investigated which thymic cell is responsible for "induction" of systemic tolerance. Thymic dendritic-enriched cells, isolated by plastic adherence, when incubated in vitro with p71-90 and injected intravenously into Lewis rats, were capable of preventing the development of EAE, but his protection was lost in thymectomized recipients. In addition, intravenous injection of thymic dendritic cells isolated from animals that had been previously injected intrathymically with p71-90 but not p21-40 also prevented the development of EAE. Second, to determine the "effector" mechanisms involved in acquired thymic tolerance, we compared TCR expression in the brains of animals with actively induced EAE with TCR expression in animals that received intrathymic injection of p71-90 or p21-40. Using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique, we found increased expression of CD4 and V beta 8.2 message in brains of immunized animals compared with those of naive animals. In animals intrathymically injected with p71-90 but not p21-40, CD4 and V beta 8.2 transcript levels were significantly reduced compared with immunized controls. Immunohistologic studies of brain tissue and spleens with specific V beta 8.2 and control V beta 10 monoclonal antibodies confirmed these observations in vivo. These findings, taken together with recent data demonstrating that activated T cells circulate through the thymus, suggest that interaction of thymic dendritic cells with specific TCR of activated peripheral T cells can lead to inactivation of these antigen-specific cells and confirm the role of V beta 8.2-expressing T cells in EAE.

Requirement of the chemokine receptor CXCR3 for acute allograft rejection.

Chemokines provide signals for activation and recruitment of effector cells into sites of inflammation, acting via specific G protein-coupled receptors. However, in vitro data demonstrating the presence of multiple ligands for a given chemokine receptor, and often multiple receptors for a given chemokine, have led to concerns of biologic redundancy. Here we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the chemokines interferon (IFN)-gamma-inducible protein of 10 kD (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T cell alpha chemoattractant (I-TAC), and by infiltration of activated T cells bearing the corresponding chemokine receptor, CXCR3. We used three in vivo models to demonstrate a role for CXCR3 in the development of transplant rejection. First, CXCR3-deficient (CXCR3(-/)-) mice showed profound resistance to development of acute allograft rejection. Second, CXCR3(-/)- allograft recipients treated with a brief, subtherapeutic course of cyclosporin A maintained their allografts permanently and without evidence of chronic rejection. Third, CXCR(+/+) mice treated with an anti-CXCR3 monoclonal antibody showed prolongation of allograft survival, even if begun after the onset of rejection. Taken in conjunction with our findings of CXCR3 expression in rejecting human cardiac allografts, we conclude that CXCR3 plays a key role in T cell activation, recruitment, and allograft destruction.

Plasma cytokines and chemokines in primary graft dysfunction post-lung transplantation.

Primary graft dysfunction (PGD) after lung transplantation causes significant morbidity and mortality. We aimed to determine the role of cytokines and chemokines in PGD. This is a multicenter case-control study of PGD in humans. A Luminex analysis was performed to determine plasma levels of 25 chemokines and cytokines before and at 6, 24, 48 and 72 h following allograft reperfusion in 25 cases (grade 3 PGD) and 25 controls (grade 0 PGD). Biomarker profiles were evaluated using a multivariable logistic regression and generalized estimating equations. PGD cases had higher levels of monocyte chemotactic protein-1 (MCP-1)/chemokine CC motif ligand 2 (CCL2) and interferon (IFN)-inducible protein (IP-10)/chemokine CXC motif ligand 10 (CXCL10) (both p < 0.05), suggesting recruitment of monocytes and effector T cells in PGD. In addition, PGD cases had lower levels of interleukin (IL-13) (p = 0.05) and higher levels of IL-2R (p = 0.05). Proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha, and IFN-gamma decreased to very low levels after transplant in both PGD cases and controls, exhibiting no differences between the two groups. These findings were independent of clinical variables including diagnosis in multivariable analyses, but may be affected by cardiopulmonary bypass. Profound injury in clinical PGD is distinguished by the upregulation of selected chemokine pathways, which may useful for the prediction or early detection of PGD if confirmed in future studies.

Effects of histone deacetylase inhibitor SAHA on effector and FOXP3+regulatory T cells in rhesus macaques.

Suberoylanilide hydroxamic acid (SAHA) a histone deacetylase inhibitor (HDACi), is clinically approved for treatment of cutaneous T-cell lymphoma. Although the exact underlying mechanisms are unknown, HDACi arrests the cell cycle in rapidly proliferating tumor cells and promote their apoptosis. HDACi were also recently shown to enhance the production and suppressive functions of Foxp3+ regulatory T (Treg) cells in rodents, leading us to begin to investigate the actions of HDACi on rhesus monkey T cells for the sake of potential preclinical applications. In this study, we show that SAHA inhibits polyclonal activation and proliferation of rhesus T cells and that the antiproliferative effects are due to inhibition of T-effector (Teff) cells and enhancement of Treg cells. Cryopreserved rhesus macaque splenocytes were CFSE labeled, stimulated with anti-CD3/anti-CD28 and cultured for 5 days in the presence of varying concentrations of SAHA. Samples were then costained to evaluate CD4 and CD8 expression. Concentrations of SAHA (10 and 5 micromol/L) were toxic to splenocytes. Proliferation was inhibited by 57% in CD4 cells and 47% in CD8 cells when unseparated splenocytes were cultured with 3 micromol/L SAHA. Effector cells alone showed decreased inhibition to proliferation when cultured with 3 micromol/L and 1 micromol/L SAHA when compared to Teff plus Treg cells. Our data suggest that SAHA can be used as part of an immunosuppressive protocol to enhance graft survival by limiting Teff cell proliferation as well as increasing Treg cells, thereby promoting tolerance.