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BACKGROUND - Many models of transplant tolerance have been found to depend on the induction of regulatory T cells (Tregs). Innate immune signals are known to suppress Tregs thereby augmenting immunity by abrogating Treg function. Such signals may also provide a barrier to transplantation tolerance mediated by Tregs. A number of cell surface molecules expressed by Tregs have been found to inhibit Treg activity, the best characterized of which is the glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein.
METHODS - By using an adoptive transfer model of allograft rejection, we can study the effects of inflammation and antigen-specific Tregs on graft survival. Inflammation resulting from the transplant procedure counter-regulates the suppressor activity of Tregs. To assess whether Treg activity could be enhanced by blocking GITR signaling, we compared the capacity of Tregs to prolong the survival of grafts in the presence or absence of activation-inducible TNF receptor (AITRL)-Fc, a novel construct that binds GITR.
RESULTS - We report that interruption of GITR-GITR ligand (GITRL) binding by AITRL-Fc resulted in long-term Treg-dependent acceptance of skin grafts in the setting of innate immune signals that otherwise interfere with Treg activity.
CONCLUSIONS - Inflammation and other innate immune signals may activate antigen presenting cells to upregulate GITRL. GITR-GITRL interaction is one pathway by which antigen presenting cells may enhance the adaptive response to foreign antigen by counter-regulating Tregs and by costimulating effector T cells. By blocking this interaction with AITRL-Fc, one can sustain the benefit conferred by graft-protective Tregs.
Inhaled immunosuppression with tacrolimus (TAC) is a novel strategy after lung transplantation. Here we investigate the feasibility of tacrolimus delivery via aerosol, assess its immunosuppressive efficacy, reveal possible mechanisms of action, and evaluate its airway toxicity. Rats received 4 mg/kg TAC via oral or inhaled (AER) administration. Pharmacokinetic properties were compared, and in vivo airway toxicity was assessed. Full-thickness human airway epithelium (AE) was grown in vitro at an air-liquid interface. Equal TAC doses (10-1,000 ng) were either added to the bottom chamber (MED) or aerosolized for gas-phase exposure (AER). Airway epithelium TAC absorption, cell toxicity, and interactions of TAC with NFκB activation were studied. Single-photon emission computed tomography demonstrated a linear tracer accumulation within the lungs during TAC inhalation. The AER TAC generated higher lung-tissue concentrations, but blood concentrations that were 11 times lower. Airway histology and gene expression did not reveal drug toxicity after 3 weeks of treatment. In vitro AE exposed to TAC at 10-1,000 ng, orally or AER, maintained its pseudostratified morphology, did not show cell toxicity, and maintained its epithelial integrity, with tight junction formation. The TAC AER-treated AE absorbed the drug from the apical surface and generated lower-chamber TAC concentrations sufficient to suppress activated lymphocytes. Tacrolimus AER was superior to TAC MED at preventing AE IFN-γ, IL-10, IL-13, monocyte chemoattractant protein-1 chemokine (C-C motif) ligand 5 (RANTES) and TNF-α up-regulation. Tacrolimus inhibited airway epithelial cell NFκB activation. In conclusion, TAC can be delivered easily and effectively into the lungs without causing airway toxicity, decreases inflammatory AE cytokine production, and inhibits NFκB activation.
Natural killer T (NKT) cells are a subset of regulatory T lymphocytes that recognize glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d. NKT cells have been implicated in regulating the progression of Type 1 diabetes (T1D) in human patients and in an animal model for T1D. In addition, glycolipid agonists of NKT cells have been successful in preventing diabetes in mice, raising enthusiasm for the development of NKT cell-based therapies for T1D.
Recently, it was shown that exogenous ubiquitin has anti-inflammatory actions in vivo and that the ubiquitin-decapeptide 50-59 has immunosuppressive effects similar to cyclosporine. Immunosuppressive effects of the native ubiquitin molecule and its therapeutic potential in transplantation are unknown. We tested the hypothesis that ubiquitin inhibits alloreactivity and increases allograft survival in a murine model of skin transplantation in fully mismatched strain combinations (C3H/HEJ-DBA2). Ubiquitin dose-dependently inhibited mixed leukocyte reaction in C3H/HEJ splenocytes in vitro. Intraperitoneal ubiquitin administration (25 microg/h for 14 days) was well-tolerated, dose-dependently increased ubiquitin serum concentrations and median allograft survival from 10 days (with albumin; control) to 17 days in DBA2 mice (survival ratio: 1.7, 95% confidence interval: 1.266-2.134; P=0.0005). The in vivo effects in this study combined with our previous work strongly indicate that ubiquitin is a potent immune modulator with broad therapeutic potential. Ubiquitin treatment could be a novel strategy to improve immunosuppressive regimens in transplantation.
Organ transplantation represents the only definitive therapy for many causes of end-organ failure. However, the universal success of this therapy is limited by chronic allograft rejection, the side effects of chronic immunosuppressive therapy, and a severe shortage of donor organs. Presently, the success of solid-organ transplantation depends on the continuous administration of toxic and nonspecific immunosuppressive agents, therapies that present risks for opportunistic infection, malignancy, and a variety of agent-specific side effects. To promote the use of transplantation with limited risk of long-term sequelae, three dominant research challenges emerge: (i) elimination of the need for exogenous immunosuppression by immunological tolerance induction; (ii) prevention of chronic rejection/graft dysfunction; and (iii) expansion of available organs for transplantation. Gene therapy may provide significant advances and solutions in each of these areas. Rejection of the graft in the immediate post-transplant period has been attacked through the transfer of immunomodulatory molecules in addition to tolerance inducing approaches. Chronic graft rejection may be similarly addressed through permanent tolerance induction or alternatively through the introduction of molecules to resist chronic graft damage. Genetic manipulation of stem cells may ultimately produce transgenic animals to serve as tissue donors to overcome the limited donor organ supply. This review will highlight ongoing developments in the translation of gene therapy approaches to the challenges inherent in transplantation.
The plant-derived cannabinoids delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD) both have immunosuppressive effects; although some effects of THC are mediated by the CB2 receptor, CB2 binds CBD weakly. In examining the effects of THC and CBD on microglial proliferation, we found that these compounds potently inhibit [3H]thymidine incorporation into a murine microglial cell line with no effect on cell cycle. Treatment with THC and CBD decreased [3H]thymidine uptake into microglia, with IC50 values that match inhibition of [3H]thymidine incorporation into DNA. CBD and, less potently, THC decreased uptake of [3H]adenosine to a similar extent as [3H]thymidine in both murine microglia and RAW264.7 macrophages. Binding studies confirm that CBD binds to the equilibrative nucleoside transporter 1 with a Ki < 250 nM. Because adenosine agonists have antiinflammatory effects, and because uptake of adenosine is a primary mechanism of terminating adenosine signaling, we tested the hypothesis that CBD is immunosuppressive because it enhances endogenous adenosine signaling. In vivo treatment with a low dose of CBD decreases TNFalpha production in lipopolysaccharide-treated mice; this effect is reversed with an A2A adenosine receptor antagonist and abolished in A2A receptor knockout mice. These studies demonstrate that CBD has the ability to enhance adenosine signaling through inhibition of uptake and provide a non-cannabinoid receptor mechanism by which CBD can decrease inflammation.
We previously demonstrated that T-regs inhibit proliferation of graft-reactive T cells in the draining lymph node (DLN), suggesting that this site may be important for regulation. TCR transgenic mice (TS1) specific for viral hemagglutinin (HA) provided antigen-specific T cells for adoptive transfer into syngeneic Balb/c hosts bearing HA+ skin grafts. T-regs were obtained from (TS1xHA28)F1 mice known to have an expanded population of HA-specific T-regs. To determine whether the lymph node is an independent site of suppression, we developed a model in which donor antigen that migrates from the allograft to the DLN drives T-cell activation after graft removal. T-regs that did not encounter the allograft itself remained able to inhibit graft antigen-specific T-cell proliferation in the DLN. Alloantigen-induced regulation can occur in the absence of the graft. This finding identifies the DLN as a potentially critical site of regulation in the early posttransplant period.
The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.
The number of patients returning to dialysis after their renal allograft fails is increasing in absolute numbers year after year. The management of the failed allograft that is not immediately symptomatic remains controversial. Surgical mortality and morbidity, a rising number of circulating antibodies, reduced erythropoietin, and diuresis are among the arguments to support simply observing the failed allograft. Chronic inflammation, potential for malignancy, infection, and the need for low-dose immunosuppression are concerns that might goad one into performing a preemptive nephrectomy. Based on the current literature and our own clinical experience, we believe allograft nephrectomies should not be routinely performed. They should be reserved for those patients who develop particular symptoms attributable to the allograft or those who require space for retransplantation. Future studies that address this issue in addition to testing various immunosuppression attrition rates may be able to discern a protocol that minimizes drug exposure while leading to reduced nephrectomy rates after returning to dialysis.
After a major illness or injury, immune status in critically ill patients may fluctuate between a marked proinflammatory response and an immunosuppressed state. Postinflammatory immunosuppression can result in increased susceptibility to infection. Alterations of cytokine production, such as suppression of IFNgamma and elevation of the anti-inflammatory cytokine IL-10, are believed to contribute to postinflammatory immunosuppression. We examined antimicrobial immunity in mice that had previously been subjected to a sublethal cecal ligation and puncture (CLP) as a model of major injury. Mice were challenged with Pseudomonas aeruginosa (5 x 10(7) CFU i.v.) on day 5 after CLP or sham surgery. Bacterial clearance in mice after CLP was impaired and associated with decreased production of IFNgamma and increased production of IL-10 in the early response to the Pseudomonas challenge. Pseudomonas-induced production of the IFNgamma-inducing factor IL-12 was also decreased in post-CLP mice. However, splenocytes from post-CLP mice remained responsive to exogenous stimulation with the IFNgamma-inducing cytokines IL-12, IL-15, and IL-18 as well as T-cell receptor activation. Furthermore, production of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 were as high, or higher, in the post-CLP group compared with sham mice after P. aeruginosa challenge. Blockade of IL-10 did not reverse IL-12 and IFNgamma suppression in splenocytes from post-CLP mice. These studies show that suppressed bacterial clearance in post-CLP mice is associated with decreased production of IFNgamma and IL-12 and with increased production of IL-10 and proinflammatory cytokines.