Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

BACKGROUND: Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. METHODS: Splenocytes from C57BL/6 J (B6) AID(−/−) mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID(−/−) mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. RESULTS: The AID(−/−) splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID(−/−) recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID(−/−) recipients with further reduction of intimal hyperplasia and fibrosis scores. CONCLUSIONS: The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.

Microarray gene expression profiling of chronic allograft nephropathy in the rat kidney transplant model.

Whole genome gene expression profiles were correlated with renal function and histology in a well-established animal model of chronic allograft nephropathy (CAN). Kidneys of F344 rats were transplanted into LEW recipients treated with a brief dose of FK506 (BFK). Blood and urine samples were collected weekly. Kidney grafts were harvested at an early (day 6) or late (days 30-90) phase after transplantation. BFK kidney grafts showed remarkable changes in function, histology, and gene expression profiles when compared to the isograft controls. In the early phase, renal function and histology were barely affected, yet the expression levels of 225 genes were significantly changed, reflecting both immune and non-immune pathways. In the late phase, however, 826 genes were affected in the BFK kidney grafts, including genes in the pathways of extracellular matrix and cell adhesion. Of these genes, 214 appear to be key factors for development of CAN, since they were affected at both early and late phases, including genes involved in the immune response, the inflammatory response, apoptosis, and metabolism. Kinetic studies with gene expression profiling can identify genes involved in the progressive development of chronic allograft rejection, leading to more detailed therapeutic approaches or useful biomarkers in clinical transplantation.

Mechanism analysis of long-term graft survival by monocarboxylate transporter-1 inhibition.

BACKGROUND: Monocarboxylate transporter (MCT)-1, a member of a family of molecules, transports monocarboxylates such as lactate. Inhibiting MCT-1 leads to long-term graft survival in rodent heart transplantation and induces tolerance. We evaluated an MCT-1 inhibitor, AS2495674, in a rat heart transplant model and analyzed its underlying mechanism. METHODS: AS2495674 was tested on rat lymphocytes to determine its effect on lactate accumulation, proliferation, and immunoglobulin production. The effect of AS2495674 on graft survival was tested on the Brown Norway to Lewis rat strain combination with a second heart transplantation to test donor-specific suppression. Histology and ex vivo analyses were done to examine the AS2495674 effects on the immune response. RESULTS: In vitro, AS2495674 resulted in lactate accumulation, inhibited lymphocyte proliferation, and prevented immunoglobulin production. AS2495674 induced long-term allograft survival with little evidence of chronic rejection and induced donor-specific suppression. Evaluation of the allograft and peripheral T lymphocytes from the AS2495674 group compared with that of vehicle showed (1) decreased donor-specific T lymphocyte response, (2) more forkhead box P3+ (Foxp3+) and CD45RA+ cells in the allograft, (3) higher gene expression of chemokines and chemokine receptors in the allograft, and (4) preferential inhibition of Foxp3(-) cells with little or no effect on Foxp3+ cells. CONCLUSIONS: AS2495674 prevents acute rejection, reduces features of chronic rejection, and induces tolerance. Our data suggest that the mechanism of AS2495674 involves generating a tolerogenic graft environment by preferentially targeting T effector cells while sparing the generation of T regulatory cells.

A blocking anti-CD28-specific antibody induces long-term heart allograft survival by suppression of the PKC theta-JNK signal pathway.

This study investigated the effects of a blocking anti-CD28 antibody (Anti-CD28-PV1-IgG3) in vitro and in vivo. Anti-CD28-PV1-IgG3, a hamster-mouse chimeric antibody against murine CD28, which does not provide CD28-positive signaling during TCR-driven T cell activation, enabled long-term survival of heart allografts across a complete mismatch of the MHC in rats. Among the T cell signaling proteins tested in the spleens from recipients, we found that recipients treated with anti-CD28-PV1-IgG3 exhibited suppression of alloantigen-initiated proximal TCR signaling events, including Lck, Zap70, Vav, and PI3K expression, and their PKC theta- and JNK-regulated expression/activation. This leads to attenuation of intragraft T cell infiltration and expression of T cell effector molecules. These results indicate that targeting the CD28 receptor with a blocking antibody leads to long-term allograft survival by reducing activation of alloantigen-mediated key signaling events in T cells that might be crucial for full T cell activation.

Unique gene expression profiles of heart allograft rejection in the interferon regulatory factor-1-deficient mouse.

Interferon regulatory factor-1 (IRF1) is a transcription factor for many genes involved in innate and adaptive immune responses. By using DNA array technology, we have previously demonstrated that IRF1 is significantly upregulated during acute rejection in rat heart allografts and is restored to isograft levels when recipients are treated with the immunosuppressants tacrolimus or cyclosporin A (CsA). To understand the precise role of IRF1 in transplant rejection, we investigated the rejection responses of mice completely deficient of IRF1 protein. Heterotopic heart transplantations were performed using C57BL/6J wild-type (WT B6) and IRF1-deficient (IRF1-/-) mice as recipients, and C3H mice as donors. Graft survival was determined by abdominal palpation and rejection was confirmed by histology. On day 6 after transplantation, isografts and allografts were harvested and subjected to gene expression analysis by a commercial nylon array and by real-time RT-PCR. Median survival time of heart allografts was 8 days in the WT B6 mice and 10 days in the IRF1-/- mice. The gene expression profiles of allografts from the WT B6 and IRF1-/- recipients were nearly identical to each other and very different from the profile of the isograft control. Both WT B6 and IRF1-/- profiles showed 13 genes upregulated (IFN-gamma, MCP-2, MIP-1alpha, MIP-1beta, CCR5, MIG, IP-10 and others) and one gene downregulated (SDF2) among the 76 genes detectable on the array. In more detailed analyses, distinct cytokine and chemokine gene expression profiles were identified in the allografts from the WT B6 and IRF1-/- recipients. Whereas IL-4, IL-6, IL-13, MCP-1, MCP-3, and MPIF-2 were upregulated, RANTES, IL-2Rgamma and gp130 were downregulated in allografts from the IRF1-/- recipients when compared to the WT B6 control. Although the inactivation of the IRF1 gene did not sufficiently prevent acute allograft rejection in this model, a unique cytokine and chemokine gene expression profile was found in the absence of IRF1.

FK778, a powerful new immunosuppressant, effectively reduces functional and histologic changes of chronic rejection in rat renal allografts.

BACKGROUND: FK778 is a new derivative of the active leflunomide metabolite A77 1726. It effectively prevented acute allograft rejection in several experimental transplant models, and it is currently in phase II trials in human transplant recipients. In this study, we examined the effects of FK778 in a well-established model of chronic renal allograft rejection in the rat. METHODS: Kidneys of Lewis (LEW) and F344 rats were orthotopically transplanted into bilaterally nephrectomized LEW recipients as the isograft and allograft control, respectively. Allograft recipients were orally administered FK778 at doses of 3 mg/kg per day, 10 mg/kg per day, and 20 mg/kg per day for 10 days. Blood and 24-hr urine samples were collected once a week after grafting for plasma creatinine, allo-specific antibodies, and proteinuria determination. Kidney grafts were harvested on the 90th day after transplantation and subjected to histologic, immunohistologic, and reverse transcriptase-polymerase chain reaction analysis. Histologic sections were semiquantitatively scored using criteria adapted from the Banff' classification for transplant pathologic conditions. RESULTS: Recipients treated with FK778 for 10 days exhibited a dose-dependent decrease in proteinuria and plasma creatinine for the entire 90-day period after transplantation when compared with the allograft control. FK778, at doses of 10 mg/kg per day and 20 mg/kg per day, remarkably reduced chronic histologic changes, including tubular atrophy, glomerulosclerosis, fibrointimal hyperplasia, and transplant glomerulopathy. In addition, FK778 treatment was associated with decreased intragraft mononuclear cell infiltration, serum allo-specific immunoglobulin (Ig)M and IgG antibody production, and intragraft transforming growth factor beta messenger RNA expression in those recipients surviving 90 days after transplantation when compared with the allograft control. CONCLUSION: FK778 effectively reduces functional and histologic chronic kidney allograft rejection in the rat.

Tacrolimus and cyclosporine differ in their capacity to overcome ongoing allograft rejection as a result of their differential abilities to inhibit interleukin-10 production.

BACKGROUND: Accumulated evidence from clinical transplantation has suggested that tacrolimus-based treatment can reverse ongoing allograft rejection in patients treated with cyclosporine (CsA)-based immunosuppression, even when a high dose of antirejection rescue therapy has failed. This evidence prompted us to investigate whether these two compounds, which share an in vitro mechanism, would differ in their abilities to regulate in situ cellular and molecular events during ongoing allograft rejection. METHODS: The equivalent effective doses of tacrolimus (3.2 mg/kg/day) and CsA (10 mg/kg/day), when administered orally to Lewis rats for 10 days (day 0-9), were predetermined and defined as the ability of the drug to induce a similar survival of Brown Norway rat heart allografts with an equal suppression of intragraft interleukin (IL)-2 mRNA expression. To investigate the ability of each drug to rescue ongoing allograft rejection, Lewis recipients of Brown Norway rat heart grafts were left untreated for the first 5 days after transplantation. Tacrolimus or CsA was then administered at the equivalent effective dose for 10 days (days 5-14). Heart grafts and blood samples, harvested on days 3, 5, 7, and 10, were analyzed by reverse transcriptase-polymerase chain reaction, real-time quantitative polymerase chain reaction, ELISA, and immunohistology. RESULTS: Ongoing allograft rejection was found to be rescued by tacrolimus but not by CsA at the equivalent dose (median survival time: untreated, 6 days; tacrolimus, 18 days; and CsA, 7 days). A significant suppression of local intragraft IL-10 mRNA expression and serum protein production along with a dramatic down-regulation of functional CD8+ T and NKR-P1a+ natural killer cell local infiltration by means of decreased of cytotoxic factor release, including granzyme B and perforin 1, was found to be associated with tacrolimus but not CsA treatment. However, both drugs inhibited other immune cells (CD4+ T cell, ED2+ macrophage) and cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-12, interferon-gamma, transforming growth factor-beta, and tumor necrosis factor-alpha) at almost the same levels. The inability of CsA to overcome ongoing allograft rejection could be rescued by cotreating recipients with neutralizing anti-IL-10 antibody on day 5 and day 6 after transplantation: anti-IL-10 antibody alone did not show such an effect. CONCLUSIONS: Inhibition of IL-10 production is a critical factor in the ability of tacrolimus to reverse ongoing allograft rejection.