Murine Cardiac Allograft Survival Research Articles

BEZ235 Prolongs Murine Cardiac Allograft Survival Through the Autophagy Pathway

BEZ235, a dual PI3K/mTOR inhibitor, has shown a critical impact in the treatment of cancers, with the ability to induce autophagy. However, the effects of BEZ235 in heart transplant have been rarely investigated. The aim of this study was to evaluate the potency of BEZ235 in cardiac allograft survival. BEZ235 was administered during the perioperative period of syngeneic or allogeneic heart transplant to assess survival time. Next, the autophagy signaling pathway and the proinflammatory cytokines were analyzed. Furthermore, a cardiomyocytes-specific ATG5 gene-ablated mouse was used to confirm the results. BEZ235 treatment significantly prolonged the survival of the cardiac graft and reduced the infiltration of inflammatory cells. The expression levels of autophagy proteins were increased in the BEZ235 treatment group compared to the control group, but the therapeutic effect of BEZ235 was weakened in the cardiomyocytes-specific ATG5 gene-ablated mice. Moreover, BEZ235 significantly downregulated the expression of IL-1β, IL-2, and TNF-α. It seems BEZ235 could induce autophagy and prolonged murine cardiac allograft survival in a mechanism that involved the autophagy pathway and changed multiple inflammatory factors. This study has proposed a theoretical foundation for the strong connection between mTOR-induced autophagy and heart transplant.

Linking erythropoietin to Treg-dependent allograft survival through myeloid cells.

Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO’s effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor–knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.

Cytosporone B (Csn-B), an NR4A1 agonist, attenuates acute cardiac allograft rejection by inducing differential apoptosis of CD4+T cells

CD4+T cell-mediated acute rejection remains a major factor that affects the early survival of transplanted organs post-transplantation. Here, we reveal that nuclear receptor subfamily 4 Group A member 1 (Nr4A1) was upregulated during cardiac allograft rejection and that the increased Nr4A1 was primarily localized in intragraft-infiltrating CD4+T cells. Nr4A1 acts as a transcription factor with an important role in CD4+T cell apoptosis, differentiation and T cell dysfunction, which indicates that Nr4A1 may play a critical role in transplant rejection. Cytosporone B (Csn-B) is a naturally occurring agonist of Nr4A1, and the role of Csn-B in the physiological process of cardiac rejection is poorly defined. This study constructed an acute rejection model of abdominal heterotopic cardiac transplantation in mice and investigated whether Csn-B could attenuate acute transplant rejection by modulating the CD4+T lymphocyte response. The results showed that Csn-B prolonged murine cardiac allograft survival and reduced inflammation in allografts. Subsequently, it was confirmed that Csn-B functions by inducing non-Treg apoptosis and promoting Treg cell differentiation. Finally, we also confirmed that Csn-B attenuates acute rejection by directly targeting Nr4A1 in CD4+T cells. Our data suggest that Csn-B is a promising novel therapeutic approach for acute cardiac allograft rejection.

A short course of tofacitinib sustains the immunoregulatory effect of CTLA4-Ig in the presence of inflammatory cytokines and promotes long-term survival of murine cardiac allografts

Costimulation blockade-based regimens are a promising strategy for management of transplant recipients. However, maintenance immunosuppression via CTLA4-Ig monotherapy is characterized by high frequency of rejection episodes. Recent evidence suggests that inflammatory cytokines contribute to alloreactive T cell activation in a CD28-independent manner, a reasonable contributor to the limited efficacy of CTLA4-Ig. In this study, we investigated the possible synergism of a combined short-term inhibition of cytokine signaling and CD28 engagement on the modulation of rejection. Our results demonstrate that the JAK/STAT inhibitor tofacitinib restored the immunomodulatory effect of CTLA4-Ig on mouse alloreactive T cells in the presence of inflammatory cytokines. Tofacitinib exposure conferred dendritic cells with a tolerogenic phenotype reducing their cytokine secretion and costimulatory molecules expression. JAK inhibition also directly affected T cell activation. In vivo, the combination of CTLA4-Ig and tofacitinib induced long-term survival of heart allografts and, importantly, it was equally effective when using grafts subjected to prolonged ischemia. Transplant survival correlated with a reduction in effector T cells and intragraft accumulation of regulatory T cells. Collectively, our studies demonstrate a powerful synergism between CTLA4-Ig and tofacitinib and suggest their combined use is a promising strategy for improved management of transplanted patients.

Absence of recipient C3aR1 signaling limits expansion and differentiation of alloreactive CD8+ T cell immunity and prolongs murine cardiac allograft survival.

Activation, differentiation, and expansion of alloreactive CD8+ T cells, the dominant effectors that mediate murine heart allograft rejection, requires allorecognition, costimulation, and cytokine-initiated signals. While previous work showed that alloreactive CD4+ T cell immunity entails immune cell-produced and locally activated complement, whether and how C3a receptor 1 (C3aR1) signaling impacts transplant outcomes and the mechanisms linking C3aR1 to alloreactive CD8+ T cell activation/expansion remain unclear. Herein we show that recipient C3aR1 deficiency or pharmacological C3aR1 blockade synergizes with tacrolimus to significantly prolong allograft survival versus tacrolimus-treated controls (median survival time 21 vs. 14days, P<.05). Recipient C3aR1-deficiency reduced the frequencies of posttransplant, donor-reactive CD8+ T cells twofold. Reciprocal adoptive transfers of naive WT or C3ar1-/- CD8+ T cells into syngeneic WT or C3ar1-/- allograft recipients showed that T cell-expressed C3aR1 induces CD8+ T proliferation, mTOR activation and transcription factor T-bet expression. Host C3aR1 indirectly facilitates alloreactive CD8+ T cell proliferation/expansion by amplifying antigen presenting cell costimulatory molecule expression and innate cytokine production. In addition to expanding mechanistic insight, our findings identify C3aR1 as a testable therapeutic target for future studies aimed at improving human transplant outcomes.

Gut microbiota-dependent modulation of innate immunity and lymph node remodeling affects cardiac allograft outcomes.

We hypothesized that the gut microbiota influences survival of murine cardiac allografts through modulation of immunity. Antibiotic pretreated mice received vascularized cardiac allografts and fecal microbiota transfer (FMT), along with tacrolimus immunosuppression. FMT source samples were from normal, pregnant (immune suppressed), or spontaneously colitic (inflammation) mice. Bifidobacterium pseudolongum (B. pseudolongum) in pregnant FMT recipients was associated with prolonged allograft survival and lower inflammation and fibrosis, while normal or colitic FMT resulted in inferior survival and worse histology. Transfer of B. pseudolongum alone resulted in reduced inflammation and fibrosis. Stimulation of DC and macrophage lines with B. pseudolongum induced the antiinflammatory cytokine IL-10 and homeostatic chemokine CCL19 but induced lesser amounts of the proinflammatory cytokines TNFα and IL-6. In contrast, LPS and Desulfovibrio desulfuricans (D. desulfuricans), more abundant in colitic FMT, induced a more inflammatory cytokine response. Analysis of mesenteric and peripheral lymph node structure showed that B. pseudolongum gavage resulted in a higher laminin α4/α5 ratio in the lymph node cortical ridge, indicative of a suppressive environment, while D. desulfuricans resulted in a lower laminin α4/α5 ratio, supportive of inflammation. Discrete gut bacterial species alter immunity and may predict graft outcomes through stimulation of myeloid cells and shifts in lymph node structure and permissiveness.

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival.

Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1-/- cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.

Gut Microbiota Determines Graft Survival Outcome Through Regulation of Anti-Inflammatory and Pro-Inflammatory Responses

Introduction The gut microbiota plays an important role on the induction and training of the host immune system. It is unknown whether microbiota from the contrasting environments of colitic or pregnant mice are pro-inflammatory or suppressive. We hypothesized that gut microbiota from different sources could influence the survival of murine cardiac allografts through regulation of immune responses. Methods C57BL/6 mice received BALB/c vascularized cardiac allografts and fecal microbiota transfer (FMT) by oral gavage. FMT samples were from normal or pregnant C57BL/6, spontaneously colitic mice, or cultured Bifidobacterium pseudolongum (Bifido) (a dominant member of the pregnant gut microbiota). Tacrolimus was administered daily. Fecal pellets were collected weekly post-transplant and analyzed by 16S rRNA gene sequencing. Grafts were assessed for survival, harvested at days 40-60 or rejection, and assessed for inflammation by H&E and fibrosis by Masson's trichrome. Macrophage (MF) and dendritic cell (DC) lines were stimulated with purified Bifido cells and cytokine responses measured. Results Pregnant FMT significantly prolonged survival, and reduced inflammation and fibrosis of cardiac allografts. Normal or colitic FMT resulted in inferior survival, along with increased fibrosis and inflammation. 16S rRNA analyses demonstrated abundant Bifido in the pregnant and normal but not the colitic FMT samples. Transfer of Bifido alone also resulted in enhanced allograft survival and reduced inflammation and fibrosis. Serial 16S rRNA analyses of gut microbiota from normal, colitic, pregnant FMT, or Bifido groups, revealed significant differences in bacterial community structure (alpha- and beta-diversity). Bifido remained abundant in pregnant FMT samples for at least 40 days. In contrast, Desulfovibrio and Deferribacteraceae were more abundant in colitic samples. Microbiota community structures of the various groups became less divergent over time, but remained distinct. DC and MF lines stimulated with Bifido expressed higher levels of the anti-inflammatory cytokine IL-10 and homeostatic chemokine CCL19, but lesser amounts of the pro-inflammatory cytokines TNFa and IL-6 compared to LPS stimulated cells. Conclusion The gut microbiota has profound effects on host immune responses with consequences for cardiac allograft outcomes. Distinct bacterial species or genera may both alter immunity and predict graft outcomes, with Bifidobacterium species inducing anti-inflammatory effects through stimulation of DC and MF. Therapeutic targeting of the microbiota or its functions could be beneficial in reducing inflammation and subsequent graft rejection.

Minocycline Prolongs Murine Cardiac Allograft Survival through Suppression of Acute Rejection

Background Acute rejection (AR) occurs in 10-20% of renal allograft recipients despite combination immunosuppression, suggesting there are pathways that are not adequately inhibited by current therapy. Large scale gene expression profiling using microarray has enabled the characterisation of the molecular changes in AR. In recent years there has been an increased focus on identifying the most highly connected genes in various disease states, rather than the most highly expressed, as these ‘key driver’ genes are more likely to assume key regulatory roles. This has not been studied in the context of AR in renal transplantation, and may serve as potential targets for novel therapeutics. Aims 1.To derive key driver genes from microarray data of human renal allograft biopsy tissue with AR 2.Identify drugs that can inhibit the key driver genes 3. Experimental validation. Methods A meta-analysis of microarray data of 735 human kidney allograft biopsy samples was performed to identify the differentially expressed genes, and the key drivers were further derived using bioinformatics methods. Drug repurposing tools were used to identify candidate therapeutics and Minocycline was selected for further evaluation in a full allo-mismatch murine heterotopic cardiac allograft model. The impact of minocycline on the allograft when used alone, or when administered with immunosuppression (CTLA4Ig), was evaluated. Allograft histology was assessed and quantitative RT-PCR was performed to measure mRNA expression of key driver genes and cytokines/chemokines. A competing risk analysis was used to assess cumulative incidence of graft rejection, with graft loss due to other causes regarded as a competing risk. Results 14 key driver genes were identified, with Caspase 1 identified as the central key driver gene. Minocycline, a tetracycline antibiotic, is a well described inhibitor of Caspase 1, the central key driver gene. Minocycline (50mg/kg IP BD) compared with saline did not prolong allograft survival, butwas able to reduce the inflammatory profile of acute rejection as demonstrated by the reduction in the mRNA expression in the allograft tissue of a number of key drivers and inflammatory cytokines and chemokines (eg IFNγ, CXCL9) at day 4 post transplant. However, when minocycline was administered in conjunction with immunosuppression (CTLA4Ig 250ug IP Day 2 alone with minocycline 50mg/kg IP BD up to 27 days post transplant), an extension in allograft survival was observed compared with the control group (CTLA4Ig Day 2 with saline) (Hazard ratio 0.27 CI 0.099-0.736 p-value <0.01). 3 treatment animals were euthanased prior to cessation of allograft heart beat for other reasons. Conclusion Minocycline alters the inflammatory profile of AR, and prolongs murine cardiac allograft survival when given in conjunction with immunosuppressive therapy. Further evaluation as to the precise mechanisms by which this occurs is required. Jacquot Research Entry Scholarship, Royal Australasian College of Physicians. KK and ZY are co-first authors.

Blockade of Extracellular HMGB1 Suppresses Xenoreactive B Cell Responses and Delays Acute Vascular Xenogeneic Rejection

Blockade of extracellular high mobility group box 1 (HMGB1) can significantly prolong murine cardiac allograft survival. Here, we determined the role of HMGB1 in xenotransplantation. Sprague-Dawley rat hearts were transplanted heterotopically into BALB/c mice. Xenografts without any treatment developed predominant acute vascular rejection within 6 days. Both passively released HMGB1 from xenografts and actively secreted HMGB1 from infiltrated immune cells were significantly increased after xenotransplantation. HMGB1-neutralizing antibody treatment significantly prolonged xenograft survival and attenuated pathologic damage, immune cell infiltration, and HMGB1 expression and release in the xenografts. Compared to control IgG treatment evaluated at study endpoint, treatment with HMGB1-neutralizing antibody markedly suppressed xenoreactive B cell responses, as evidenced by the significant inhibition of anti-rat antibody production and deposition in xenografts at Day 6 posttransplant. Furthermore, treatment with anti-HMGB1 antibody suppressed B cell activation and reduced IFN-γ and IL-17A production after xenotransplantation. These results demonstrate for the first time that HMGB1 plays an important role in mediating acute xenograft rejection. Thus, we have shown that neutralization of extracellular HMGB1 can significantly inhibit xenoreactive B cell responses and delay xenograft rejection in a rat-to-mouse model of xenotransplantation, uncovering new insights in the role of HMGB1 in transplantation.

Treadmill exercise induces murine cardiac allograft survival and generates regulatory T cell.

Exercise therapy has been associated with improvement in functional capacity and quality of life. The role of exercise therapy in heart transplant recipients is of great interest for the transplant society, although concerning the effect of exercise therapy, there is little knowledge at present. We analyzed the effects of exercise on alloimmune responses in murine cardiac allograft transplantation. CBA mice (H2k) underwent transplantation of C57Bl/6 (H2b) hearts and exercised on a treadmill. Untreated CBA recipients rejected C57Bl/6 cardiac grafts acutely (median survival time [MST], 7 days). CBA recipients treated with treadmill for 1 week after transplantation, and for 1 week both before and after transplantation prolonged allograft survivals (MSTs, 35 and 18 days, respectively). However, treadmill exercise recipients for 1 week before transplantation were not effective to allograft survival (MST, 8 days). Adoptive transfer of whole splenocytes and CD4+ cells from treadmill exercise recipients significantly prolonged allograft survival in naive secondary recipients (MSTs, 30 and 52 days, respectively), suggesting that regulatory cells was generated after treadmill exercise. Moreover, flow cytometry studies showed that CD4+CD25+Foxp3+ cell population increased in treadmill exercise recipients. Therefore, postoperative but not pre-operative exercise could induce prolongation of survival of fully allogeneic cardiac allografts and generate CD4+CD25+Foxp3+ regulatory T cells.

Targeting sirtuin-1 improves murine renal allograft survival and function (TRAN3P.901)

Abstract Pharmacologic inhibition of sirtuin-1 (Sirt1) augments suppressive functions of Foxp3+ regulatory T cells (Treg) and prolongs murine cardiac allograft survival. We investigated if Sirt1 targeting produced longer allograft function and allograft-dependent survival in a MHC-mismatched renal transplant model. After transplantation of BALB/c kindey allografts into C57Bl/6 wild type or fl-Sirt1/CD4cre recipients, native kidneys were removed three days post-operatively. We followed the mice for survival, and allograft function by weekly serum electrolyte and renal function (BUN, creatinine), as well as hematocrit. We observed, that loss of Sirt1 in T cells led to prolonged median renal allograft-dependent survival from 14 days (IQR: 11-91.25) to 143 days (IQR: 27-147) in wild type controls vs. fl-Sirt1/CD4cre recipients (Mantel-Cox, p&amp;lt;0.0004). In addition, by comparing mice that survived until 13 weeks, loss of Sirt1 in the recipient’s T cells led to improved renal allograft function, with 34.2 ±20 vs. 90.8 ±37.6 mg/dL BUN (p&amp;lt;0.001), and lower creatinine 0.33 ±0.14 vs. 0.61 ±0.3 mg/dL (p&amp;lt;0.001) in fl-Sirt1/CD4cre vs. wild type control mice, respectively. Long-term surviving fl-Sirt1/CD4cre renal transplant recipients (&amp;gt;100 days) became tolerant to antigen re-challenge with a BALB/c, but rejected third-party C3H cardiac allografts. In conclusion, deletion of Sirt1 in T cells is effective at prolonging allograft function and allograft-dependent survival.

Flt3L Combined with Rapamycin Promotes Cardiac Allograft Tolerance by Inducing Regulatory Dendritic Cells and Allograft Autophagy in Mice

The induction of immune tolerance is still a formidable challenge in organ transplantation. Dendritic cells (DCs) play an important role in orchestrating immune responses by either mediating protective immune responses or inducing antigen specific tolerance. Previous studies demonstrated that the fms-like tyrosine kinase 3 receptor (Flt3) and its ligand (Flt3L) play an essential role in the regulation of DC commitment and development. Here, we report a synergic effect between Flt3L and low-dose rapamycin (Rapa) in the protection of allograft rejction. It was found that Flt3L combined with Rapa significantly prolonged murine cardiac allograft survival time as compared with that of untreated recipients or recipients treated with Rapa or Flt3L alone. Mechanistic studies revealed that Flt3L combined with low-dose of Rapa induced the generation of tolerogenic DCs along with the production of CD25+ Foxp3+ regulatory T cells and IL-10 secretion. We also observed enhanced autophagy in the cardiac allograft, which could be another asset contributing to the enhanced allograft survival. All together, these data suggest that Flt3L combined with low-dose of Rapa could be an effective therapeutic approach to induce tolerance in clinical setting of transplantation.

Auditory stimulation of opera music induced prolongation of murine cardiac allograft survival and maintained generation of regulatory CD4+CD25+ cells

BackgroundInteractions between the immune response and brain functions such as olfactory, auditory, and visual sensations are likely. This study investigated the effect of sounds on alloimmune responses in a murine model of cardiac allograft transplantation.MethodsNaïve CBA mice (H2k) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to one of three types of music--opera (La Traviata), classical (Mozart), and New Age (Enya)--or one of six different single sound frequencies, for 7 days. Additionally, we prepared two groups of CBA recipients with tympanic membrane perforation exposed to opera for 7 days and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment). An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Immunohistochemical, cell-proliferation, cytokine, and flow cytometry assessments were also performed.ResultsCBA recipients of a B6 cardiac graft that were exposed to opera music and Mozart had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to a single sound frequency (100, 500, 1000, 5000, 10,000, or 20,000 Hz) or Enya did not (MSTs, 7.5, 8, 9, 8, 7.5, 8.5 and 11 days, respectively). Untreated, CBA mice with tympanic membrane perforations and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment) rejected B6 cardiac grafts acutely (MSTs, 7, 8 and 8 days, respectively). Adoptive transfer of whole splenocytes, CD4+ cells, or CD4+CD25+ cells from opera-exposed primary allograft recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and > 100 days, respectively). Proliferation of splenocytes, interleukin (IL)-2 and interferon (IFN)-γ production was suppressed in opera-exposed mice, and production of IL-4 and IL-10 from opera-exposed transplant recipients increased compared to that from splenocytes of untreated recipients. Flow cytometry studies showed an increased CD4+CD25+ Forkhead box P3 (Foxp3)+ cell population in splenocytes from those mice.ConclusionOur findings indicate that exposure to opera music, such as La traviata, could affect such aspects of the peripheral immune response as generation of regulatory CD4+CD25+ cells and up-regulation of anti-inflammatory cytokines, resulting in prolonged allograft survival.

High-mobility group box 1 promotes early acute allograft rejection by enhancing IL-6-dependent Th17 alloreactive response

Previously, we reported that extracellular high-mobility group box 1 (HMGB1) functions as an innate alarmin implicated in cardiac allograft acute rejection. We now present evidence suggesting that HMGB1 is pivotal in inducing interleukin-17 (IL-17)-producing alloreactive T cells by stimulating dendritic cells secretion of IL-6. Those IL-17+ T cells are likely to be the major effector cells responsible for the early stage of cardiac allograft rejection through mediating an influx of neutrophils into allografts, and therefore, blockade of IL-17A significantly prolonged murine cardiac allograft survival. In contrast to the classical model for a dominant role of IFN-γ+-Th1 cells have in acute allograft rejection, our data suggest that IFN-γ+-Th1 cells are responsible for the late stage of graft destruction by inducing monocyte infiltration when IL-17+ T-cell response recedes. Blockade of HMGB1 significantly decreased splenic alloreactive Th17 cells and IFN-γ-producing CD8+ T cells in the recipients, leading to less infiltration of neutrophils along with lower IL-6 and IL-17 expression levels in the grafts as well as prolongation of cardiac allograft survival. Together, these data support a novel model in which HMGB1 induces IL-17-producing alloreactive T cells to mediate early stage of allograft rejection, whereas IFN-γ-producing alloreactive Th1 cells provoke graft destruction after Th17 response recedes.

Cyclic AMP prolongs graft survival by suppressing apoptosis and inflammatory gene expression in acute cardiac allograft rejection

This study was designed to investigate the effects of cAMP on immune regulation and apoptosis during acute rat cardiac allograft rejection. We found that the production of immune markers such as inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), iNOS expression, and nitric oxide (NO) production, was significantly increased in the blood and transplanted hearts of allograft recipients, but not of isograft controls. These increases were effectively suppressed by the administration of the membrane permeable cAMP analog dibutyryl cAMP (db-cAMP). Administration of db-cAMP reduced allograft-induced elevation of several biochemical markers, such as adhesion molecule expression, iron-nitrosyl complex formation, caspase-3 activation, and apoptotic DNA fragmentation in an animal model. Furthermore, treatment of allograft recipients with db-cAMP prolonged median graft survival to 11 days compared with a median graft survival time of 8 days in saline-treated allograft recipients. These results suggest that db-cAMP exerts a beneficial effect on murine cardiac allograft survival by modulating allogeneic immune response and cytotoxicity.

Prolongation of Allograft Survival by Administration of Anti-CD45RB Monoclonal Antibody Is Due to Alteration of CD45RBhi: CD45RBlo T-Cell Proportions

CD45RB monoclonal antibody (mAb) therapy is capable of prolonging allograft survival. We have previously shown that CD45RB mAb enriches the CD45RBlo T-cell population in vitro and in vivo by preferentially depleting CD45RBhi T cells. The present study assessed the importance of CD45RBhi T-cell depletion in murine cardiac allograft survival by infusion of naive CD45RB T-cell subsets. Here we show that naturally occurring CD45RBloCD4+ T cells express regulatory transcription factor Foxp3 and have regulatory function, whereas CD45RBhiCD4+ T cells express low levels of Foxp3 and have effector function. Infusion of syngeneic CD45RBhi T cells significantly reduced graft survival after depletion of CD45RBhi T cells by CD45RB mAb. Reduction of graft survival also occurred when syngeneic CD45RBhi T cells were infused into rapamycin-treated mice, whereas survival was prolonged when CD45RBlo T cells were added. This indicates that an alteration in the balance between regulatory CD45RBlo and effector CD45RBhi T cells is critical to the immunologic function of CD45RB mAb. A strategy to eliminate effector T cells with consequent enrichment of the regulatory T-cell compartment may be an important new strategy in the prevention of rejection.

Gene therapy using adenoviral vector encoding 4-1BBIg gene significantly prolonged murine cardiac allograft survival

4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, interacts with 4-1BBL expressed on APC and delivers a costimulatory signal for T cell activation and growth. In this study, we investigated the efficacy of an adenoviral vector encoding murine 4-1BB extracellular domain and human IgG1 Fc (Ad4-1BBIg) fusion gene on murine cardiac allograft survival. Abdomen heterotopical heart graft model was performed from Balb/c to C57BL/6 mice. The adenoviral vectors, Ad4-1BBIg or an adenoviral vector containing EGFP gene (AdEGFP), were administered intravenously to recipient animals after cardiac grafting. The cardiac allograft survival was monitored by daily palpation. The serum level of 4-1BBIg and graft histology was assessed. Cytokine profiles in the grafts were detected by RT-PCR. IFN-γ producing cells in recipient spleen were examined by flow cytometry. 4-1BBIg gene expression was achieved highly level at 72 h after vector injection. The proportion of IFN-γ producing cells in recipient spleen was significantly reduced after administration of Ad4-1BBIg, compared to the group given AdEGFP or to the untreated control group. Unlike in controls, cardiac allograft expression of mRNA coding for IL-2 and IFN-γ remained low in the Ad4-1BBIg group. Ad4-1BBIg therapy markedly reduced T cell infiltration into the graft and significantly prolonged recipient survival time (13.5 days), compared to the untreated group (7.5 days) and the AdEGFP-treated group (8.0 days) ( P < 0.05). These results indicate that blockade of 4-1BB/4-1BB ligand interactions by Ad4-1BBIg inhibited alloreactive T-cell activation and attenuated T-cell infiltration into the graft, resulting in significant prolongation of murine cardiac allograft survival. Therefore, Ad4-1BBIg may be useful for preventing allograft rejection.

Lymphoid Sequestration of Alloreactive Memory CD4 T Cells Promotes Cardiac Allograft Survival

Memory T cells specific for donor Ags present a unique challenge in transplantation. In addition to expressing robust immune responses to a transplanted organ, memory T cells may be resistant to the effects of immunosuppressive therapies used to prolong graft survival. In this study, we explore the possibility of controlling deleterious donor-reactive memory CD4 T cells through lymphoid sequestration. We showed that sphingosine 1-phosphate receptor-1 agonist FTY720 induces relocation of circulating memory CD4 T cells into secondary lymphoid organs. Lymphoid sequestration of these donor-reactive memory CD4 T cells prolonged survival of murine heterotopic cardiac allografts and synergizes with conventional costimulatory blockade to further increase graft survival. Despite limited trafficking, memory CD4 T cells remain capable of providing help for the induction of anti-donor CD8 T cell and alloantibody responses. Elimination of antidonor humoral immunity resulted in indefinite allograft survival proving the pathogenicity of alloantibody under these conditions. Overall, this is the first demonstration that FTY720 influences memory CD4 T cell trafficking and attenuates their contribution to allograft rejection. The data have important implications for guiding FTY720 therapy and for designing combinatorial strategies aimed at prolonging allograft survival in sensitized transplant patients with donor-specific memory T cells.

Cre/loxP-mediated CTLA4IgG gene transfer induces clinically relevant immunosuppression via on–off gene recombination in vivo

Transfer of the CTLA4IgG gene induces long-term and high levels of CTLA4IgG expression, which can result in generalized immunosuppression. In this study, we utilized Cre/loxP-mediated on-off switch recombination to eliminate transgene expression of CTLA4IgG following acceptance of murine cardiac allografts. Fully MHC-mismatched hearts from BALB/c donor mice were transplanted into C3H/He recipient mice. Adenovirus-containing CTLA4IgG flanked between two loxP sites was administered via a recipient tail vein immediately after transplantation. Cre-recombinase gene was subsequently transferred at day 30 posttransplantation. Long-term allograft survival was observed in recipients that received the CTLA4IgG gene. Cre-mediated recombination reduced CTLA4IgG gene expression without any adverse effect on the graft survival. Secondary skin grafts of donor type and of third party were promptly rejected in the recipients that accepted cardiac allografts. In addition, the B cell response against ovalbumin was suppressed during high levels of serum CTLA4IgG, but recovered after Cre-mediated inactivation of CTLA4IgG gene. CTLA4IgG gene transfer promoted long-term survival of murine cardiac allografts; however, this was not sufficient to induce tolerance. Cre/loxP-mediated on-off switch recombination was useful to inactivate the CTLA4IgG gene so that recipients' immune responses against neoantigens were restored without an influence on the allograft survival. This system may open novel strategies to orchestrate clinically relevant immunosuppression.