Prolonged Allograft Survival Research Articles

Therapeutic effect of multiple functional minicircle vector encoding anti-CD25/IL-10/CXCR3 in allograft rejection model.

Background/AimsWe previously proposed minicircle vector technology as the potential platform for the development and production of new biologics. In this study, we have designed a novel target molecule for the treatment of allograft rejection and evaluated its feasibility as the therapeutic agent in this disease using the minicircle vector system.MethodsWe engineered vectors to carry cassette sequences for anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) fusion protein, and then isolated minicircle vectors from the parent vectors. We verified the substantial production of anti-CD25/IL-10/CXCR3 fusion protein from minicircles and their duration in HEK293T cells and mice models. We also evaluated whether minicircle-derived anti-CD25/IL-10/CXCR3 has therapeutic effects in a skin allograft in mice model.ResultsWe confirmed the production of anti-CD25/IL-10/CXCR3 from minicircle by its significant availability in cells transfected with the minicircle and in its conditioned media. After a single injection of minicircle by hydrodynamic injection via mouse tail vein, luminescence or red fluorescence was maintained until 40 days in the liver tissue, suggesting the production of anti-CD25/IL-10/CXCR3 protein from minicircles via protein synthesis machinery in the liver. Mice treated with the minicircle encoding anti-CD25/IL-10/CXCR3 showed prolonged skin allograft survival times accompanied by improved immunologic regulation e.g., reduction of the lymphocyte population of Th1, Th2, and Th17 and an induction of regulatory T cells.ConclusionsThese findings implied that self-generated anti-CD25/IL-10/CXCR3 protein drug by minicircle technology is functionally active and relevant for reducing allograft rejection. The minicircle vector system may be useful for developing new biological drugs, avoiding manufacturing or practical problems.

Small extracellular vesicles derived from PD-L1-modified mesenchymal stem cell promote Tregs differentiation and prolong allograft survival.

We aimed to explore whether programmed cell death protein-1 ligand (PD-L1) modification on small extracellular vesicles (sEVs) could promote T regulatory cells (Tregs) differentiation. In this study, it was confirmed that under physiological conditions, PD-L1 expression was minimal in the MSCs and absent in the MSC-sEVs. A vector harboring the PD-L1 gene was constructed and transfected into bone marrow mesenchymal stem cells (BM-MSCs). By extracting the sEVs of these modified BM-MSCs and monitoring the expression of the PD-L1 protein, however, PD-L1 expression was substantially increased in the MSCs and concentrated in the sEVs. Then, the rat naïve CD4 + T cells were cocultured with the sEVs derived from the PD-L1-modified MSCs (sEVsPD-L1). By flow cytometry, a higher percentage of Tregs and anti-inflammatory downstream cytokines (including IL-2, IFN-γ, TGF-β, IL-10) was detected in the sEVsPD-L1 group than that in the control group treated by either sEVs in wild type, modified by empty vector, or blank control. Suppressive effect on CD4 + T cell proliferation serves as additional evidence to support the immunoregulation capacity of sEVsPD-L1. The animal model of vascularized composite allograft further confirmed that PD-L1-modified sEVs induce an immune tolerance, by clinically observation, histopathology, T cell fate and cell product. In conclusion, sEVsPD-L1 efficiently promotes Treg cell differentiation in vitro and in vivo, which suggests their therapeutic potential in the treatment of allograft rejection.

Antibody-suppressor CXCR5+CD8+ T cellular therapy ameliorates antibody-mediated rejection following kidney transplant in CCR5 KO mice

CCR5KO kidney transplant (KTx) recipients are extraordinarily high alloantibody producers and develop pathology that mimics human antibody-mediated rejection (AMR). C57BL/6 and CCR5KO mice (H-2b ) were transplanted with A/J kidneys (H-2a ); select cohorts received adoptive cell therapy (ACT) with alloprimed CXCR5+ CD8+ T cells (or control cells) on day 5 after KTx. ACT efficacy was evaluated by measuring posttransplant alloantibody, pathology, and allograft survival. Recipients were assessed for the quantity of CXCR5+ CD8+ T cells and CD8-mediated cytotoxicity to alloprimed IgG+ B cells. Alloantibody titer in CCR5 KO recipients was four-fold higher than in C57BL/6 recipients. The proportion of alloprimed CXCR5+ CD8+ T cells 7days after KTx in peripheral blood, lymph node, and spleen was substantially lower in CCR5 KO compared to C57BL/6 recipients. In vivo cytotoxicity towards alloprimed IgG+ B cells was also reduced six-fold in CCR5 KO recipients. ACT with alloprimed CXCR5+ CD8+ T cells (but not alloprimed CXCR5- CD8+ or third-party primed CXCR5+ CD8+ T cells) substantially reduced alloantibody titer, ameliorated AMR pathology, and prolonged allograft survival. These results indicate that a deficiency in quantity and function of alloprimed CXCR5+ CD8+ T cells contributes to high alloantibody and AMR in CCR5KO recipient mice, which can be rescued with ACT.

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.

Ablation of BATF Alleviates Transplant Rejection via Abrogating the Effector Differentiation and Memory Responses of CD8+ T Cells.

Allogeneic CD8+ T cells are prominently involved in allograft rejection, but how their effector differentiation and function are regulated at a transcriptional level is not fully understood. Herein, we identified the basic leucine zipper ATF-like transcription factor (BATF) as a key transcription factor that drives the effector program of allogeneic CD8+ T cells. We found that BATF is highly expressed in graft-infiltrating CD8+ T cells, and its ablation in CD8+ T cells significantly prolonged skin allograft survival in a fully MHC-mismatched transplantation model. To investigate how BATF dictates allogeneic CD8+ T cell response, BATF–/– and wild-type (WT) CD8+ T cells were mixed in a 1:1 ratio and adoptively transferred into B6.Rag1 –/– mice 1 day prior to skin transplantation. Compared with WT CD8+ T cells at the peak of rejection response, BATF–/– CD8+ T cells displayed a dysfunctional phenotype, evident by their failure to differentiate into CD127–KLRG1+ terminal effectors, impaired proliferative capacity and production of pro-inflammatory cytokines/cytotoxic molecules, and diminished capacity to infiltrate allografts. In association with the failure of effector differentiation, BATF–/– CD8+ T cells largely retained TCF1 expression and expressed significantly low levels of T-bet, TOX, and Ki67. At the memory phase, BATF-deficient CD8+ T cells displayed impaired effector differentiation upon allogeneic antigen re-stimulation. Therefore, BATF is a critical transcriptional determinant that governs the terminal differentiation and memory responses of allogeneic CD8+ T cells in the transplantation setting. Targeting BATF in CD8+ T cells may be an attractive therapeutic approach to promote transplant acceptance.

Treg tissue stability depends on lymphotoxin beta-receptor- and adenosine-receptor-driven lymphatic endothelial cell responses.

SUMMARYRegulatory T cell (Treg) lymphatic migration is required for resolving inflammation and prolonging allograft survival. Focusing on Treg interactions with lymphatic endothelial cells (LECs), we dissect mechanisms and functional consequences of Treg transendothelial migration (TEM). Using three genetic mouse models of pancreatic islet transplantation, we show that Treg lymphotoxin (LT) αβ and LEC LTβ receptor (LTβR) signaling are required for efficient Treg migration and suppressive function to prolong allograft survival. Inhibition of LT signaling increases Treg conversion to Foxp3loCD25lo exTregs. In a transwell-based model of TEM across polarized LECs, non-migrated Tregs become exTregs. Such conversion is regulated by LTβR nuclear factor κB (NF-κB) signaling in LECs, which increases interleukin-6 (IL-6) production and drives exTreg conversion. Migrating Tregs are ectonucleotidase CD39hi and resist exTreg conversion in an adenosine-receptor-2A-dependent fashion. Human Tregs migrating across human LECs behave similarly. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.

Tumor necrosis factor receptor superfamily member 25 (TNFRSF25) agonists in islet transplantation: Endogenous in vivo regulatory T cell expansion promotes prolonged allograft survival

Regulatory T cells (Tregs) modulate alloimmune responses and may facilitate minimization or withdrawal of immunosuppression posttransplant. Current approaches, however, rely on complex ex vivo Treg expansion protocols. Herein, we explore endogenous in vivo Treg expansion through antibody-mediated agonistic stimulation of the tumor necrosis factor receptor superfamily member 25 (TNFRSF25) pathway and its potential to prolong graft survival in a mouse model of islet allotransplantation. C57BL/6male mice were treated with a single dose of TNFRSF25 agonistic antibodies (4C12 or mPTX-35) or IgG control. Diabetes was induced using streptozotocin. Four days later, flow cytometry was completed to corroborate Treg expansion, and 500 islets (CBA/J male mice) were transplanted. Glycemia was assessed thrice weekly until rejection/endpoint. Early intra-graft Treg infiltration was assessed 36h posttransplant. TNFRSF25 antibodies enabled pronounced Treg expansion and treated mice had significantly prolonged graft survival compared with controls (p<.001). Additionally, the degree of Treg expansion significantly correlated with graft survival (p<.001). Immunohistochemistry demonstrated marked Treg infiltration in long-term surviving grafts; intra-graft Treg infiltration occurred early posttransplant. In conclusion, a single dose of TNFRSF25 antibodies enabled in vivo Treg expansion, which promotes prolonged graft survival. TNFRSF25-mediated in vivo Treg expansion could contribute to achieving lasting immunological tolerance in organ transplantation.

Adenosinergic Pathway and Linked Suppression: Two Critical Suppressive Mechanisms of Human Donor Antigen Specific Regulatory T Cell Lines Expanded Post Transplant.

Regulatory T cells are an important component of an immune response shaping the overall behavior to potential antigens including alloantigens. Multiple mechanisms have been shown to contribute towards developing and sustaining a immunological regulatory response. One of the described contact dependent suppressive mechanisms regulatory cells have been shown to utilize is through the production of adenosine from extracellular ATP mediated by CD39 and CD73. In this study we demonstrate that the adenosinergic pathway plays a major role in the suppressive/regulatory effects antigen specific regulatory T cell enriched lines (ASTRLs) that have been of expanded ex vivo from stable kidney transplant patients. We have previously shown that these ASTRL cells are capable of suppressing alloimmune responses in vitro and significantly prolonging allograft survival in an animal model of kidney transplantation. For this study nineteen ASTRLs were expanded from 17 kidney transplant patients by repeated stimulation of recipient peripheral blood mononuclear cells with donor specific HLA-DR peptides. All 19 ASTRLs showed upregulation of numerous markers associated with regulatory cells and were able to inhibit donor antigen specific T cell proliferation in a dose dependent fashion. ASTRLs suppressed indirect and direct alloimmune responses compatible with our previous animal study findings. Upregulation of both CD39 and CD73 was observed post expansion and ASTRLs demonstrated extracellular hydrolysis of ATP, indicating functionality of the upregulated proteins. We also showed that inhibition of the adenosinergic pathway using inhibitors of CD39 resulted in abrogation of suppression and increased antigen specific T cell proliferation. This demonstrates that the main mechanism of action of the suppressive activity donor peptide driven ASTRLs generated from kidney transplant patients is the adenosinergic pathway. Furthermore this suggests the possibility that combining infusion of Tregs with other treatments, such as adenosine receptor agonists or increasing CD39 expression in the grafts may further enhance a regulatory response to the allograft and possibly achieve transplantation tolerance.

Novel Technology Has Made It Possible To Diagnose A New Clinical Phenotype Of Rejection called Early Antibody Mediated Rejection, With Significant Therapeutic Implications For Renal Allograft Survival

Novel technology in transplant, specifically, the Molecular Microscope Diagnostic System, has made it possible to diagnose a new clinical phenotype of rejection called "early antibody-mediated rejection." Here, we present 2 kidney transplant recipients who had normal serum creatinine levels but elevated donor-derived cell-free DNA. Allograft biopsies did not show antibody-mediated rejection, but the Molecular Microscope Diagnostic System reported early antibody -mediated rejection. Once considered as an isolated incident occurring after kidney transplant, antibody-mediated rejection is now recognized to be a progressive condition that waxes and wanes over time and may ultimately lead to chronic allograft damage and allograft loss. Hence, if it can be diagnosed early before causing allograft injury, the early diagnosis can represent a paradigm shift in the management of antibody-mediated rejection in kidney transplant recipients, with better treatment outcomes and prolonged allograft survival.

Saireito (TJ-114) Suppressed Donor-Specific Antibody Through Immunomodulatory Effects and Regulatory T Cell Induction in a Murine Heart Transplant Model

Saireito (Tsumura Japan [TJ]-114) could induce long-term cardiac allograft survival through the generation of CD4+CD25+Foxp3+ cells (regulatory T cells, Tregs). However, little is known regarding the effects of TJ-114 on the suppression of donor-specific antibody (DSA). Therefore, we aimed to further investigate the suppressive properties of TJ-114 and its effects on DSA production in a murine cardiac allograft model. CBA mice underwent transplantation of C57BL/6 hearts and were subsequently administered TJ-114 (2g/kg/d) from the day of transplantation until 7 days afterward. TJ-114-treated recipients demonstrated upregulation of splenic Tregs and suppressed DSA production at postoperative day 10 relative to untreated controls. This effect was sustained at postoperative day 20 even when TJ-114 administration was stopped at day 7. To then investigate the involvement of Tregs in the suppression of DSA production, anti-interleukin-2 receptor alpha antibody (PC-61) was administered to deplete Treg populations in TJ-114-treated CBA recipients on postoperative days 0, 3, 6, and 9 or 20, 23, and 26. At day 10, CBA recipients that received PC-61 with TJ-114 demonstrated suppression of DSA production similar to those receiving only TJ-114. Nonetheless, when mice were treated with PC-61 at days 20, 23, and 26, DSA levels gradually increased to levels comparable to those of untreated mice by day 29, suggesting that Tregs are necessary to sustain the suppression of DSA once the effects of TJ-114 have subsided. Taken together, TJ-114 may be a promising therapeutic strategy to prolong allograft survival through its combined immunosuppressive effects in inducing Tregs and suppressing DSA production.

Interferon-β acts directly on T cells to prolong allograft survival by enhancing regulatory T cell induction through Foxp3 acetylation

Type I interferons (IFNs) are pleiotropic cytokines with potent antiviral properties that also promote protective Tcell and humoral immunity. Paradoxically, type I IFNs, including the widely expressed IFNβ, also have immunosuppressive properties, including promoting persistent viral infections and treating T-cell-driven, remitting-relapsing multiple sclerosis. Although associative evidence suggests that IFNβ mediates these immunosuppressive effects by impacting regulatory T (Treg) cells, mechanistic links remain elusive. Here, we found that IFNβ enhanced graft survival in a Treg-cell-dependent murine transplant model. Genetic conditional deletion models revealed that the extended allograft survival was Treg cell-mediated and required IFNβ signaling on Tcells. Using an in silico computational model and analysis of human immune cells, we found that IFNβ directly promoted Treg cell induction via STAT1- and P300-dependent Foxp3 acetylation. These findings identify a mechanistic connection between the immunosuppressive effects of IFNβ and Treg cells, with therapeutic implications for transplantation, autoimmunity, and malignancy.

Taurodeoxycholic acid and valine reverse obesity-associated augmented alloimmune responses and prolong allograft survival

Obesity initiates a chronic inflammatory network linked to perioperative complications and increased acute rejection rates in organ transplantation. Bariatric surgery is the most effective treatment of obesity recommended for morbidly obese transplant recipients. Here, we delineated the effects of obesity and bariatric surgery on alloimmunity and transplant outcomes in diet-induced obese (DIO) mice. Allograft survival was significantly shorter in DIO-mice. When performing sleeve gastrectomies (SGx) prior to transplantation, we found attenuated T cell-derived alloimmune responses resulting in prolonged allograft survival. Administering taurodeoxycholic acid (TDCA) and valine, metabolites depleted in DIO-mice and restored through SGx, prolonged graft survival in DIO-mice comparable with SGx an dampened Th1 and Th17 alloimmune responses while Treg frequencies and CD4+ T cell-derived IL-10 production were augmented. Moreover, in recipient animals treated with TDCA/valine, levels of donor-specific antibodies had been reduced. Mechanistically, TDCA/valine restrained inflammatory M1-macrophage polarization through TGR5 that compromised cAMP signaling and inhibited macrophage-derived T cell activation. Consistently, administering a TGR5 agonist to DIO-mice prolonged allograft survival. Overall, we provide novel insights into obesity-induced inflammation and its impact on alloimmunity. Furthermore, we introduce TDCA/valine as a noninvasive alternative treatment for obese transplant patients.

Modulating donor mitochondrial fusion/fission delivers immunoprotective effects in cardiac transplantation

Early insults associated with cardiac transplantation increase the immunogenicity of donor microvascular endothelial cells (ECs), which interact with recipient alloreactive memory T cells and promote responses leading to allograft rejection. Thus, modulating EC immunogenicity could potentially alter T cell responses. Recent studies have shown modulating mitochondrial fusion/fission alters immune cell phenotype. Here, we assess whether modulating mitochondrial fusion/fission reduces EC immunogenicity and alters EC‐T cell interactions. By knocking down DRP1, a mitochondrial fission protein, or by using the small molecules M1, a fusion promoter, and Mdivi1, a fission inhibitor, we demonstrate that promoting mitochondrial fusion reduced EC immunogenicity to allogeneic CD8+ T cells, shown by decreased T cell cytotoxic proteins, decreased EC VCAM‐1, MHC‐I expression, and increased PD‐L1 expression. Co‐cultured T cells also displayed decreased memory frequencies and Ki‐67 proliferative index. For in vivo significance, we used a novel murine brain‐dead donor transplant model. Balb/c hearts pretreated with M1/Mdivi1 after brain‐death induction were heterotopically transplanted into C57BL/6 recipients. We demonstrate that, in line with our in vitro studies, M1/Mdivi1 pretreatment protected cardiac allografts from injury, decreased infiltrating T cell production of cytotoxic proteins, and prolonged allograft survival. Collectively, our data show promoting mitochondrial fusion in donor ECs mitigates recipient T cell responses and leads to significantly improved cardiac transplant survival.

Effects of Each Domain in Recombinant Human Soluble Thrombomodulin on Prolongation of Murine Cardiac Allograft Survival

Thrombomodulin is used to manage disseminated intravascular coagulation. In our murine heart transplantation model, the administration of recombinant human soluble thrombomodulin (rTM) could induce the prolongation of cardiac allograft survival. However, there are limited data on the graft protective effects of each r domain (D1, D2, and D3). In this study, we investigated the effects of each domain of rTM on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients using microsurgical techniques. CBA mice that underwent transplantation of C57BL/6 cardiac allografts were assigned to 4 groups: no treatment and each domain-exposed group. The dosage of each domain was determined based on our previous experiments. Flow cytometry and histologic studies were performed to determine whether Foxp3+ regulatory T cells were generated. Untreated and D2-exposed CBA recipients acutely rejected C57BL/6 cardiac allografts within 9 days. Administration of D3 resulted in modest prolongation of allograft survival, and administration of D1 significantly prolonged allograft survival. Histologic studies showed that myocardial damage of allografts from D1- and D3-exposed CBA recipients was controlled compared with that of untreated recipients. In particular, the CD4+CD25+Foxp3+ cell population in the splenocytes of D1-exposed CBA recipients was increased. In conclusion, D1 in rTM could help prolong cardiac allograft survival through regulatory T cell induction and graft protective effects.

Induction of Endotoxin Tolerance Delays Acute Rejection in a Hindlimb Transplantation Model in Rats.

Acute rejection is seen in 85 percent of composite vascular allogeneic transplants despite long-term immunosuppression. Recently, it was reported that the induction of endotoxin tolerance prolonged heart allograft survival in mice. However, it produced side effects in all the animals secondary to the inflammatory reaction. Galactomannan has shown endotoxin tolerance without this side effect in vitro. The authors hypothesized that galactomannan-induced endotoxin tolerance delays acute rejection in vascular allogeneic transplantation without the side effects produced by lipopolysaccharide. Twenty-four rat hindlimb transplants were divided into four groups according to the preconditioning received: control, lipopolysaccharide (0.16 ml/kg), galactomannan 72 hours before (galactomannan-72) (8 ml/kg), and galactomannan 24 hours before (galactomannan-24) (8 ml/kg). Median acute rejection time, weight loss, and diarrheal episodes were monitored. Blood samples were collected at 0, 7, 21, 30, 45, and 60 days. Plasma cytokines (i.e., tumor necrosis factor alpha, interferon gamma), peripheral chimerism, and lymphocyte percentages were analyzed. Median allograft survival was 40 days (range, 40 to 44 days) in the control group, 68 days (range, 61 to 71 days) in the lipopolysaccharide group, and 70 days (range, 69 to 73 days) in both galactomannan groups (p = 0.001). Weight loss was higher in the lipopolysaccharide group (p < 0.001), as was the 83.3 percent rate of diarrheal episodes (control, 0 percent, p = 0.015; galactomannan-72, 0 percent, p = 0.015; and galactomannan-24, 16.7 percent, p = 0.02). Preconditioned rats had higher peripheral blood chimerism (lipopolysaccharide, 2.30 ± 0.13 percent; galactomannan-72, 2.63 ±1.46 percent; and galactomannan-24, 2.47 ± 0.19 percent) compared to the control group (2.06 ± 0.36 percent) (lipopolysaccharide, p = 0.04; galactomannan-72, p = 0.002; and galactomannan-24, p = 0.002). Induction of endotoxin tolerance delays acute rejection in the rat hindlimb transplantation model. Galactomannan preconditioning has no lipopolysaccharide side effects and was equally effective in delaying acute rejection. The contributions of this experimental work are very incipient. Although the use of galactomannan in clinical practice requires more studies to assess its safety, there is no doubt that immunomodulation may be one of the responses that solve the problem of long-term immunosuppression.

Rocaglamide Prolonged Allograft Survival by Inhibiting Differentiation of Th1/Th17 Cells in Cardiac Transplantation.

Background Aglaia (Meliaceae) species are used for treating autoimmune disorders and allergic diseases in Asian countries. Rocaglamide, an extract obtained from Aglaia species, exhibits suppressive effect by regulating the T cell subset balance and cytokine network in cancer. However, whether it can be used in organ transplantation is unknown. In this study, we investigated the antirejection effect and mechanism of action of rocaglamide in a mouse cardiac allograft model. Methods Survival studies were performed by administering mice with phosphate-buffered saline (PBS) (n = 6) and rocaglamide (n = 8). Heart grafts were monitored until they stopped beating. After grafting, the mice were sacrificed on day 7 for histological, mixed lymphocyte reaction (MLR), enzyme-linked immunosorbent assay (ELISA), and flow cytometric analyses. Results Rocaglamide administration significantly prolonged the median survival of the grafts from 7 to 25 days compared with PBS treatment (P < 0.001). On posttransplantation day 7, the rocaglamide-treated group showed a significant decrease in the percentage of Th1 cells (7.9 ± 0.9% vs. 1.58 ± 0.5%, P < 0.001) in the lymph nodes and spleen (8.0 ± 2.5% vs. 2.4 ± 1.3%, P < 0.05). Rocaglamide treatment also significantly inhibited the production of Th17 cells (6.4 ± 1.0% vs. 1.8 ± 0.4%, P < 0.01) in the lymph nodes and spleen (5.9 ± 0.3% vs. 2.9 ± 0.8%, P < 0.01). Furthermore, the prolonged survival of the grafts was associated with a significant decrease in IFN-γ and IL-17 levels. Our results also showed that NF-AT activation was inhibited by rocaglamide, which also induced p38 and Jun N-terminal kinase (JNK) phosphorylation in Jurkat T cells. Furthermore, by using inhibitors that suppressed p38 and JNK phosphorylation, rocaglamide-mediated reduction in NF-AT protein levels was prevented. Conclusion We identified a new immunoregulatory property of rocaglamide, wherein it was found to regulate oxidative stress response and reduce inflammatory cell infiltration and organ injury, which have been associated with the inhibition of NF-AT activation in T cells.

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.

NFAT-dependent and -independent exhaustion circuits program maternal CD8 T cell hypofunction in pregnancy.

Pregnancy is a common immunization event, but the molecular mechanisms and immunological consequences provoked by pregnancy remain largely unknown. We used mouse models and human transplant registry data to reveal that pregnancy induced exhausted CD8 T cells (Preg-TEX), which associated with prolonged allograft survival. Maternal CD8 T cells shared features of exhaustion with CD8 T cells from cancer and chronic infection, including transcriptional down-regulation of ribosomal proteins and up-regulation of TOX and inhibitory receptors. Similar to other models of T cell exhaustion, NFAT-dependent elements of the exhaustion program were induced by fetal antigen in pregnancy, whereas NFAT-independent elements did not require fetal antigen. Despite using conserved molecular circuitry, Preg-TEX cells differed from TEX cells in chronic viral infection with respect to magnitude and dependency of T cell hypofunction on NFAT-independent signals. Altogether, these data reveal the molecular mechanisms and clinical consequences of maternal CD8 T cell hypofunction and identify pregnancy as a previously unappreciated context in which T cell exhaustion may occur.

CXCR4 blockade reduces the severity of murine heart allograft rejection by plasmacytoid dendritic cell-mediated immune regulation

Allograft-specific regulatory T cells (Treg cells) are crucial for long-term graft acceptance after transplantation. Although adoptive Treg cell transfer has been proposed, major challenges include graft-specificity and stability. Thus, there is an unmet need for the direct induction of graft-specific Treg cells. We hypothesized a synergism of the immunotolerogenic effects of rapamycin (mTOR inhibition) and plerixafor (CXCR4 antagonist) for Treg cell induction. Thus, we performed fully-mismatched heart transplantations and found combination treatment to result in prolonged allograft survival. Moreover, fibrosis and myocyte lesions were reduced. Although less CD3+ T cell infiltrated, higher Treg cell numbers were observed. Noteworthy, this was accompanied by a plerixafor-dependent plasmacytoid dendritic cells-(pDCs)-mobilization. Furthermore, in vivo pDC-depletion abrogated the plerixafor-mediated Treg cell number increase and reduced allograft survival. Our pharmacological approach allowed to increase Treg cell numbers due to pDC-mediated immune regulation. Therefore pDCs can be an attractive immunotherapeutic target in addition to plerixafor treatment.

In vivo Treg expansion under costimulation blockade targets early rejection and improves long-term outcome

CTLA4Ig has been shown to improve kidney allograft function, but an increased frequency of early rejection episodes poses a major obstacle for more widespread clinical use. The deleterious effect of CTLA4Ig on Treg numbers provides a possible explanation for graft injury. Therefore, we aimed at improving CTLA4Ig's efficacy by therapeutically increasing the number of Tregs. Murine cardiac allograft transplantation (BALB/c to B6) was performed under CTLA4Ig therapy modeled after the clinically approved dosing regimen and Tregs were transferred early or late after transplant. Neither early nor late Treg transfer prolonged allograft survival. Transferred Tregs were traceable in various lymphoid compartments but only modestly increased overall Treg numbers. Next, we augmented Treg numbers in vivo by means of IL2 complexes. A short course of IL2/anti‐IL2‐complexes administered before transplantation reversed the CTLA4Ig‐mediated decline in Tregs. Of note, the addition of IL2/anti‐IL2‐complexes to CTLA4Ig therapy substantially prolonged heart allograft survival and significantly improved graft histology on day 100. The depletion of Tregs abrogated this effect and resulted in a significantly diminished allograft survival. The increase in Treg numbers upon IL2 treatment was associated with a decreased expression of B7 on dendritic cells. These results demonstrate that therapy with IL2 complexes improves the efficacy of CTLA4Ig by counterbalancing its unfavorable effect on Tregs.