Prolonged Cardiac Allograft Survival Research Articles

Protein Phosphatase 2A Activation Promotes Heart Transplant Acceptance in Mice.

Although heart transplantation is the definitive treatment for heart failure in eligible patients, both acute and chronic transplant rejection frequently occur. Protein phosphatase 2A (PP2A) activity is critical in maintaining tissue and organ homeostasis. In this study, we evaluated the effect of a novel class of small molecule activators of PP2A (SMAPs) on allograft rejection in a mouse heterotopic heart transplantation model. Recipient mice were administered with DT-061 (a pharmaceutically optimized SMAP) or vehicle by oral gavage beginning 1 d after transplantation. Histological and immunofluorescence analyses were performed to examine allograft rejection. Regulatory T cells (Treg) from recipient spleens were subjected to flow cytometry and RNA sequencing analysis. Finally, the effect of DT-061 on smooth muscle cells (SMCs) migration and proliferation was assessed. DT-061 treatment prolonged cardiac allograft survival. SMAPs effectively suppressed the inflammatory immune response while increasing Treg population in the allografts, findings corroborated by functional analysis of RNA sequencing data derived from Treg of treated splenic tissues. Importantly, SMAPs extended immunosuppressive agent cytotoxic T lymphocyte-associated antigen-4-Ig-induced cardiac transplantation tolerance and allograft survival. SMAPs also strongly mitigated cardiac allograft vasculopathy as evidenced by a marked reduction of neointimal hyperplasia and SMC proliferation. Finally, our in vitro studies implicate suppression of MEK/ERK pathways as a unifying mechanism for the effect of PP2A modulation in Treg and SMCs. PP2A activation prevents cardiac rejection and prolongs allograft survival in a murine model. Our findings highlight the potential of PP2A activation in improving alloengraftment in heart transplantation.

Immune Regulation of the Liver Through the PCSK9/CD36 Pathway During Heart Transplant Rejection.

PCSK9 (proprotein convertase subtilisin/kexin 9), which is mainly secreted by the liver, is not only a therapeutic target for hyperlipidemia and cardiovascular disease, but also has been implicated in the immune regulation of infections and tumors. However, the role of PCSK9 and the liver in heart transplant rejection (HTR) and the underlying mechanisms remain unclear. We assessed serum PCSK9 expression in both murine and human recipients during HTR and investigated the effect of PCSK9 ablation on HTR by using global knockout mice and a neutralizing antibody. Moreover, we performed multiorgan histological and transcriptome analyses, and multiomics and single-cell RNA-sequencing studies of the liver during HTR, as well. We further used hepatocyte-specific Pcsk9 knockout mice to investigate whether the liver regulated HTR through PCSK9. Last, we explored the regulatory effect of the PCSK9/CD36 pathway on the phenotype and function of macrophages in vitro and in vivo. Here, we report that murine and human recipients have high serum PCSK9 levels during HTR. PCSK9 ablation prolonged cardiac allograft survival and attenuated the infiltration of inflammatory cells in the graft and the expansion of alloreactive T cells in the spleen. Next, we demonstrated that PCSK9 was mainly produced and significantly upregulated in the recipient liver, which also showed a series of signaling changes, including changes in the TNF-α (tumor necrosis factor α) and IFN-γ (interferon γ) signaling pathways and the bile acid and fatty acid metabolism pathways. We found mechanistically that TNF-α and IFN-γ synergistically promoted PCSK9 expression in hepatocytes through the transcription factor SREBP2 (sterol regulatory element binding protein 2). Moreover, in vitro and in vivo studies indicated that PCSK9 inhibited CD36 expression and fatty acid uptake by macrophages and strengthened the proinflammatory phenotype, which facilitated their ability to promote proliferation and IFN-γ production by donor-reactive T cells. Last, we found that the protective effect of PCSK9 ablation against HTR is dependent on the CD36 pathway in the recipient. This study reveals a novel mechanism for immune regulation by the liver through the PCSK9/CD36 pathway during HTR, which influences the phenotype and function of macrophages and suggests that the modulation of this pathway may be a potential therapeutic target to prevent HTR.

337.10: Monotherapy With TNX-1500, a Fc-Modified Anti-CD154mAb, Prolongs Cardiac Allograft Survival in Cynomolgus Monkeys

Purpose: Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to replace conventional clinical immunosuppressive therapy. TNX-1500 (TNX)* is a novel humanized anti-CD154 mAb that contains the hu5c8 Fab region and an Fc region engineered to modulate FcγR2 binding to reduce the risk of thromboembolic events seen with hu5c8 IgG1 in previous clinical trials. Its efficacy was evaluated in cynomolgus monkey heterotopic heart allograft recipients. Methods: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with TNX 30mg/kg iv 2x weekly for 2 weeks, then either a ‘standard-dose’ regimen of 20mg/kg weekly from days 21-180 (sTNX, n=5) or a reduced-dose TNX maintenance regimen (10 mg/kg weekly x6, then 20mg/kg monthly; loTNX), with (loTNX+MMF, n=4) or without mycophenolate mofetil (MMF) at 200mg/d po (loTNX, n=4). In 3 sTNX animals therapy was discontinued on day 175. Results were compared to historical data with hu5c8 monotherapy dosed as for loTNX (n=5). Biopsies or explanted hearts were evaluated at three protocol-defined time-points (d45, d90, d180), at earlier explant. Results: sTNX (MST >232d, range 116-305) significantly prolonged cardiac allograft survival relative to hu5c8 alone (MST 133d; p=0.016), loTNX (MST 99d; p=0.014), or loTNX+MMF (MST 88d; p=0.011). All grafts treated with loTNX () or with loTNX+MMF () exhibited acute rejection histologically within 90 days, and 7 of 8 grafts failed before day 180. One sTNX graft rejected at 116d in association with bacterial infection; one graft was electively explanted at 181d; and three grafts survived for 57, 90, and 130 days after discontinuing TNX-1500. Neither sustained thrombocytopenia nor micro- or macro-vascular thrombosis were observed. At 90 days, loTNX+MMF had a significantly higher median ISHLT score (2.0) than sTNX grafts (0.2; p=0.016); loTNX+MMF also had a significantly higher median CAV score (1.8) at d90 than either loTNX (0.2; p=0.022) or sTNX (0.3; p=0.026). At subsequent graft explant or end-of-study biopsy (180d), loTNX+MMF had a higher median CAV score (2.4) than loTNX (0.4; p=0.002) or sTNX (0.8; p=0.002). sTNX suppressed the rise in Teff/Tregs ratio at the final time-point (180d or <180d if graft failure) more effectively than loTNX+MMF (3.5 ± 0.9 vs 9.5 ± 6.1, p=0.031). sTNX (5/5) but not loTNX (1/4) or loTNX+MMF (2/4) consistently prevented anti-donor antibody elaboration. Conclusions: Blockade of CD154 with sTNX monotherapy was not associated with thrombotic events, well tolerated, and consistently prevented pathogenic alloimmunity in this stringent preclinical model. Lower TNX maintenance dosing, with or without MMF, was less effective. *TNX-1500 is a pre-IND molecule and not approved for any clinical indication. NIH grant P01 HL158504. Tonix Pharmaceuticals. RC is supported by the Benjamin Research Fellowship from the German Research Foundation.

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.

Lactobacillus rhamnosus HN001 Ameliorates BEZ235-Induced Intestinal Dysbiosis and Prolongs Cardiac Transplant Survival.

ABSTRACTCardiac allograft rejection remains a major factor limiting long-term engraftment after transplantation. A novel phosphoinositide 3-kinase (PI3K)/mTOR dual inhibitor, BEZ235, prolonged cardiac allograft survival by effectively suppressing activation of the PI3K/serine/threonine kinase (AKT)/mTOR pathway. However, long-term usage of pharmacological immunosuppressant drugs can cause intestinal microbiota dysbiosis. We established mouse models of allogeneic heterotopic heart transplantation with different treatments. Fecal samples were collected and subjected to 16S rRNA sequencing and targeted fecal metabolomic analysis. Graft samples were taken for immune cell detection by flow cytometry. Inflammatory cytokines in serum were quantified by enzyme-linked immunosorbent assay (ELISA). Compared to single-target approaches (IC-87114 and rapamycin), BEZ235 more efficiently prolongs cardiac transplant survival. Interestingly, BEZ235 reduces the diversity and abundance of the intestinal microbiota community. We demonstrated that Lactobacillus rhamnosus HN001 rescues the intestinal microbiota imbalance induced by BEZ235.IMPORTANCE Our data confirmed that the combination of BEZ235 and Lactobacillus rhamnosus HN001 significantly prolongs cardiac transplant survival. A main metabolic product of Lactobacillus rhamnosus HN001, propionic acid (PA), enriches regulatory T (Treg) cells and serves as a potent immunomodulatory supplement to BEZ235. Our study provides a novel and efficient therapeutic strategy for transplant recipients.

TNX-1500, an Fc-Modified Anti-CD154 Antibody, Prolongs Nonhuman Primate Cardiac Allograft Survival

<h3>Purpose</h3> Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to replace current clinical immunosuppression. TNX-1500 (TNX) is a novel humanized anti-CD154 mAb that contains the hu5c8 Fab region and an Fc region engineered to modulate FcγR2 binding to reduce the risk of pro-thrombotic platelet activation and aggregation, and thus potentially prevent the thromboembolic events seen with hu5c8 IgG1 in preclinical models and clinical trials. <h3>Methods</h3> Cynomolgus monkey heterotopic cardiac allograft recipients were treated with TNX either using a ‘standard' regimen (sTNX; 30 mg/kg iv twice weekly x4, then 20 mg/kg weekly from d21, n=5), using a reduced-dose TNX maintenance regimen (loTNX; 30 mg/kg iv twice weekly x4, 10 mg/kg weekly x6, then 20 mg/kg monthly; n=4), or loTNX with mycophenolate mofetil (loTNX+MMF, 200mg/d po, n=4); results were compared to historical data using hu5c8 (n=5). Graft function was monitored by telemetry and ultrasound until graft failure or elective experimental termination at 6 months. Protocol biopsy and explanted graft histology and anti-donor antibody (alloAb) were evaluated. <h3>Results</h3> Four of five grafts treated with sTNX maintained good graft function (MST >154d, range 114-180, 4 of 5 ongoing); one graft rejected at 114d. All grafts treated with loTNX, alone or with MMF, experienced acute cellular rejection and/or antibody-mediated rejection; 7 of 8 grafts failed before 6 months. Relative to hu5c8 alone (MST 123d), loTNX (MST 99d) and sTNX (MST>154d) showed a statistically similar prolongation of cardiac allograft survival. Although loTNX+MMF attenuated IgG alloAb production during treatment (4/4 animals) more consistently than loTNX alone (2/4), cardiac allograft vasculopathy (CAV) tended to be more prominent with loTNX+MMF; alloAb and CAV in the sTNX group after study completion will be presented. Neither sustained thrombocytopenia nor micro- or macrovascular thrombosis were observed in TNX-treated animals. <h3>Conclusion</h3> Blockade of CD154 with TNX monotherapy consistently and safely prevents pathologic alloimmunity in this stringent preclinical model, similar to hu5c8 monotherapy. Lower maintenance dosing with TNX, with or without MMF, was less effective.

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.

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.

How Can Immuno suppression After Organ Transplantation be Made More Effective and Safer? – A Review on Liposomal Formulations with Consideration of Archaeal Tetraetherlipid

Immune-suppressive agents such as methylprednisolone and cyclosporine exert tremendous side effects, because of high dosage and long-term application required for immune suppression after organ transplantation. Major side effects of methylprednisolone include bleeding of the gastro-intestinal tract, hypertension, and osteoporosis, whereas cyclosporine is nephrotoxic. Liposomes are phospholipid particles that allow delivery of drugs preferentially to the reticuloendothelial system. They can be prepared from phospholipids such as lecithin from soybean or egg yolk, other specific or modified lipids or from membrane-spanning tetraether lipid (TEL), which can be extracted and purified from archaeal cell membranes. One advantage in the use of liposomal application is reduced toxicity of many drugs. We report on various liposomal preparations of cyclosporine, methylprednisolone (L-MPL) and its palmitate derivative (L-MPLP). It has been documented that liposomal cyclosporine A (L-CsA), 1.75 mg/kg/day for seven days has potential for use as an immune-suppressive agent in rats with increased efficacy and decreased nephrotoxicity compared to commercially available forms of intravenous CsA. Liposomal methylprednisolone (L-MPL) 2 mg/kg, intravenously (IV), twice a week shows significantly prolonged cardiac allograft survival in rats and tissue-selective sequestration of the drug in comparison with the same dosage regimen of methylprednisolone in solution, administered daily. We report on organ distribution of L-MPLP in rats after intraperitoneal (IP) administration. Conclusion: Liposomal preparations of immunosuppressants have significantly higher immune-suppressive potential and lower toxicity than non-liposomal preparations. Bipolar TEL can be extracted, fractionated and purified from archaea to form stable liposomes which are extremely resistant, even to gastric fluid. Hence, TEL liposomes allow (besides IV and IP) for oral administration of immuno suppressants after organ transplantation with pharmacological and toxicological advantages as common liposomal phospholipid bilayer preparations.

CD73 expression is critical to therapeutic effects of human endometrial regenerative cells in inhibition of cardiac allograft rejection in mice.

The newly found mesenchymal‐like endometrial regenerative cells (ERCs) have been proved to induce immune tolerance in cardiac allograft transplantation. However, the therapeutic mechanism is not clear. The present study was undertaken to investigate whether ecto‐5′‐nucleotidase (CD73) expression on ERCs is critical to cardiac allograft protection. C57BL/6 mouse recipients receiving BALB/c mouse cardiac allografts were treated with unmodified ERCs or anti‐CD73 monoclonal antibodies (mAb) pretreated ERCs, respectively. It has been found that CD73 expression was critical to ERC‐induced attenuation of graft pathology. The blockage of CD73 expression on ERCs was related to the percentage decline of tolerogenic dendritic cells (Tol‐DCs), macrophages type 2 (M2), and regulatory T cells (Tregs). As compared with anti‐CD73 mAb pretreated ERCs group, CD73 expressing ERCs significantly increased the level of anti‐inflammatory cytokine IL‐10 but decreased levels of pro‐inflammatory cytokines including IFN‐γ and TNF‐α. In addition, CD73 expressing ERCs showed tissue protective function via the regulation of adenosine receptor expression which was related to the infiltration of CD4+ and CD8+ cells in the allografts. Furthermore, significant increase of A2B receptors in the cardiac allograft was also associated with CD73 expressing ERC‐induced prolongation of cardiac allograft survival.

Circular RNA circSnx5 Controls Immunogenicity of Dendritic Cells through the miR-544/SOCS1 Axis and PU.1 Activity Regulation

Dendritic cells (DCs) can orchestrate either immunogenic or tolerogenic responses to relay information on the functional state. Emerging studies indicate that circular RNAs (circRNAs) are involved in immunity; however, it remains unclear whether they govern DC development and function at the transcriptional level. In this study, we identified a central role for a novel circRNA, circSnx5, in modulating DC-driven immunity and tolerance. Ectopic circSnx5 suppresses DC activation and promotes the development of tolerogenic functions of DCs, while circSnx5 knockdown promotes their activation and inflammatory phenotype. Mechanistically, circSnx5 can act as a miR-544 sponge to attenuate miRNA-mediated target depression on suppressor of cytokine signaling 1 (SOCS1) and inhibit nuclear translocation of PU.1, regulating DC activation and function. Furthermore, the main splicing factors (SFs) were identified in DCs, of which heterogeneous nuclear ribonucleoprotein (hnRNP) C was essential for circSnx5 generation. Moreover, our data demonstrated that vaccination with circSnx5-conditioned DCs prolonged cardiac allograft survival in mice and alleviated experimental autoimmune myocarditis. Taken together, our results revealed circSnx5 as a key modulator to fine-tune DC function, suggesting that circSnx5 may serve as a potential therapeutic avenue for immune-related diseases.

BET Protein Inhibition Prolongs Cardiac Transplant Survival via Enhanced Myocardial Autophagy.

Graft rejection continues to be a major barrier to long-term engraftment after transplantation. Autophagy plays an important role in cardiac injury pathogenesis. The bromodomain and extraterminal protein inhibitor (S)-tert-butyl2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (JQ1) inhibits inflammatory responses. However, the beneficial effect of JQ1 on transplant and the potential role of autophagy in the protective effect of graft survival are yet to be investigated. Syngeneic or allogeneic heterotopic heart transplantation was performed using C57BL/6 or BALB/c donors for C57BL/6 recipients through different treatments. Some mice were used to observe the survival of the grafts. The other mice were euthanized on the third, fifth, and seventh days after surgery. The graft samples were taken for cytokines and autophagy pathway analyses. Our study revealed that JQ1 treatment prolonged cardiac allograft survival. JQ1 increased the expression levels of liver kinase beta 1, autophagy-specific gene 5, and microtubule-associated protein light chain3-II (LC3-II) and potentiated the phosphorylation of AMP-activated protein kinase, unc-51-like kinase 1 (ULK1), and autophagy-specific gene 14 in allografts. A conditional autophagy-specific gene 5 deletion donor was utilized to abrogate the effect induced by JQ1. The combined use of JQ1 with bafilomycin A1 partially reversed the effect of JQ1, suggesting that autophagy is involved in the signaling pathway in graft survival. JQ1 downregulated the expression of inflammatory cytokines, such as interleukin-6, interleukin-1β, tumor necrosis factor-α, and interferon-γ, which was abrogated when autophagy was inhibited. JQ1 prolonged cardiac allograft survival by potentiating myocardial autophagy through the liver kinase beta 1-AMP-activated protein kinase-ULK1 signaling pathway and inhibiting the subsequent release of inflammatory cytokines. This result might provide novel insights for extending transplant survival.

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts

BackgroundWe previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model. MethodsCBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS). ResultsITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α+ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α− DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS. ConclusionsITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.

Administration of Interleukin-35-Conditioned Autologous Tolerogenic Dendritic Cells Prolong Allograft Survival After Heart Transplantation

Background/Aims: IL-35, a powerful suppressor of inflammation and autoimmunity, is primarily secreted by regulatory T cells (Tregs) and can, in turn, promote Treg differentiation. However, the precise effect of IL-35 on dendritic cells (DCs) remains to be clarified. Methods: In this study, we investigated the expression of IL-35 in DCs after stimulation with LPS utilizing enzyme linked immunosorbent assay(ELISA), quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting, and the influence of IL-35 on the maturation and function of DCs by mixed lymphocyte reaction assay and flow cytometry. We further examined the regulation of IL-35 in DCs by the microRNA let-7i (let-7i) via transfected with let-7i mimic, inhibitor or suppressor of cytokine signalling 1 (SOCS1) siRNA. IL-35-overexpressing DCs were transfused into BALB/c recipients with C57BL/6 heart transplantations to verify the role of immune tolerance in transplantation. Results: The results showed that IL-35 expression was significantly up-regulated following lipopolysaccharide (LPS)-induced DC maturation. Overexpression of IL-35 suppressed DC maturation, promoted the secretion of anti-inflammatory cytokines, and subsequently affected the balance between Treg and Th17 cells. IL-35 expression in DCs was regulated by let-7i, which targets SOCS1. The transfusion of IL-35-transfected DCs induced Treg generation in mice and prolonged cardiac allograft survival. Conclusion: Our data demonstrated that IL-35 induces tolerogenic DCs which are capable of alleviating allograft rejection. Clinical application of IL-35-treated DCs might be a promising approach for eliciting cardiac allograft immune tolerance.

Yogurt Feeding Induced the Prolongation of Fully Major Histocompatibility Complex–Mismatched Murine Cardiac Graft Survival by Induction of CD4+Foxp3+ Cells

Yogurt is a nutrient-rich food and the beneficial effects of yogurt on both health and immunomodulatory effects are well documented. In this pilot study, we investigated the effects of commercially produced yogurt R-1 on alloimmune responses in a murine cardiac transplantation model. The R-1 is produced by Meiji Co., Ltd., and contains live and active lactic acid bacteria (lactobacillus bulgaricus OLL1073R-1) mainly. CBA (H2k) mice underwent transplantation of a C57BL/6 (H2b; B6) heart and received oral administration of 1 mL, 0.1 mL, and 0.01 mL of R-1 from the day of transplantation until 7 days afterward. Additionally, we prepared one group of CBA recipients given 1 mL of R-1 sterilized by microwave for 7 days. Histological and immunohistochemical studies were performed. Naïve CBA mice rejected B6 cardiac graft acutely (median survival time [MST]: 7 days). CBA recipients given of 1 mL of R-1 had significantly prolonged B6 allograft survival (MST, 27 days). However, other doses of 0.1 mL and 0.01 mL of R-1 did not prolonged allograft survival (MSTs, 9 days and 8.5 days, respectively). Also, CBA recipients administered microwaved R-1 had no prolongation of B6 allograft (MST, 9 days). Histological and immunohistochemical studies showed the cardiac allograft from R-1–exposed CBA recipients had preserved graft and vessel structure and the number of infiltrated CD4+, CD8+, and Foxp3+ cells in R-1–exposed CBA recipients increased, respectively. In conclusion, our findings imply that yogurt containing active lactic acid bacteria could change alloimmune responses partially and induce the prolongation of cardiac allograft survival via CD4+Foxp3+ regulatory cells.

AS2553627, a novel JAK inhibitor, prevents chronic rejection in rat cardiac allografts

Janus family kinases (JAKs) are essential molecules for cytokine responses and attractive targets for the treatment of transplant rejection and autoimmune diseases. Several JAK inhibitors have shown demonstrable effects on acute rejection in experimental cardiac transplant models. However, little is known about the potential benefits of JAK inhibitors on chronic rejection outcomes such as vasculopathy and fibrosis. Here, we examined the pharmacological profile of a novel JAK inhibitor, AS2553627, and explored its therapeutic potential in chronic rejection as well as acute rejection in a rat cardiac transplant model. AS2553627 potently inhibited JAK kinases but showed no inhibition of other kinases, including TCR-associated molecules. The compound also suppressed proliferation of IL-2 stimulated human and rat T cells. In a rat cardiac transplant model, oral administration of AS2553627 alone or co-administration with a sub-therapeutic dose of tacrolimus effectively prolonged cardiac allograft survival, suggesting the efficacy in treating acute rejection. To evaluate the effect on chronic rejection, recipient rats were administered a therapeutic dose of tacrolimus for 90 days. In combination with tacrolimus, AS2553627 significantly reduced cardiac allograft vasculopathy and fibrosis that tacrolimus alone did not inhibit. AS2553627 at the effective dose in rat transplantation models did not significantly reduce reticulocyte counts in peripheral whole blood after in vivo erythropoietin administration, indicating a low risk for anemia. These results suggest that AS2553627 may be a therapeutic candidate for the prevention of not only acute but also chronic rejection in cardiac transplantation.

Prolongation of Cardiac Allograft Survival by Endometrial Regenerative Cells: Focusing on B-Cell Responses.

Endometrial regenerative cells (ERCs) have been recently evaluated as an attractive candidate source for emerging stem cell therapies in immunosuppression, but their role in immunoregulation is not fully understood. The present study was designed to investigate their effects, especially on B‐cell responses in heart transplantation. In this study, ERCs were noninvasively obtained from menstrual blood. Heart transplantation was performed between C57BL/6 (H‐2b) donor mice and BALB/c (H‐2d) recipients. B‐cell activation and antibody levels were determined using fluorescence‐activated cell sorting, enzyme‐linked immunosorbent assay and ELISpot. In this study, we demonstrated that ERCs negatively regulated B‐cell maturation and activation in vitro without affecting their viability. ERC treatment prolonged cardiac allograft survival in mice, which was correlated with a decrease in IgM and IgG deposition and circulating antidonor antibodies, as well as with reduction in frequencies of antidonor antibody‐secreting CD19+ B cells. In addition, upon ex vivo stimulation, B cells from ERC‐treated heart transplant recipients had impaired proliferation capacity and produced less IgM and IgG antibody. Moreover, ERC treatment of mice receiving ovalbumin (OVA)‐aluminum hydroxide vaccine resulted in significant lower numbers of anti‐OVA IgG antibody‐secreting splenic B cells and lower anti‐OVA antibody titres. Our results indicate that therapeutic effects of ERCs may be attributed at least in part by their B‐cell suppression and humoral response inhibition, suggesting the potential use of ERCs for attenuating antibody‐mediated allograft rejection. Stem Cells Translational Medicine 2017;6:778–787

What's Hot, What's New From the 2016 American Transplant Congress.

From June 11-15, 2016 the American Transplant Congress, the joint meeting of the American Society of Transplantation and the American Society of Transplant Surgeons, held its annual meeting in Boston, MA. The meeting, attended by 5200 registrants, included pre-meeting conferences, focused topic sessions, and hundreds of high-quality presentations from the transplant field. This meeting report highlights key findings from specific basic science, translational, and clinical research presentations deemed to have notable impact in thematic areas. In particular, there were a number of transformative studies indicating important advances in the understanding of alloimmunity, chronic rejection, tolerance, and organ-specific outcomes. Many of these results are discussed in the context of the published literature to showcase rapid advances in the transplant field.

MicroRNA let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently induces prolonged cardiac allograft survival in rats

Abstract Objective In this study, we investigated whether microRNA (MiR) let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently prolongs rat cardiac allograft survival. Methods QRT-PCR, ELISA, dual luciferase assay were performed to verify whether IL-10 was the target of let 7i. Tregs were assessed by flow cytometry and immunohistochemical study, and JAK1, STAT3 and pSTAT3 expressions were detected by western bolt. Lewis recipients of DA heart were transfused with PBS, LPS-mDCs, or let 7i inhibitor-mDCs. Allograft survival times were recorded, and histologic study were performed. Results Expression of IL-10 mRNA level and production of IL-10 were increased in let 7i inhibitor-mDCs as compared to LPS-mDCs. Luciferase activity showed that the translational level of the IL-10 luciferase reporter was decreased by let 7i mimic, but increased by let 7i inhibitor. MiR let 7i inhibitor suppressed DC maturation by depressing CD80 and CD86, however， pretreatment of IL-10 SiRNA attenuated the suppression. Expressions of JAK1, STAT3, and pSTAT3 in mDCs were suppressed by let 7i mimic and pretreatment of IL-10 SiRNA, however up regulated by let 7i inhibitor. Lewis recipients transfused with let 7i inhibitor-mDCs significantly prolonged DA cardiac allograft survival. The allografts transfused with let 7i inhibitor-mDCs showed slight cell infiltration and significantly preserved graft structure. Inhibition of let 7i increased Tregs and modulated cytokine profiles both in vivo and in vitro. Conclusion MiR let 7i regulated DCs maturation and function targeting the IL-10 through JAK1-STAT3 pathway. Moreover, transfusion of let 7i inhibitor-mDCs induced prolonged cardiac allograft survival and generated Tregs.

Glutamate Receptor Interacting Protein 1 Regulates CD4(+) CTLA-4 Expression and Transplant Rejection.

PDZ domains are common 80- to 90-amino-acid regions named after the first three proteins discovered to share these domains: postsynaptic density 95, discs large, and zonula occludens. PDZ domain-containing proteins typically interact with the C-terminus of membrane receptors. Glutamate receptor interacting protein 1 (GRIP1), a seven-PDZ domain protein scaffold, regulates glutamate receptor surface expression and trafficking in neurons. We have found that human and mouse T cells also express GRIP1. T cell-specific GRIP1(-/-) mice >11weeks old had prolonged cardiac allograft survival. Compared with wild-type T cells, invitro stimulated GRIP1(-/-) T cells had decreased expression of activation markers and increased apoptotic surface marker expression. Surface expression of the strong T cell inhibitory molecule cytotoxic T lymphocyte antigen-4 (CTLA-4) was increased on GRIP1(-/-) T cells from mice >11weeks old. CTLA-4 increases with T cell stimulation and its surface expression on GRIP1(-/-) T cells remained high after stimulation was removed, indicating a possible internalization defect in GRIP1-deficient T cells. CTLA-4-blocking antibody treatment following heart transplantation led to complete rejection in T cell GRIP1(-/-) mice, indicating that increased CTLA-4 surface expression contributed to the extended graft survival. Our data indicate that GRIP1 regulates T cell activation by regulating CTLA-4 surface expression.