Cause Of Late Graft Loss Research Articles

#5108 SUSTAINED IL-6 SECRETION CAN LEAD TO AN AMPLIFIED INFLAMMATORY RESPONSE, MEDIATED BY THE ACTIVATION OF STAT3 AND NFKB VIA THE IL-6 AMPLIFIER LOOP

Abstract Background and Aims Organ transplants are the preferred treatment for end-stage organ failure. Potent and specific immunosuppressive agents have significantly decreased acute allograft rejection following renal transplantation. A significant barrier to long-term kidney allograft outcomes is chronic antibody-mediated rejection (CABMR). Chronic inflammation is a major cause of late graft loss that is mediated by soluble mediators released by immune and non-immune cells. IL-6 is a crucial cytokine that plays a central role in developing chronic inflammation. Non-immune cells, such as fibroblasts, have recently been identified as mediating chronic allograft rejection by activating the IL-6 amplifier loop (IL-6+IL-17). Our Aim of the study: -to study the effect of IL-6+IL-17 on secretion of IL-6 in the culture supernatant of fibroblasts derived from renal biopsy of chronic antibody mediated rejection (CABMR) patients; -to see the effect of anti-IL-6 (Tocilizumab)and anti-IL-17 on the secretion of IL-6 from the fibroblasts derived from kidney tissue of chronic antibody mediated rejection (CABMR) patients; and -to elucidate the pro-inflammatory pathway leading to increased IL-6 secretion by induction of Amplifier loop. Method Fibroblasts from grafted kidney from CABMR patients (n = 6) were cultured and stimulated with IL-6 (20ng/ µl), IL-17(50ng/ µl), IL-6 plus IL-17 for 24 hours. Levels of IL-6, MCP-1 and CCL20 were estimated in culture supernatants by ELISA as marker of IL-6 amplifier loop activation. mRNA expression of IL-6, MCP1, CCL20, and SOCS3 genes were measured in the stimulated fibroblasts. Stimulated Renal fibroblast cells from CABMR patients were lysed with Lysis buffer and subjected to SDS–PAGE and western blotting with anti-phospho-STAT3 and anti-phospho-NFκB p65. Additionally, IL-6, MCP1, and CCL20 levels were measured in Healthy control (n = 10), CABMR (n = 20), and non-CABMR (n = 30) patients. Results IL-6 and IL-17 synergistically induced more IL-6, CCL-20 & MCP-1 production from fibroblasts in culture supernatant. Gene expression analysis of IL-6, MCP1, and CCL20 was significantly higher with synergistic activation of IL-6 and IL-17 as compared to either IL-6 or IL-17 alone, while SOCS3 gene expression was downregulated. Our results also suggested that IL-6 Amplifier loop activation induces the NFκB and STAT3 signalling pathway activation in the non-immune cells like fibroblast derived from CABMR patients. Additionally, concentrations of IL-6, CCL-20 & MCP-1 in sera were significantly higher in CABMR patients compared to non-CABMR patients (p<0.001). There was a significant reduction in IL-6 concentration in culture supernatant with IL-6 and IL-17 inhibitor together and mRNA expression of IL-6, CCL20 and MCP-1 was significantly reduced while SOCS3 gene expression was upregulated. Conclusion In humans after kidney transplantation, IL-6 amplifier activation plays an active role in chronic rejection responses. Inhibition of IL-6 with Anti-IL-6 (Tocilizumab) and inhibition of IL-17 with Anti-IL-17 together reduces markers of tissue injury (IL-6, MCP1, CCL20) and rejection of allografts. so, IL-6 amplifier may be a therapeutic target for Chronic transplant rejection.

Evolution of kidney allograft loss causes over 40 years (1979–2019)

IntroductionThe improvement of kidney allograft recipient and graft survival showed a decrease over the last 40 years. Long-term graft loss rate remained stable during a 25-year time span. Knowing the changing causes and the risk factors associated with graft loss requires special attention.The present study aimed to assess the causes of graft loss and kidney allograft recipient death. Also, we aimed to compare two different periods (1979−1999 and 2000−2019) to identify changes in the characteristics of the failed allografts and recipient and donors profile. Methods and patientsWe performed a single-center cohort study. We included all the kidney transplant recipients at the Hospital del Mar (Barcelona) between May 1979 and December 2019. Graft loss was defined as recipient death with functioning graft and as loss of graft function (return to dialysis or retransplantation). We assessed the causes of graft loss using clinical and histological information. We also analyzed the results of the two different transplant periods (1979−1999 and 2000−2019). ResultsBetween 1979 and 2019, 1522 transplants were performed. The median follow-up time was 56 (IQR 8–123) months. During follow-up, 722 (47.5%) grafts were lost: 483 (66.9%) due to graft failure and 239 (33.1%) due to death with functioning graft. The main causes of death were cardiovascular (25.1%), neoplasms (25.1%), and infectious diseases (21.8%). These causes were stable between the two periods of time. Only the unknown cause of death has decreased in the last period.The main cause of graft failure (loss of graft function) was the allograft chronic dysfunction (75%). When histologic information was available, antibody-mediated rejection (ABMR) and interstitial fibrosis/tubular atrophy (IF/TA) were the most frequent specific causes (15.9% and 12.6%). Of the graft failures, 213 (29.5%) were early (<1 year of transplantation). Vascular thrombosis was the main cause of early graft failure in the second period (2000−2019) (46.7%) and T-cell-mediated rejection (TCMR) was the main cause (31.3%) in the first period (1979−1999). The causes of late graft loss were similar between the two periods. ConclusionsThe causes of kidney allograft recipient death are still due to cardiovascular and malignant diseases. Vascular thrombosis has emerged as a frequent cause of early graft loss in the most recent years. The evaluation of the causes of graft loss is necessary to improve kidney transplantation outcomes.

Rationale for the IMAGINE study for chronic active antibody-mediated rejection (caAMR) in kidney transplantation

Chronic active antibody-mediated rejection (caAMR) in kidney transplantation is a major cause of late graft loss and despite all efforts to date, there is no proven effective therapy. Indeed, the Transplant Society (TTS) consensus opinion called for a conservative approach optimizing baseline immunosuppression and supportive care focused on blood pressure, blood glucose, and lipid control. This review provides the rationale and early evidence in kidney transplant recipients with caAMR that supported the design of the IMAGINE study whose goal is to evaluate the potential impact of targeting the IL6/IL6R pathway.

P8.007: Single Nuclei RNA-Sequencing Unravels Complex Renal And Immune Cells Signaling Driving Fibrosis in Kidney Allografts

Introduction: Chronic allograft dysfunction (CAD), characterized by interstitial fibrosis and tubular atrophy (IFTA), is the main cause of late graft loss in kidney transplantation. This study dissects the molecular and cellular heterogeneity of the human kidney stroma and immune cells driving CAD. Methods: Using single-nuclei RNA sequencing, 41,893 nuclei were evaluated from 8 rare kidney allograft biopsies (6 CAD and 3 normal patients). These needle biopsies were collected on average at ≥15-months posttransplanation and were stored in the cryopreservative, RNAlater. Following, integrative analysis was applied including pathway and enrichment analysis, pseudotime trajectories, and ligand receptor analyses. XY chromosome linked gene expression analysis was also used to determine the origin of both immune and nonimmune cells in sex-mismatch transplants. Results: Two states of fibrosis were discovered (low and high ECM) that differ based on significant alterations in kidney subclusters, immune cell proportions and phenotypes, and intercellular communication. Pseudotime trajectories revealed a mixed tubule cluster, a key intermediate of injured proximal tubular cells, that transitioned to an activated fibroblast cluster also enriched in myofibroblasts markers. Such differentiation of proximal tubular cells was the main driver of fibrosis. Unique to high ECM, MT1 performed replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. Conversely, low ECM did not show a strong replicative repair signature. Differences in immune cell populations highlighted the dynamic nature of immune responses, some of which, promoted severe metabolic dysfunction and limited repair in low ECM or perpetual tissue regeneration and sustained injury in high ECM. Low ECM was characterized by increased dendritic cells (cDCs, pDCs), mast cells, and monocytes (MO1, MO2) whereas high ECM was characterized by increased B cells, T cells (CD8+ T cells, NKs, and Tregs), and plasma cells. Moreover, 65 ligand-receptor pairs between kidney cells and donor-derived macrophages (previously unidentified after several years post-transplantation) played a novel role in injury propagation. Conclusion: This work improves our understanding of targetable cell types and regulatory mechanisms involved in tissue repair. Interventions aimed at graft fibrogenesis likely require a more targeted approach based on the unique molecular pathways characterizing each condition. National Institutes of Health grant R01DK109581. National Institutes of Health grant R01DK122682 (VRM).

Antibody-mediated rejection: prevention, monitoring and treatment dilemmas.

Purpose of reviewAntibody-mediated rejection (AMR) has emerged as the leading cause of late graft loss in kidney transplant recipients. Donor-specific antibodies are an independent risk factor for AMR and graft loss. However, not all donor-specific antibodies are pathogenic. AMR treatment is heterogeneous due to the lack of robust trials to support clinical decisions. This review provides an overview and comments on practical but relevant dilemmas physicians experience in managing kidney transplant recipients with AMR.Recent findingsActive AMR with donor-specific antibodies may be treated with plasmapheresis, intravenous immunoglobulin and corticosteroids with additional therapies considered on a case-by-case basis. On the contrary, no treatment has been shown to be effective against chronic active AMR. Various biomarkers and prediction models to assess the individual risk of graft failure and response to rejection treatment show promise.SummaryThe ability to personalize management for a given kidney transplant recipient and identify treatments that will improve their long-term outcome remains a critical unmet need. Earlier identification of AMR with noninvasive biomarkers and prediction models to assess the individual risk of graft failure should be considered. Enrolling patients with AMR in clinical trials to assess novel therapeutic agents is highly encouraged.

Everolimus Alleviates Renal Allograft Interstitial Fibrosis by Inhibiting Epithelial-to-Mesenchymal Transition Not Only via Inducing Autophagy but Also via Stabilizing IκB-α.

Chronic allograft dysfunction (CAD) is the major cause of late graft loss in long-term renal transplantation. In our previous study, we found that epithelial–mesenchymal transition (EMT) is a significant event in the progression of renal allograft tubulointerstitial fibrosis, and impaired autophagic flux plays a critical role in renal allograft fibrosis. Everolimus (EVR) has been reported to be widely used to prevent the progression of organ fibrosis and graft rejection. However, the pharmacological mechanism of EVR in kidney transplantation remains to be determined. We used CAD rat model and the human kidney 2 (HK2) cell line treated with tumor necrosis factor-α (TNF-α) and EVR to examine the role of EVR on TNF-α-induced EMT and transplanted renal interstitial fibrosis. Here, we found that EVR could attenuate the progression of EMT and renal allograft interstitial fibrosis, and also activate autophagy in vivo. To explore the mechanism behind it, we detected the relationship among EVR, autophagy level, and TNF-α-induced EMT in HK2 cells. Our results showed that autophagy was upregulated upon mTOR pathway inhibition by EVR, which could significantly reduce expression of TNF-α-induced EMT. However, the inhibition of EVR on TNF-α-induced EMT was partly reversed following the addition of autophagy inhibitor chloroquine. In addition, we found that TNF-α activated EMT through protein kinase B (Akt) as well as nuclear factor kappa B (NF-κB) pathway according to the RNA sequencing, and EVR’s effect on the EMT was only associated with IκB-α stabilization instead of the Akt pathway. Together, our findings suggest that EVR may retard impaired autophagic flux and block NF-κB pathway activation, and thereby prevent progression of TNF-α-induced EMT and renal allograft interstitial fibrosis.

Evolución de las causas de pérdida del injerto en trasplante renal durante 40 años (1979-2019)

IntroductionThe improvement of kidney allograft recipient and graft survival showed a decrease over the last 40 years. Long-term graft loss rate remained stable during a 25-year time span. Knowing the changing causes and the risk factors associated with graft loss requires special attention.The present study aimed to assess the causes of graft loss and kidney allograft recipient death. Also, we aimed to compare two different periods (1979-1999 and 2000-2019) to identify changes in the characteristics of the failed allografts and recipient and donors profile. Methods and patientsWe performed a single-center cohort study. We included all the kidney transplant recipients at the Hospital del Mar (Barcelona) between May 1979 and December 2019. Graft loss was defined as recipient death with functioning graft and as loss of graft function (return to dialysis or retransplantation). We assessed the causes of graft loss using clinical and histological information. We also analyzed the results of the two different transplant periods (1979-1999 and 2000-2019). ResultsBetween 1979 and 2019, 1522 transplants were performed. The median follow-up time was 56 (IQR: 8-123) months. During follow-up, 722 (47.5%) grafts were lost: 483 (66.9%) due to graft failure and 239 (33.1%) due to death with functioning graft. The main causes of death were cardiovascular (25.1%), neoplasms (25.1%), and infectious diseases (21.8%). These causes were stable between the two periods of time. Only the unknown cause of death has decreased in the last period.The main cause of graft failure (loss of graft function) was the allograft chronic dysfunction (75%). When histologic information was available, antibody-mediated rejection (ABMR) and interstitial fibrosis/tubular atrophy (IF/TA) were the most frequent specific causes (15.9% and 12.6%). Of the graft failures, 213 (29.5%) were early (<1 year of transplantation). Vascular thrombosis was the main cause of early graft failure in the second period (2000-2019) (46.7%) and T-cell-mediated rejection (TCMR) was the main cause (31.3%) in the first period (1979-1999). The causes of late graft loss were similar between the two periods. ConclusionsThe causes of kidney allograft recipient death are still due to cardiovascular and malignant diseases. Vascular thrombosis has emerged as a frequent cause of early graft loss in the most recent years. The evaluation of the causes of graft loss is necessary to improve kidney transplantation outcomes.

Cardiac allograft vasculopathy: current review and future research directions.

Cardiac allograft vasculopathy (CAV) is a pathologic immune-mediated remodelling of the vasculature in transplanted hearts and, by impairing perfusion, is the major cause of late graft loss. Although best understood following cardiac transplantation, similar forms of allograft vasculopathy occur in other vascularized organ grafts and some features of CAV may be shared with other immune-mediated vasculopathies. Here, we describe the incidence and diagnosis, the nature of the vascular remodelling, immune and non-immune contributions to pathogenesis, current therapies, and future areas of research in CAV.

Clinical significance of de novo donor-specific antibody in kidney transplant recipients with chronic antibody-mediated rejection.

BackgroundChronic antibody-mediated rejection (CABMR) is an important cause of late graft loss. De novo donor-specific antibody (dnDSA) is an important prognostic factor for long-term allograft outcomes. However, the prognosis of CABMR based on the presence of dnDSA is uncertain.MethodsWe retrospectively analyzed 35 kidney transplant recipients with CABMR between 2010 and 2018. Fourteen recipients had no detectable DSA, and 21 recipients had detectable DSA. We investigated the pathologic findings at diagnosis of CABMR, allograft function 12 months later, related factors for allograft failure, and allograft survival rate based on the presence of dnDSA.ResultsThe pathologic findings showed that acute and chronic changes were more severe in the dnDSA (+) group than in the dnDSA (–) group. There was no significant difference in the allograft function 12 months after the diagnosis of CABMR and in the amount of proteinuria at diagnosis between the two groups. However, the death-censored graft survival rate was lower in the high-proteinuria group than in the low-proteinuria group in both groups. The treatment rate of recipients was higher in the dnDSA (+) group than in the dnDSA (–) group; however, there was no significant difference in the death-censored graft survival rate between the two groups.ConclusionsAlthough the effect of dnDSA on the prognosis of CABMR is not clear, it would be important not to neglect treatment for CABMR with risk factors for allograft failure even without dnDSA. Continuous and rigorous surveillance of DSA and allograft function is needed in patients with CABMR.

Late graft loss after intestinal transplantation.

Despite improvement in short-term outcomes after intestinal transplantation in the last 20 years, long-term rates of graft attrition and patient survival remain unchanged, with worse outcomes compared with other solid organ transplants. This review investigates the multiple causes of late graft loss, including chronic rejection, infection, graft-versus-host disease, posttransplant lymphoproliferative disorder and postsurgical complications. New insights into immunology of the intestine and evolution of immunosuppression, as well as review of current persistent causes of late graft loss, shed light on findings that may help improve long-term intestinal allograft survival. Although intestinal transplantation remains a life-saving intervention with significant advancements since its inception, further understanding of mechanisms of injury is needed to improve long-term outcomes and prevent late intestinal graft loss.

Risk factors for early development of cardiac allograft vasculopathy by intravascular ultrasound.

Cardiac allograft vasculopathy (CAV) is the leading cause of late graft loss. While there are numerous post-transplant factors which may increase the risk of the development of CAV, there is a paucity of data on the impact of donor-derived atherosclerosis (DA), early discontinuation of prednisone, and early initiation of proliferation signal inhibitors (PSI) as assessed by intravascular ultrasound (IVUS). Retrospective single-center study of all adult transplant patients (2008-2017) with serial IVUS at baseline and annually for 5years. DA was defined as a baseline maximal intimal thickness (MIT) ≥0.5mm, and CAV development was defined as MIT ≥1mm or an increase in MIT ≥0.5mm at year 1 compared with baseline or an increase in 0.3mm annually thereafter. Clinical risk factors for CAV were identified using multivariable hazard regression. Separate multistate models were applied to assess the association of prednisone discontinuation and PSI initiation and CAV. Of 282 patients screened, 186 patients had a 1-year angiogram. The mean age of those included in the cohort was 51±11years, 70% were male, 58% were Caucasian, and 27% were supported by a left ventricular assist device. Donor atherosclerosis was present in 40%. The cumulative incidence of CAV at 5years is 41% in DA- vs. 59% in DA + (p=.012). Donor age was a strong predictor of DA (p=.016). Significant risk factors for CAV included male sex (HR=4.141, p=.001), non-Caucasian race (HR=1.98, p=.011), BMI<18kg/m2 (HR=4.596, p=.042), longer ischemic time (HR=1.374, p=.028), older donor age (HR=1.158, p=.009), and rejection with hemodynamic compromise within the first year (HR=2.858, p=.043). Prednisone discontinuation within 1year was associated with a lower risk of CAV (HR 0.58 p=.047). Initiation of proliferation signal inhibitors (PSI) within 2years resulted in fewer cases of CAV (HR 0.397 p<.001). In patients with an angiogram at 1year, those with DA were significantly more likely to develop CAV. Lower incidence of CAV by IVUS was seen in patients who discontinued prednisone in the first year or had initiation of a PSI within two years of transplantation. Knowledge of early IVUS may allow a more tailored approach to patient management.

What have we learned about how to prevent and treat antibody-mediated rejection in kidney transplantation?

Antibody-mediated rejection (ABMR) in kidney transplantation is a major cause of late graft loss, and despite all efforts to date the "standard of care" remains plasmapheresis, IVIg, and steroids, which itself is based on low quality evidence. This review focuses on the risk factors leading to memory and de novo donor-specific antibody (DSA)-associated ABMR, the optimal prevention strategies for ABMR, and advances in adjunctive and emerging therapies for ABMR. Because new agents require regulatory approval via a Phase 3 randomized control trial (RCT), an overview of progress in innovative trial design for ABMR is provided. Finally, based on the insights gained in the biology of ABMR, current knowledge gaps are identified for future research that could significantly affect our understanding of how to optimally treat ABMR.

Involvement of heme oxygenase-1 in suppression of T cell activation by quercetin

Aim Acute rejection is still a major problem in transplantation and one of the most important causes of late graft loss. Cyclosporine and tacrolimus are widely used for suppression of T cell function to avoid graft rejection, but long-term use of these compounds is associated with serious toxicities. Quercetin, a flavonoid found in fruits and vegetables, has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO) -1, an enzyme involved in heme catabolism. We hypothesized that quercetin induces HO-1 in T cells and suppresses T cell function via HO-1. In the present study, we showed that quercetin suppressed the A23187-mediated expression of interleukin (IL) -2 in T cells. Methods Mouse splenocytes, enriched T cells, and EL4 cells, a mouse T cell line, were treated with quercetin, and then stimulated with A23187, a calcium ionophore, concanavalin A, or anti-CD3ε and anti-CD28 antibodies. Cell proliferation, expression of IL-2, calcium mobilization, apoptosis, cell cycle, and phosphorylation of extracellular signal-regulated kinase (ERK) were investigated. Results Quercetin induced HO-1, and this induction of HO-1 was implicated in the suppression of IL-2 production. Furthermore, the induction of HO-1 by quercetin suppressed the influx of calcium ions, a known trigger of IL-2 production. Additionally, quercetin suppressed T cell proliferation through promotion of cell cycle arrest via HO-1 induction, but quercetin did not induce apoptosis. To investigate the role of the signal transduction pathway in quercetin’s effect on cell proliferation, we evaluated the phosphorylation of ERK in T cells. Quercetin suppressed the A23187-mediated stimulation of ERK, an effect that was mediated through HO-1. These results suggested that HO-1 is involved in the suppressive effects of quercetin on T cell activation and proliferation. Conclusion Our findings indicate that the quercetin may be a promising candidate for inducing HO-1 in T cells, thereby facilitating immunosuppressive effects.

Bioinformatic analysis of peripheral blood RNA-sequencing sensitively detects the cause of late graft loss following overt hyperglycemia in pig-to-nonhuman primate islet xenotransplantation

Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3+ T cells. Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.

Autotaxin Inhibitor Protects from Chronic Allograft Injury in Rat Kidney Allotransplantation

Background: Tissue fibrosis is the final common phase of chronic allograft injury, the leading cause of late graft loss in kidney transplantation. Preclinical evidence points to the involvement of lysophosphatidic acid (LPA), a bioactive phospholipid, in the development of renal fibrosis. Objectives: We assessed whether treatment with an orally available inhibitor of autotaxin (ATXi), the main LPA-producing enzyme, could slow the progression of chronic allograft injury in a fully major histocompatibility complex-mismatched rat kidney transplant model and compared its effects with those of the angiotensin-converting enzyme inhibitor lisinopril. Methods: Kidney allograft recipients were given ciclosporin for the first 15 postoperative days to prevent early acute rejection. Thereafter, they received either no treatment or ATXi or lisinopril and were followed for 180 days after transplantation. Results: Renal LPA levels were increased in allograft rats, providing the rationale for using ATXi in this model. Chronic treatment with ATXi or lisinopril limited progressive proteinuria and ameliorated tubulointerstitial fibrosis compared with allograft rats, although the effects were more robust under ATX inhibition. The administration of ATXi, but not lisinopril, attenuated systemic hypertension, reduced intragraft T cell infiltration, and eventually improved renal graft survival. Conclusions: In summary, ATXi had protective effects on indices of chronic allograft injury and could be of therapeutic add-on value in the kidney transplant setting. Notably, an ATX inhibitor is currently being investigated in 2 large phase 3 studies in idiopathic pulmonary fibrosis, underscoring the clinical relevance of our findings.

Notch-1 Inhibition Promotes Immune Regulation in Transplantation Via Regulatory T Cell-Dependent Mechanisms.

Transplantation is the treatment of choice for many patients with end-stage organ disease. Despite advances in immunosuppression, long-term outcomes remain suboptimal, hampered by drug toxicity and immune-mediated injury, the leading cause of late graft loss. The development of therapies that promote regulation while suppressing effector immunity is imperative to improve graft survival and minimize conventional immunosuppression. Notch signaling is a highly conserved pathway pivotal to T-cell differentiation and function, rendering it a target of interest in efforts to manipulate T cell-mediated immunity. We investigated the pattern of Notch-1 expression in effector and regulatory T cells (Tregs) in both murine and human recipients of a solid-organ transplant. Using a selective human anti-Notch-1 antibody (aNotch-1), we examined the effect of Notch-1 receptor inhibition in full major histocompatibility complex-mismatch murine cardiac and lung transplant models, and in a humanized skin transplant model. On the basis of our findings, we further used a genetic approach to investigate the effect of selective Notch-1 inhibition in Tregs. We observed an increased proportion of Tregs expressing surface and intracellular (activated) Notch-1 in comparison with conventional T cells, both in mice with transplants and in the peripheral blood of patients with transplants. In the murine cardiac transplant model, peritransplant administration of aNotch-1 (days 0, 2, 4, 6, 8, and 10) significantly prolonged allograft survival in comparison with immunoglobulin G-treated controls. Similarly, aNotch-1 treatment improved both histological and functional outcomes in the murine lung transplant model. The use of aNotch-1 resulted in a reduced proportion of both splenic and intragraft conventional T cells, while increasing the proportion of Tregs. Furthermore, Tregs isolated from aNotch-1-treated mice showed enhanced suppressive function on a per-cell basis, confirmed with selective Notch-1 deletion in Tregs (Foxp3EGFPCreNotch1fl/fl). Notch-1 blockade inhibited the mammalian target of rapamycin pathway and increased the phosphorylation of STAT5 (signal transducer and activator of transcription 5) in murine Tregs. Notch-1low Tregs isolated from human peripheral blood exhibited more potent suppressive capacity than Notch-1high Tregs. Last, the combination of aNotch-1 with costimulation blockade induced long-term tolerance in a cardiac transplant model, and this tolerance was dependent on CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4) signaling. Our data reveal a promising, clinically relevant approach for immune modulation in transplantation by selectively targeting Notch-1.

Indoleamine 2,3-dioxygenase suppresses humoral alloimmunity via pathways that different to those associated with its effects on T cells.

Chronic antibody-mediated rejection remains a major cause of late graft loss. Regarding cellular alloimmunity, the immunosuppressive properties of indoleamine 2,3-dioxygenase (IDO) have been well investigated; however, little is known of its effects on humoral alloimmunity. Therefore, the present study aimed to evaluate the effects of IDO on humoral alloimmunity. We developed a method for the induction of humoral alloimmunity in a one-way mixed lymphocyte reaction (MLR), which was measured with an antibody-mediated complement-dependent cytotoxicity assay using resting cells, which are similar to the stimulator cells of the aforementioned MLR. In parallel, cellular alloimmunity was assessed in two-way MLRs. The IDO inhibitor 1-methyl-DL-tryptophan was used for evaluating the role of IDO. In order to investigate whether the pathways known to serve a role in the effects of IDO on T cells are applied in humoral alloimmunity, the general control nonderepressible-2 (GCN-2) kinase activator tryptophanol and the aryl hydrocarbon receptor (AhR) inhibitor CH223191 were employed. The IDO inhibitor was revealed to increased cellular autoimmunity, but was decreased by the GCN-2 kinase activator. Unexpectedly, the AhR inhibitor decreased cellular alloimmunity. In addition, the IDO inhibitor was observed to suppress humoral alloimmunity, which may occur in manners independent of GCN-2 kinase AhR. The present study proposed that IDO may decrease humoral alloimmunity in primary human peripheral blood mononuclear cells via pathways that differ to those associated with its effect on T cells.

Outstanding questions in transplantation: B cells, alloantibodies, and humoral rejection.

Over the past three decades, improved immunosuppression has significantly reduced T cell-mediated acute rejection rates, but long-term graft survival rates have seen only marginal improvement. The cause of late graft loss has been under intense investigation, and chronic antibody-mediated rejection (AMR) has been identified as one of the leading causes, thus providing a strong rationale for basic science investigation into donor-specific B cells and antibodies in transplantation and ways to mitigate their pathogenicity. In 2018, the American Society of Transplantation launched a community-wide online discussion of Outstanding Questions in Transplantation, and the topic of B cell biology and donor-specific antibody prevention emerged as a major area of interest to the community, leading to a highly engaged dialogue, with comments from basic and translational scientists as well as physicians (http://community.myast.org/communities/community-home/digestviewer). We have summarized this discussion from a bedside to bench perspective and have organized this review into outstanding questions within the paradigm that AMR is a leading cause of graft loss in the clinic, and points of view that challenge aspects of this paradigm. We also highlight opportunities for basic and translational scientists to contribute to the resolution of these questions, mapping important future directions for the transplant research field.

The Gift That Keeps on Giving? The Impact of Donor Coronary Atherosclerosis on the Development of Coronary Allograft Vasculopathy by IVUS

Purpose Cardiac allograft vasculopathy (CAV) is a leading cause of late graft loss . While there are numerous risk factors for the development of CAV, there is a paucity of data on the impact of donor derived atherosclerosis (DA), particularly as assessed by intravascular ultrasound (IVUS). Methods Retrospective single center study of all adult transplant patients (2008-2017) with serial IVUS at baseline and annually upto 5 years. DA was defined as a baseline maximal intimal thickness (MIT) ≥ 0.5 mm, CAV development was defined as MIT ≥ 1 mm or an increase in MIT ≥ 0.5 mm at year 1 or an increase in 0.3 mm annually thereafter. Clinical risk factors for CAV were identified using parametric hazard regression. Separate multistate models were applied to assess the association of prednisone discontinuation, proliferation signal inhibitors (PSI) initiation with CAV. Results Of 284 patients transplanted, 186 patients survived and had an IVUS at 1 year. Mean age of 51 ± 11 years, 70% male, 58% Caucasian with 27% supported by LVAD and 40% had DA. Patients who developed CAV at 1-year had longer ischemic times (4 vs. 3.7 hours, p=0.03), DA+ (65% vs. 39%, p=0.047) and older donor age (40 vs. 33 years, p=0.03). The 5-year cumulative incidence of CAV 41% in DA- vs. 59% in DA+ (Fig 1A). Significant risk factors for CAV were male sex HR 4.1, non-Caucasian race HR 2, underweight (BMI hemodynamic rejection within 1 year HR 2.9. Initiation of PSI within 2 years for reasons other than CAV resulted in fewer cases of CAV HR 0.40 p Conclusion In patients with a baseline and yearly IVUS, DA was more prevalent HR 2 in recipients who developed CAV. Reductions in CAV were seen in patients who discontinued prednisone in the first year or had early PSI initiation for reasons other than CAV. Early IVUS results may allow physicians to tailor immunosuppression and potentially delay progression of CAV.

Detection of Angiotensin II type I‐receptor antibodies in transplant glomerulopathy

Transplant glomerulopathy (TG) is an important cause of late graft loss. The role of angiotensin type 1-receptor antibodies (AT1 R-Ab) in TG is not known. All the TG cases (N=137) between January 2007 and December 2014 (N=1410) were analyzed. Donor-specific anti-HLA antibodies (DSA) at the time of biopsy and AT1 R-Ab IgG (positive, >17UI/mL; "at risk," 10-17UI/mL; negative, <10UI/mL) in pre-transplant sera (PT-Ab) and at biopsy time (BT-Ab) were studied. AT1 R-PT-Ab+ and AT1 R-BT-Ab+ patients were 16.5% (51.5% "at risk") and 11.5% (27.4% "at risk"), respectively. Clinical correlations were found between AT1 R-Ab and HCV infection, number of transplants, and age. Considering Banff scores, ptc was higher in DSA+ patients vs AT1 R-PT-Ab+ (P=0.002) or AT1 R-BT-Ab+ (P=0.001) without differences in g and chronicity score (ci+ct); cg showed lower scores in DSA+ patients vs AT1 R-BT-Ab+ (P=0.001). Graft survival was not influenced by the presence of AT1 R-Ab, AT1-R-Ab titer or MFI, but we observed a longer graft survival in patients with both AT1 R-BT-Ab+ or "at risk" and DSA+ vs patients positive only for DSA (P=0.02), for AT1 R-BT-Ab (P=0.019) or AT1 R-BT-Ab "at risk" (P=0.039). AT1 R-Ab showed no independent prognostic role in TG in this pilot analysis.