Preoperative carfilzomib and lulizumab based desensitization prolongs graft survival in a sensitized non-human primate model.

Sensitized patients are difficult to transplant due to pre-formed anti-donor immunity. We have previously reported successful desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated selective blockade of the co-stimulatory signal (CD28-B7) with Lulizumab, which preserves the co-inhibitory signal (CTLA4-B7). Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to either desensitization with once-weekly Carfilzomib (27mg/m2 IV) and Lulizumab (12.5mg/kg SC) over four weeks, or no desensitization (Control). NHPs then underwent life-sustaining kidney transplantation from their previous skin donor. Rhesus-specific anti-thymocyte globulin was used as induction therapy and immunosuppression maintained with tacrolimus, mycophenolate, and methylprednisolone. Desensitized subjects demonstrated a significant reduction in donor-specific antibody, follicular helper T cells (CD4+PD-1+ICOS+), and proliferating B cells (CD20+Ki67+) in the lymph nodes. Interestingly, regulatory T cell (CD4+CD25+CD127lo) frequency was maintained after desensitization in addition to increased frequency of naïve CD4 T cells (CCR7+CD45RA+) and naïve B cells (IgD+CD27-CD20+) in circulation. This was associated with significant prolongation in graft survival (MST = 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05) and lower antibody-mediated rejection scores compared to control animals. However, all desensitized animals eventually developed AMR and graft failure. Desensitization with CFZ and Lulizumab improves allograft survival in allosensitized NHPs, by transient control of the germinal center and shifting of the immune system to a more naive phenotype. This regimen may translate into clinical practice to improve outcomes of highly sensitized transplant patients.

B cells in transplant tolerance and rejection: friends or foes?

Our understanding of the role of B cells in organ transplantation remains incomplete and continues to grow. The majority of research has focused on the detrimental role of antibodies that drive the development of pathogenesis of the transplanted organ. However, it has been shown that not all donor-specific antibodies are harmful and in some circumstances can even promote tolerance through the mechanism of accommodation. Furthermore, B cells can have effects on transplanted organs through their interaction with T cells, namely antigen presentation, cytokine production, and costimulation. More recently, the role and importance of Bregs was introduced to the field of transplantation. Due to this functional and ontogenetic heterogeneity, targeting B cells in transplantation may bring undesired immunologic side effects including increased rejection. Therefore, the selective control of B cells that contribute to the humoral response against donor antigens will continue to be an important and challenging area of research and potentially lead to improved long-term transplant outcomes.

Pretransplant Desensitization with Costimulation Blockade and Proteasome Inhibitor Reduces DSA and Delays Antibody-Mediated Rejection in Highly Sensitized Nonhuman Primate Kidney Transplant Recipients.

BACKGROUND: Patients with broad HLA sensitization have poor access to donor organs, high mortality while waiting for kidney transplant, and inferior graft survival. Although desensitization strategies permit transplantation via lowering of donor-specific antibodies, the B cell-response axis from germinal center activation to plasma cell differentiation remains intact. METHODS: To investigate targeting the germinal center response and plasma cells as a desensitization strategy, we sensitized maximally MHC-mismatched rhesus pairs with two sequential skin transplants. We administered a proteasome inhibitor (carfilzomib) and costimulation blockade agent (belatacept) to six animals weekly for 1 month; four controls received no treatment. We analyzed blood, lymph node, bone marrow cells, and serum before desensitization, after desensitization, and after kidney transplantation. RESULTS: The group receiving carfilzomib and belatacept exhibited significantly reduced levels of donor-specific antibodies (P=0.05) and bone marrow plasma cells (P=0.02) compared with controls, with a trend toward reduced lymph node T follicular helper cells (P=0.06). Compared with controls, carfilzomib- and belatacept-treated animals had significantly prolonged graft survival (P=0.02), and renal biopsy at 1 month showed significantly reduced antibody-mediated rejection scores (P=0.02). However, four of five animals with long-term graft survival showed gradual rebound of donor-specific antibodies and antibody-mediated rejection. CONCLUSIONS: Desensitization using proteasome inhibition and costimulation blockade reduces bone marrow plasma cells, disorganizes germinal center responses, reduces donor-specific antibody levels, and prolongs allograft survival in highly sensitized nonhuman primates. Most animals experienced antibody-mediated rejection with humoral-response rebound, suggesting desensitization must be maintained after transplantation using ongoing suppression of the B cell response.

Once-daily prolonged-release tacrolimus versus twice-daily tacrolimus in liver transplantation.

OBJECTIVE: For patients who have received a kidney transplant, studies have shown that once-daily prolonged-release tacrolimus (TAC) has similar efficacy and safety to standard twice-daily dosing. The purpose of this study was to perform a meta-analysis to compare the effectiveness and safety of daily TAC (TAC qd) versus standard twice-daily TAC (TAC bid) administration in liver transplantation (LT). DESIGN: Meta-analysis. SETTING AND PARTICIPANTS: We systematically searched the PubMed/MEDLINE, Web of Science, and Cochrane Library databases for studies comparing outcomes of LT patients who received TAC qd versus TAC bid. OUTCOME MEASURES: Results were reported as odds ratios (ORs) with 95% CIs. RESULTS: Six studies, which included 5179 LT recipients (TAC qd = 951; TAC bid = 4228) were included in the analysis. The TAC qd group had a low 1-year graft loss rate (OR 0.70 [95% CI 0.54-0.91], P = 0.008) and lower rate of biopsy-proven acute rejection (BPAR) at 90 days (OR 0.46 [95% CI 0.24-0.89], P = 0.02) compared with the TAC bid group. There was no significant difference in 1-year mortality or the incidence of adverse events after LT between the 2 groups. CONCLUSIONS: Current evidence suggests that TAC qd is safe and effective for LT patients during the first year after transplantation. Longer-term follow-up studies are necessary to determine if TAC qd is safe and effective beyond the first year after LT.

The past, present, and future of costimulation blockade in organ transplantation.

PURPOSE OF REVIEW: Manipulating costimulatory signals has been shown to alter T cell responses and prolong graft survival in solid organ transplantation. Our understanding of and ability to target various costimulation pathways continues to evolve. RECENT FINDINGS: Since the approval of belatacept in kidney transplantation, many additional biologics have been developed targeting clinically relevant costimulation signaling axes including CD40-CD40L, inducible costimulator-inducible costimulator ligand (ICOS-ICOSL), and OX40-OX40L. Currently, the effects of costimulation blockade on posttransplant humoral responses, tolerance induction, and xenotransplantation are under active investigation. Here, we will discuss these pathways as well as preclinical and clinical outcomes of biologics targeting these pathways in organ transplantation. SUMMARY: Targeting costimultion is a promising approach for not only controlling T cell but also B cell responses. Consequently, costimulation blockade shows considerable potential for improving outcomes in antibody-mediated rejection and xenotransplantation.

Improved survival in simultaneous lung-liver recipients and candidates in the modern era of lung allocation.

BACKGROUND: Liver-lung transplantation (LLT) is a rare procedure performed for patients with end-stage liver and lung disease. The lung allocation score (LAS), introduced in 2005, guides lung allocation including those receiving LLT. However, the impact of the LAS on outcomes in LLT is currently unknown. MATERIALS AND METHODS: The OPTN/United Network for Organ Sharing STAR file was queried for LLT candidates and recipients from 1988 to 2016. Demographic characteristics before (historic) and after (modern) the LAS were compared. Survival was analyzed with the Kaplan-Meier method and log-rank test. RESULTS: In total, 167 candidates were listed for LLT, and 62 underwent LLT. The historic cohort had a higher FEV1% (48.22% versus 29.82%, P = 0.014), higher creatinine (1.22 versus 0.72, P < 0.001), and a higher percentage with pulmonary hypertension as the indication for transplantation (40% versus 0%, P = 0.003) compared with the modern cohort. LLT candidates in the historic cohort had a lower rate of transplant per 100 candidates (10.87 versus 33.33, P < 0.0001) and worse waitlist survival (1 y: 69.6% versus 80.9%, 3 y: 39.1% versus 66.8%, P = 0.004). Post-transplant survival was significantly lower in the historic cohort (1 y: 50.0% versus 82.7%, 5 y: 40.0% versus 69.0%, 10 y: 20.0% versus 55.5%, P = 0.0099). CONCLUSIONS: Most analyses of LLT have included patients before and after the introduction of the LAS. Our study shows that LLT candidates and recipients before the modern allocation system had distinct baseline characteristics and worse overall survival. Although many factors contributed to recent improved outcomes, these cohorts are significantly different and should be treated as such in future studies.

Donor liver apoptosis is associated with early allograft dysfunction and decreased short-term graft survival after liver transplantation.

BACKGROUND: The clinical significance of apoptosis in assessing the quality of donor liver grafts remains unknown. AIMS: This study aimed to determine whether apoptosis in a donor liver is predictive of early allograft dysfunction (EAD) and graft survival after liver transplantation (LT). METHODS: Donor liver specimens were analyzed for apoptosis using TUNEL assays. The prognostic factors for EAD were identified through logistic regression analyses, and a nomogram was developed. RESULTS: The apoptosis index of donor livers in EAD patients was significantly higher than that of donors livers in non-EAD patients (median 5.3; interquartile range [IQR] 3.4 vs 3.5; 3.6, P < 0.001). Multivariate analyses identified the apoptosis index of the donor liver (HR = 6.927, P < 0.001) and five other characteristics as independent predictors of EAD. A nomogram built on these predictive variables showed good calibration and discriminatory abilities, with a c-index value of 0.847. The 30-day graft survival rates in the high apoptosis index (apoptosis index >4.4%) group were significantly lower than those in the low apoptosis index (apoptosis index ≤4.4%) group (84.4% vs 97.6%, P = 0.004). CONCLUSIONS: Donor liver apoptosis plays a significant role in predicting EAD after LT. Furthermore, a high apoptosis index in the donor liver was associated with inferior graft survival in the short-term.

The early outcomes of candidates with portopulmonary hypertension after liver transplantation.

BACKGROUND: Portopulmonary hypertension (PPH) was once regarded as a contraindicaton to liver transplantation (LT). However, growing evidence has indicated that PPH patients undergoing LT may show similar outcomes compared to those without PPH, and researchers have recommended it not be an absolute contraindication. Given this controversy, we aimed to identify and review the current evidence on this topic and to provide a comparison of the outcomes after LT between candidates with PPH and those without. METHODS: We systematically searched the MEDLINE, EMBASE and Cochrane Library databases for all studies that compared the outcomes of PPH patients and those without PPH after LT. All studies reporting outcomes of PPH patients versus those without PPH (Control) were further considered for inclusion in this meta-analysis. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to compare the pooled data between PPH and Control groups. RESULTS: Eleven retrospective trials and one prospective, randomized, controlled trial, involving 37,686 transplant recipients were included. The PPH patients had increased 1-year mortality with an OR of 1.59 (95% CI = 1.26-2.01, P = 0.0001) compared to the control group. There was no significant difference in graft loss and 30-day mortality after LT between the two groups. CONCLUSIONS: Patients with PPH who underwent LT had increased 1-year mortality compared to those without PPH, while graft loss and 30-day mortality were similar. Nevertheless, LT may be a reasonable therapeutic option for some patients with PPH, but further studies are needed to identify those select patients with PPH who would benefit most from LT.

Improved contemporary outcomes of liver transplantation for pediatric hepatoblastoma and hepatocellular carcinoma.

PURPOSE: Improvement in outcomes of LT for pediatric HB and HCC has been reported in small series. We analyzed national outcomes and changes in donor, recipient, and perioperative factors over time that may contribute to survival differences. METHODS: The UNOS database was queried for patients age <21 years that underwent LT for a primary diagnosis of HB or HCC (1987-2017). Subjects were divided into historic (transplant before 2010) and contemporary (transplant after 2010) cohorts. Baseline characteristics were compiled and examined. Survival was estimated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: In total, 599 children with HB received LT (320 historic vs 279 contemporary). Concurrently, 141 children with HCC received LT (92 historic vs 49 contemporary). For both tumors, waitlist time decreased (HB 56.2 days historic vs 33.2 days contemporary, P = 0.017; HCC 189.3 days historic vs 71.7 days contemporary, P = 0.012). In the historic cohorts, patients with HB had a 1-year and 5-year OS of 84.6% and 75.1%, respectively. Survival for HCC was 84.4% and 59.9%, respectively. Outcomes improved in the contemporary era to 89.1% and 82.6% for HB, and 94.7% and 80.8% for HCC, respectively (both log-rank test P < 0.0001). CONCLUSION: Outcomes of LT have improved significantly, with contemporary survival now equivalent between these tumors and exceeding 80% 5-year OS. Future studies are needed to explore whether offering LT in patients that are resectable is justifiable.

Accuracy of MR Imaging and MR Spectroscopy for Detection and Quantification of Hepatic Steatosis in Living Liver Donors: A Meta-Analysis.

Purpose To determine the accuracy of magnetic resonance (MR) imaging for detection and quantification of hepatic steatosis (HS) in living liver donor candidates. Materials and Methods A systematic search of the literature was performed to find studies on the diagnostic and quantitative accuracy of MR imaging for assessment of HS in liver donors. The Quality Assessment of Diagnostic Accuracy Studies 2 tool was used, and patient selection, index text, reference standard, and study flow and timing were assessed to evaluate the quality of each included study. Pooled sensitivity, specificity, positive and negative likelihood ratios, hierarchical summary receiver operating characteristic (ROC) curves, and the area under the curve were estimated by using hierarchical summary ROC and bivariate random-effects models. Results Eight studies involving 934 subjects were eligible for the meta-analysis. For detection of HS with MR imaging and/or MR spectroscopy in living liver donors, the pooled sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio, respectively, were 0.89 (95% confidence interval [CI]: 0.75, 0.95), 0.84 (95% CI: 0.76, 0.89), 5.53 (95% CI: 3.71, 8.25), and 0.14 (95% CI: 0.06, 0.31). The area under the curve was 0.92 (95% CI: 0.89, 0.94). For detection of substantial HS (>10% to >30% HS at liver pathologic examination, as defined in each study), these corresponding diagnostic estimates were 0.91 (95% CI: 0.82, 0.95), 0.89 (95% CI: 0.84, 0.93), 8.30 (95% CI: 5.47, 12.59), 0.10 (95% CI: 0.05, 0.21), and 0.96 (95% CI: 0.93, 0.97), respectively. Moderate heterogeneity was detected. No publication bias was detected (P = .12). Conclusion MR imaging and MR spectroscopy show high sensitivity and specificity for detection of HS, especially when HS is substantial, and may be useful for noninvasive evaluation of HS in living liver donors. © RSNA, 2016 Online supplemental material is available for this article.

PD-1-dependent restoration of self-tolerance in the NOD mouse model of diabetes after transient anti-TCRß mAb therapy.

AIMS/HYPOTHESIS: T cells play a major role in the pathogenesis of type 1 diabetes, and there is great interest in developing curative immunotherapies targeting these cells. In this study, a monoclonal antibody (mAb) targeting the T cell receptor ß-chain (TCRß) was investigated for its ability to prevent and reverse disease in mouse models of diabetes. METHODS: RIP-OVA(hi) (C57BL/6-Tg(Ins2-OVA)59Wehi/WehiJ) mice adoptively transferred with ovalbumin-specific T cells (an induced model of diabetes) and NOD mice (a spontaneous model of diabetes) were used to test anti-TCRß mAb therapy as a means of preventing and reversing type 1 diabetes. RESULTS: A single dose of anti-TCRß completely prevented disease in RIP-OVA(hi) mice without inducing the release of inflammatory cytokines. Transient anti-TCRß therapy prevented diabetes in 90% of NOD mice and reversed the disease after its onset in 73% of NOD mice. Long after the remission of type 1 diabetes, the anti-TCRß treated mice were able to reject BALB/c skin allografts with normal kinetics while maintaining normoglycaemia. Treatment did not cause significant reductions in lymphocyte numbers in the spleen or pancreatic lymph nodes, but did result in a decreased percentage of chemokine receptor 9 (CCR9) positive, CD8(+) T cells. Notably, anti-TCRß therapy increased the expression of programmed death 1 (PD-1) on the surface of the T cells; PD-1 expression is important for maintaining anti-TCRß-induced self-tolerance, as type 1 diabetes recurs in mice following a blockade of PD-1 signalling. CONCLUSIONS/INTERPRETATION: Anti-TCRß mAb is a safe and effective immunotherapy that results in reduced numbers of CCR9(+) T cells, an increased expression of PD-1 on T cells and the restoration of self-tolerance in NOD mice.

Adjuvant chemotherapy for patients with primary hepatocellular carcinoma: a meta-analysis.

Numerous studies have investigated the effects of adjuvant chemotherapy for primary hepatocellular carcinoma (HCC) patients. We conducted this analysis to evaluate the efficacy of adjuvant chemotherapy in HCC patients after hepatectomy. PubMed/MEDLINE, EMBASE, Cochrane, and other databases were searched for eligible studies. The major endpoints were overall survival (OS) and disease-free survival (DFS). The pooled odds ratio (OR) was calculated using a random-effects model to summarize the results. In the meta-analysis of 13 randomized control trials (RCTs) and 35 observational studies with 4747 patients, hepatectomy plus adjuvant chemotherapy showed superiority over hepatectomy alone in 1-year DFS (OR = 1.86, 1.38-2.51, p < 0.001), 3-year DFS (OR = 2.37, 1.73-3.24, p < 0.001) and 5-year DFS (OR = 1.99, 1.55-2.55, p < 0.001), as well as 1-year OS (OR = 2.16, 95% confidence interval 1.75-2.68, p < 0.001), 3-year OS (OR = 1.77, 1.48-2.13, p < 0.001) and 5-year OS (OR = 1.92, 1.44-2.56, p < 0.001). Subgroup and sensitivity analysis revealed that only adjuvant TACE had significant survival benefits. The meta-analysis of studies involving patients with portal vein tumor thrombus (PVTT), but not other factors related to recurrence risk, revealed favorable outcomes of the Treatment arm over the Control arm. The present study shows that adjuvant chemotherapy can improve outcomes for HCC patients. The benefits of adjuvant TACE have been confirmed whereas the effects of other adjuvant chemotherapy modalities remain uncertain. Adjuvant chemotherapy is likely to be more applicable to certain patient populations for instance those with PVTT, but further research in identifying these patient factors is of importance for tailoring adjuvant therapies to individual patients in the future.

Expanded criteria donor kidneys for retransplantation United Network for Organ Sharing update: proceed with caution.

This study analyzed outcomes of retransplantation from expanded criteria donors (ECD) over the last two decades to determine the benefits and risks of using ECD kidneys for retransplantation. Data from the United Network for Organ Sharing database were collected and analyzed. Graft survival, death-censored graft survival, and patient survival for retransplantation with ECD kidneys (re-ECD) were reported and compared with primary transplantation with ECD kidneys (prim-ECD) and retransplantation with standard criteria donor kidneys (re-SCD). Re-ECD kidneys had higher risk of graft failure compared with prim-ECD (hazard ratio [HR] = 1.19) and to re-SCD (HR = 1.76). Patient survival was better in re-ECD compared with prim-ECD (HR = 0.89) but was worse than re-SCD (HR = 1.82). After censoring the patients who died with a functioning graft, re-ECD had a higher mortality risk compared with prim-ECD (HR = 1.45) and re-SCD (HR = 1.79). Transplantation improves quality of life and reduces healthcare costs, and due to the risk associated with resumption of hemodialysis and the longer waiting list times for SCD kidneys, there is a benefit to accepting ECD kidneys for select patients requiring retransplantation. Although this benefit exists for select patients, retransplantation with ECD kidneys should be undertaken with trepidation, and appropriate informed consent should be obtained.

A dynamic dual role of IL-2 signaling in the two-step differentiation process of adaptive regulatory T cells.

The molecular mechanism of the extrathymic generation of adaptive, or inducible, CD4(+)Foxp3(+) regulatory T cells (iTregs) remains incompletely defined. We show that exposure of splenic CD4(+)CD25(+)Foxp3(-) cells to IL-2, but not other common γ-chain cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ∼10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4(+)CD25(+)Foxp3(-) iTreg precursors. In this study, to further define the role of IL-2 in the formation of iTreg precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg differentiation model. During the initial "conditioning" step, CD4(+)CD25(-)Foxp3(-) naive T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3-Stat5 was required during this step to generate CD4(+)CD25(+)Foxp3(-) cells containing iTreg precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg precursors. This two-step method generated a large number of iTregs with relatively stable expression of Foxp3, which were able to prevent CD4(+)CD45RB(high) cell-mediated colitis in Rag1(-/-) mice. In consideration of this information, whereas initial inhibition of IL-2 signaling upon T cell priming generates iTreg precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg differentiation and may facilitate the therapeutic use of iTregs in immune disorders.

Premalignant Lesions in the Kidney Transplant Candidate.

End-stage kidney disease patients who are referred for transplant undergo an extensive evaluation process to ensure their health prior to transplant due in part to the shortage of available organs. Although management and surveillance guidelines exist for malignancies identified in the transplant and waitlist populations, less is written about the management of premalignant lesions in this population. This review covers the less common premalignant lesions (intraductal papillary mucinous neoplasm, gastrointestinal stromal tumor, thymoma, and pancreatic neuroendocrine tumor) that can be found in the transplant candidate population. High-level evidence for the management of these rarer premalignant lesions in the transplant population is lacking, and this review extrapolates evidence from the general population and should not be a substitute for a multidisciplinary discussion with medical and surgical oncologists.

Living donor liver transplantation versus deceased donor liver transplantation for hepatocellular carcinoma: a meta-analysis.

Because of the severe organ shortage, living donor liver transplantation (LDLT) offers a timely alternative to deceased donor liver transplantation (DDLT) for patients with hepatocellular carcinoma (HCC). However, the higher recurrence rate of HCC after LDLT and the indication criteria remain controversial. By conducting a quantitative meta-analysis, we sought to compare the survival outcomes and recurrence rates with LDLT and DDLT for patients with HCC. Comparative studies of LDLT and DDLT for HCC, which were identified by a comprehensive literature search, were included in this study. The evaluated outcomes included patient survival, recurrence-free survival (RFS), and recurrence rates at defined time points. Seven studies with a total of 1310 participants were included in this study. For LDLT and DDLT recipients, we found comparable patient survival rates [1 year, odds ratio (OR) = 1.03, 95% confidence interval (CI) = 0.62-1.73; 3 years, OR = 1.07, 95% CI = 0.77-1.48; and 5 years, OR = 0.64, 95% CI = 0.33-1.24] and RFS rates (1 year, OR = 0.86, 95% CI = 0.54-1.38; 3 years, OR = 1.04, 95% CI = 0.69-1.58; and 5 years, OR = 1.11, 95% CI = 0.70-1.77). Moreover, we found no significant differences in the 1-, 3-, or 5-year recurrence rates between LDLT and DDLT recipients (1 year, OR = 1.55, 95% CI = 0.36-6.58; 3 years, OR = 2.57, 95% CI = 0.53-12.41; and 5 years, OR = 1.21, 95% CI = 0.44-3.32). A subgroup analysis revealed similar outcomes for patients with HCC meeting the Milan criteria. These findings demonstrate that for HCC patients (especially those within the Milan criteria), LDLT represents an acceptable option that does not compromise patient survival or increase HCC recurrence in comparison with DDLT.

The anti-apoptotic protein HAX-1 is a regulator of cardiac function.

The HS-1 associated protein X-1 (HAX-1) is a ubiquitously expressed protein that protects cardiomyocytes from programmed cell death. Here we identify HAX-1 as a regulator of contractility and calcium cycling in the heart. HAX-1 overexpression reduced sarcoplasmic reticulum Ca-ATPase (SERCA2) pump activity in isolated cardiomyocytes and in vivo, leading to depressed myocyte calcium kinetics and mechanics. Conversely, downregulation of HAX-1 enhanced calcium cycling and contractility. The inhibitory effects of HAX-1 were abolished upon phosphorylation of phospholamban, which plays a fundamental role in controlling basal contractility and constitutes a key downstream effector of the beta-adrenergic signaling cascade. Mechanistically, HAX-1 promoted formation of phospholamban monomers, the active/inhibitory units of the calcium pump. Indeed, ablation of PLN rescued HAX-1 inhibition of contractility in vivo. Thus, HAX-1 represents a regulatory mechanism in cardiac calcium cycling and its responses to sympathetic stimulation, implicating its importance in calcium homeostasis and cell survival.

A novel method for in vitro culture and expansion of nonhuman primate B cells.

BACKGROUND: Given the role of B cells in sensitization and antibody-mediated rejection pathogenesis, the ability to identify, isolate, and study B cells in vitro is critical for understanding these processes and developing novel therapeutics. While in vivo nonhuman primate models have been used to this end, an in vitro nonhuman primate model of B cell activation and proliferation has not been developed. METHODS: CD20+ B cells and CD3+ T cells were isolated using magnetic bead separation from the peripheral blood of naive and skin allograft sensitized nonhuman primates. Allogeneic B and T cells were co-cultured in plates pre-coated with murine stromal cells engineered to express human CD40L and stimulated with cytokines. Cells and supernatants were harvested every 2 days for immune phenotyping and donor specific antibody quantification by flow cytometry. RESULTS: The optimized culture system consisted of MS40L cells co-cultured with B and allogenic T cells and stimulated with cytokines. This culture system resulted in increased memory cells and plasmablasts over time compared to other culture systems. Comparison of culture of naïve and sensitized nonhuman primate samples revealed faster B cell exhaustion and marginally increased plasmablast differentiation in sensitized culture. Donor-specific antibody production was not observed in either culture group. CONCLUSIONS: This study describes the first in vitro nonhuman primate model of B cell activation and proliferation using both naïve and allosensitized samples. This model provides an opportunity for exploration of B cell mechanisms and novel therapeutics and is a preliminary step in the development of an in vitro germinal center model.

Belatacept-Based Maintenance Immunosuppression Controls the Post-Transplant Humoral Immune Response in Highly Sensitized Nonhuman Primates.

Preexisting donor-specific antibodies (DSA) to MHC antigens increase the risk of antibody-mediated rejection (AMR) in sensitized transplant recipients and reduces graft survival. Pretransplant desensitization with costimulation blockade and proteasome inhibition has facilitated transplantation in our preclinical nonhuman primate (NHP) model. However, long-term graft survival is limited by rebound of DSA after transplantation. In this study, we performed kidney transplants between highly sensitized, maximally MHC-mismatched NHPs (n=14). At kidney transplantation, primates received T cell depletion with rhesus-specific anti-thymocyte globulin (rhATG; n=10) or monoclonal anti-CD4 and anti-CD8 antibodies (n=4). Maintenance immunosuppression consisted of belatacept and tacrolimus (n=5) or belatacept and rapamycin (n=9) with steroids. Rebound of DSA post-kidney transplantation was significantly reduced compared with maintenance immunosuppression with tacrolimus, mycophenolate, and steroids. Protocol lymph node biopsy specimens showed a decrease in germinal center activity, with low frequencies of T follicular helper cells and class-switched B cells after kidney transplantation. Combined belatacept and rapamycin was superior in controlling viral reactivation, enabling weaning of ganciclovir prophylaxis. Tacrolimus was associated with increased morbidity that included cytomegalovirus and parvovirus viremia and post-transplant lymphoproliferative disorder. All primates in the tacrolimus/belatacept group failed discontinuation of antiviral therapy. Overall, belatacept-based immunosuppression increased AMR-free graft survival by controlling post-transplant humoral responses in highly sensitized NHP recipients and should be further investigated in a human clinical trial.

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.