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Pediatric patients with recurrent metastatic neuroblastoma (NB) have a dismal 5-year survival. Novel therapeutic approaches are urgently needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is expressed in a variety of pediatric solid tumors, including NB, and constitutes a novel target for immunotherapy. Here, we developed a chimeric antigen receptor (CAR) expressing natural killer (NK) cell-targeting MCAM by non-viral electroporation of CAR mRNA into ex vivo expanded NK cells. Expression of anti-MCAM CAR significantly enhanced NK cell cytotoxic activity compared to mock NK cells against MCAM high but not MCAM low/knockout NB cells in vitro . Anti-MCAM-CAR-NK cell treatment significantly decreased tumor growth and prolonged animal survival in an NB xenograft mouse model. NKTR-255, a polymer-conjugated recombinant human interleukin-15 agonist, significantly stimulated NK cell proliferation and expansion and further enhanced the in vitro cytotoxic activity and in vivo anti-tumor efficacy of anti-MCAM-CAR-NK cells against NB. Our preclinical studies demonstrate that ex vivo expanded and modified anti-MCAM-CAR-NK cells alone and/or in combination with NKTR-255 are promising novel alternative therapeutic approaches to targeting MCAM high malignant NB., Competing Interests: M.S.C. has served as a consultant for Jazz Pharmaceuticals, Omeros Pharmaceuticals, Servier Pharmaceuticals, Abbvie, and Novartis Pharmaceuticals; with the Speakers Bureau for Jazz Pharmaceuticals, Servier Pharmaceuticals, Amgen, Inc., Sanofi, and Sobi; and on the Advisory Board for Astra Zeneca and receives research funding from Celularity, Merck, Miltenyi Biotec, Servier, Omeros, Jazz, and Janssen. D.A.L. reports personal fees and others from Kiadis Pharma, CytoSen Therapeutics, Courier Therapeutics, and Caribou Biosciences outside of the submitted work. In addition, D.A.L. has a patent broadly related to NK cell therapy of cancer with royalties paid to Kiadis Pharma. T.P.C. recently served as a one-time consultant to Blueprint, Incyte, and Oncopeptides and as a DSMB chair for SpringWorks and is a cofounder of Vironexis Biotherapeutics, Inc., (© 2024 The Author(s).)

Transcatheter pulmonary valve replacement (TPVR) is now frequently performed in patients with adult congenital heart disease. As the life expectancy of the population with adult congenital heart disease continues to improve, more patients will require pulmonary valve intervention. This study details the short-term and midterm clinical outcomes of patients aged ≥40 years who underwent TPVR. We performed an institutional retrospective cohort study that included patients aged ≥40 years who underwent TPVR (and clinical follow-up) from January 1, 2012 to January 1, 2024. Descriptive analyses, Kaplan-Meier survival analysis, and Cox proportional hazard modeling were used to determine outcomes and risk factors affecting survival. The study included 67 patients, and median age at TPVR was 48 years (43 to 57). Median hospital length of stay after TPVR was 1 day (1 to 3); periprocedural complications occurred in 5 patients, and acute kidney injury occurred in 1 patient. Median duration of follow-up was 3.5 years (0.1 to 9.7). There were 9 total deaths, and 1-, 3-, and 5-year Kaplan-Meier survival after TPVR was 95%, 91%, and 82%, respectively. Moderate or worse right ventricular dysfunction was present in 22 patients before TPVR and in 20 patients after TPVR. Inpatient status before TPVR negatively affected survival (hazard ratio 24.7, 3.3 to 186.1, p = 0.002). In conclusion, TPVR was performed in patients aged ≥40 years with favorable periprocedural and midterm follow-up outcomes including survival, but right ventricular dysfunction did not improve, and further exploration of the ideal timing of TPVR in this age group is warranted., Competing Interests: Declaration of competing interest Drs. Greenbaum and Babaliaros have received institutional research support from Edwards Lifesciences, Gore Medical, and Medtronic; and consulting fees from Edwards Lifesciences and Medtronic. Dr. Ligon is a consultant for Abbott Vascular, Inc., B. Braun Interventional Systems, and Medtronic. The remaining authors have no competing interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Background: Pediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient tumors, in large part due to the immunosuppressive tumor microenvironment (TME). Melanoma cell adhesion molecule (MCAM) is highly expressed on ES and associated with ES metastasis. NKTR-255 is a polymer-conjugated recombinant human interleukin-15 (IL-15) agonist improving NK cell activity and persistence. Magrolimab (MAG) is a CD47 blockade that reactivates the phagocytic activity of macrophages., Methods: Transcriptome profiling coupled with CIBERSORT analyses in both ES mouse xenografts and human patient tumors were performed to identify mechanisms of NK resistance in ES TME. A chimeric antigen receptor (CAR) NK cell targeting MCAM was engineered by CAR mRNA electroporation into ex vivo expanded NK cells. In vitro cytotoxicity assays were performed to investigate the efficacy of anti-MCAM-CAR-NK cell alone or combined with NKTR-255 against ES cells. Interferon-γ and perforin levels were measured by ELISA. The effect of MAG on macrophage phagocytosis of ES cells was evaluated by in vitro phagocytosis assays. Cell-based and patient-derived xenograft (PDX)-based xenograft mouse models of ES were used to investigate the antitumor efficacy of CAR-NK alone and combined with NKTR-255 and MAG in vivo., Results: We found that NK cell infiltration and activity were negatively regulated by tumor-associated macrophages (TAM) in ES TME. Expression of anti-MCAM CAR significantly and specifically enhanced NK cytotoxic activity against MCAM high but not MCAM-knockout ES cells in vitro, and significantly reduced lung metastasis and extended animal survival in vivo. NKTR-255 and MAG significantly enhanced in vitro CAR-NK cytotoxicity and macrophage phagocytic activity against ES cells, respectively. By combining with NKTR-255 and MAG, the anti-MCAM-CAR-NK cell significantly decreased primary tumor growth and prolonged animal survival in both cell- and PDX-based ES xenograft mouse models., Conclusions: Our preclinical studies demonstrate that immunotherapy via the innate immune system by combining tumor-targeting CAR-NK cells with an IL-15 agonist and a CD47 blockade is a promising novel therapeutic approach to targeting MCAM high malignant metastatic ES., Competing Interests: Competing interests: MSC has served as a consultant for Jazz Pharmaceuticals, Omeros Pharmaceuticals and Abbvie; Speakers Bureau for Jazz Pharmaceuticals and Sobi; and research funding from Merck, Miltenyi Biotec, Servier, Omeros, Jazz and Janssen. DL reports personal fees and other from Kiadis Pharma, CytoSen Therapeutics, Courier Therapeutics, and Caribou Biosciences outside the submitted work. In addition, DL has a patent broadly related to NK cell therapy of cancer with royalties paid to Kiadis Pharma. TC recently served as a one-time consultant to Blueprint, Incyte, Oncopeptides, DSMB chair for SpringWorks and is a cofounder of Vironexis Biotherapeutics., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Ewing sarcoma (ES) is a malignant tumor of bone and soft tissue that most often occurs in children, adolescents, and young adults. Debate and controversy remain in the management of relapsed/refractory ES (RR-ES). The authors leveraged the expertise assembled by the National Ewing Sarcoma Tumor Board, a multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES. In this review, they focus on select topics that apply to the management of patients with RR-ES. The specific topics covered include the initial approach of such patients and discussion of the goals of care, the role of molecular testing, chemotherapy regimens and novel agents to consider, the role of maintenance therapy, and the use of high-dose chemotherapy with autologous stem cell rescue. The data referenced are often limited to subgroup analyses and/or compiled from multiple sources. Although not intended to replace the clinical judgement of treating physicians, these guidelines are intended to support clinicians and provide some clarity and recommendations for the management of patients with RR-ES. PLAIN LANGUAGE SUMMARY: Ewing sarcoma (ES) is a bone and soft tissue cancer that most often occurs in teenagers and young adults. This article uses the experience of the National Ewing Sarcoma Tumor Board, a multi-institution, multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES and to address questions related to the treatment of patients with relapsed ES. Although not intended to replace the clinical judgement of treating physicians and limited by available data, these consensus recommendations will support clinicians who treat patients with this challenging malignancy, made even more difficult when it recurs., (© 2024 The Author(s). Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Background: The outcomes of single-ventricle palliation in unbalanced atrioventricular canal defect with coarctation of aorta (uAVC+CoA) have not been well studied. Systemic ventricle outflow tract obstruction has a propensity to develop in these patients after aortic arch repair with pulmonary artery banding (arch-PAB), which may adversely affect survival and Fontan candidacy., Methods: A retrospective review was performed of patients who underwent single-ventricle palliation for uAVC+CoA from 2000 to 2022. Patients were divided into 2 groups based on initial palliation: (1) arch-PAB and (2) Norwood procedure. Demographic and clinical characteristics were analyzed and compared along with survival data., Results: Stage 1 palliation for uAVC+CoA was performed in 41 patients. Arch-PAB was performed in 14 infants and Norwood in 27 infants. Arch-PAB patients had more chromosomal abnormalities (28.6 vs 7.4%, P < .009) and less severe systemic ventricle outflow tract obstruction on baseline echocardiogram (0.0 vs 70.4%, P < .001). Survival to stage 3 palliation was lower for the arch-PAB group (28.6% vs 66.6%, P = .02). Arch-PAB remained a significant risk factor for mortality (hazard ratio, 2.93; 95% CI, 1.05-8.53; P = .04) after adjusting for chromosomal abnormalities and atrioventricular valve regurgitation. After arch-PAB, systemic ventricle outflow tract obstruction was diagnosed in 13 of 14 patients. Echocardiography underestimated the degree of outflow tract obstruction in 10 of 13 arch-PAB patients., Conclusions: Arch-PAB has worse outcomes than Norwood for uAVC+CoA. Systemic ventricle outflow tract obstruction develops in almost all patients after arch-PAB. Outflow tract obstruction is underestimated by the echocardiogram and requires a high index of suspicion, along with advanced imaging, to ensure timely diagnosis and management., Competing Interests: Disclosures Fawwaz R. Shaw is a member of The Annals of Thoracic Surgery Editorial Board. The other authors have no conflicts of interest to disclose., (Copyright © 2024 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Success of atrioventricular septal defect repair is defined by post-operative atrioventricular valve function and presence of residual intracardiac shunting. We evaluated differences in interpretation of atrioventricular valve function and residual defects between transesophageal and transthoracic echocardiography in a contemporary cohort of infants undergoing atrioventricular septal defect repair. Among 106 patients, we identified an increase in left and right atrioventricular valve regurgitation, right atrioventricular valve inflow gradient, and increased detection rate of residual intracardiac shunting on transthoracic compared to transesophageal echocardiograms, although residual shunts identified only on transthoracic echocardiogram were not haemodynamically significant. Findings may help inform expectation of post-operative transthoracic echocardiogram findings based on intraoperative assessment.

Background: Heart transplantation in the neonatal period is associated with excellent survival. However, outcomes data are scant and have been obtained primarily from two single-center reports within the United States. We sought to analyze the outcomes of all neonatal heart transplants performed in the United States using the United Network for Organ Sharing (UNOS) dataset., Methods: The UNOS dataset was queried for patients who underwent infant heart transplantation from 1987 to 2021. Patients were divided into two groups based on age - neonates (<=31 days), and older infants (32 days-365 days). Demographic and clinical characteristics were analyzed and compared, along with follow up survival data., Results: Overall, 474 newborns have undergone heart transplantation in the United States since 1987. Freedom from death or re-transplantation for neonates was 63.5%, 58.8% and 51.6% at 5, 10, and 20 years, respectively. Patients in the newborn group had lower unadjusted survival compared to older infants (p < .001), but conditional 1-year survival was higher in neonates (p = .03). On multivariable analysis, there was no significant difference in survival between the two age groups (p = .43). Black race, congenital heart disease diagnosis, earlier surgical era, and preoperative mechanical circulatory support use were associated with lower survival among infant transplants (p < .05)., Conclusions: Neonatal heart transplantation is associated with favorable long-term clinical outcomes. Neonates do not have a significant survival advantage over older infants. Widespread applicability is limited by the small number of available donors. Efforts to expand the donor pool to include non-standard donor populations ought to be considered., (© 2024 Wiley Periodicals LLC.)

Background: Macrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo., Methods: Macrophages were derived from human peripheral blood monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors., Results: We found that MAG significantly increased macrophage phagocytosis of ES cells in vitro (p < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors., Conclusions: By turning "two keys" simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES., (© 2024. The Author(s).)

Ewing sarcoma (ES) is a malignant tumor of bone and soft tissue that most often occurs in adolescents and young adults. Despite an international coordinated approach, several nuances, discrepancies, and debates remain in defining the standard of care for treating ES. In this review, the authors leverage the expertise assembled by formation of the National Ewing Sarcoma Tumor Board, a multi-institution, multidisciplinary virtual tumor board that meets monthly to discuss complicated and challenging cases of ES. This report is focused on select topics that apply to the management of patients with newly diagnosed ES. The specific topics covered include indications for bone marrow aspirate and biopsy for initial evaluation compared with fluorodeoxyglucose-positron emission tomography, the role of interval compressed chemotherapy in patients aged 18 years and older, the role of adding ifosfamide/etoposide to vincristine/doxorubicin/cyclophosphamide for patients with metastatic disease, the data on and role of high-dose chemotherapy with autologous stem cell transplantation, maintenance therapy, and whole-lung irradiation. The data referenced are often limited to subgroup analyses and/or compiled from multiple sources. Although not intended to replace the clinical judgement of treating physicians, the guidelines are intended to provide clarity and recommendations for the upfront management of patients with ES. PLAIN LANGUAGE SUMMARY: Ewing sarcoma is a malignant tumor of bone and soft tissue that most often occurs in adolescents and young adults. For this review, the authors used the experience of the National Ewing Sarcoma Tumor Board, a multi-institution, multidisciplinary virtual tumor board that meets monthly to discuss complicated and challenging cases of Ewing sarcoma. Although not intended to replace the clinical judgement of treating physicians, the guidelines will focus on the development of consensus statements for the upfront management of patients with Ewing sarcoma., (© 2023 American Cancer Society.)

Background: We aim to identify the incidence and timing of dysfunction and failure of stented bioprosthetic valves in the pulmonary position in congenital heart disease patients., Methods: A total of 482 congenital heart disease patients underwent 484 stented bioprosthetic pulmonary valve implantations between 2008 and 2018. There were 164 porcine valves (Porcine) and 320 bovine pericardial valves (Pericardial) implanted. Primary endpoints were survival, valve dysfunction, and valve failure., Results: Pericardial valves were implanted in older patients (22.0, interquartile range [IQR] 14-33 vs 16.0, IQR 11-23 years, P < 0.001). Five-year survival (96.7% vs 97.9%) for the Pericardial and Porcine groups, respectively, were similar, P > 0.05. Forty-six (34%) Porcine and 75 (27%) Pericardial group patients met criteria for valve dysfunction at a median echocardiographic follow-up time of 7.43 years (IQR 4.1-9.5 years) and 3.26 years (IQR 1.7-4.7 years), respectively. More Pericardial group patients suffered from at least mild late PR while late median peak gradient was higher in the Porcine group, P < .001 for both. Risk factors for valve dysfunction included decreasing patient age for the entire cohort (hazard ratio [HR] 1.02, 95% confidence interval [CI] 1.00-1.04, P = .015) and lack of anticoagulation at discharge for the Porcine group (HR 3.06, 95% CI 1.03-9.10, P = .044) but not the Pericardial group. Five-year cumulative incidence of dysfunction was 39% for the Pericardial group and 17% for the Porcine group., Conclusions: Porcine stented and bovine pericardial stented valves can be implanted in the pulmonary position in all age groups safely. However, despite similar rates of valve failure, bovine pericardial stented valves have a higher incidence of valve dysfunction at mid-term follow-up., (Copyright © 2023 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Pain following surgery for cardiac disease is ubiquitous, and optimal management is important. Despite this, there is large practice variation. To address this, the Paediatric Acute Care Cardiology Collaborative undertook the effort to create this clinical practice guideline., Methods: A panel of experts consisting of paediatric cardiologists, advanced practice practitioners, pharmacists, a paediatric cardiothoracic surgeon, and a paediatric cardiac anaesthesiologist was convened. The literature was searched for relevant articles and Collaborative sites submitted centre-specific protocols for postoperative pain management. Using the modified Delphi technique, recommendations were generated and put through iterative Delphi rounds to achieve consensus., Results: 60 recommendations achieved consensus and are included in this guideline. They address guideline use, pain assessment, general considerations, preoperative considerations, intraoperative considerations, regional anaesthesia, opioids, opioid-sparing, non-opioid medications, non-pharmaceutical pain management, and discharge considerations., Conclusions: Postoperative pain among children following cardiac surgery is currently an area of significant practice variability despite a large body of literature and the presence of centre-specific protocols. Central to the recommendations included in this guideline is the concept that ideal pain management begins with preoperative counselling and continues through to patient discharge. Overall, the quality of evidence supporting recommendations is low. There is ongoing need for research in this area, particularly in paediatric populations.

Objective: Surgery for heart defects in children with trisomy 13 or 18 is controversial. We analyzed our 20-year experience., Methods: Since 2002, we performed 21 operations in 19 children with trisomy 13 (n = 8) or trisomy 18 (n = 11). Age at operation was 4 days to 12 years (median, 154 days). Principal diagnosis was ventricular septal defect in 10 patients, tetralogy of Fallot in 7 patients, arch hypoplasia in 1 patient, and patent ductus arteriosus in 1 patient., Results: The initial operation was ventricular septal defect closure in 9 patients, tetralogy of Fallot repair in 7 patients, pulmonary artery banding in 1 patient, patent ductus arteriosus ligation in 1 patient, and aortic arch/coarctation repair in 1 patient. There were no operative or hospital deaths. Median postoperative intensive care and hospital stays were 189 hours (interquartile range, 70-548) and 14 days (interquartile range, 8.0-37.0), respectively, compared with median hospital stays in our center for ventricular septal defect repair of 4.0 days and tetralogy of Fallot repair of 5.0 days. On median follow-up of 17.4 months (interquartile range, 6.0-68), 1 patient was lost to follow-up after 5 months. Two patients had reoperation without mortality. There have been 5 late deaths (4 with trisomy 18, 1 with trisomy 13) predominately due to respiratory failure from 4 months to 9.4 years postoperatively. Five-year survival was 66.6% compared with 24% in a group of unoperated patients with trisomy 13 or 18., Conclusions: Cardiac operation with an emphasis on complete repair can be performed safely in carefully selected children with trisomy 13 or trisomy 18. Hospital resource use measured by postoperative intensive care and hospital stays is considerably greater compared with nontrisomy 13 and 18., (© 2022 The Author(s).)

Objectives: Readmission within 30 days of discharge after coronary artery bypass grafting is a measure of quality and a driver of cost in health care. Traditional predictive models use time-independent variables. We developed a new model to predict time to readmission after coronary artery bypass grafting using both time-independent and time-dependent preoperative and perioperative data., Methods: Adults surviving to discharge after isolated coronary artery bypass grafting at a multi-hospital academic health system from January 2017 to September 2018 were included in this study. Two distinct data sources were used: the institutional cardiac surgical database and the clinical data warehouse, which provided more granular data points for each patient. Patients were divided into training and validation sets in an 80:20 ratio. We evaluated 82 potential risk factors using Cox survival regression and machine learning techniques. The area under the receiver operating characteristic curve was used to estimate model predictive accuracy., Results: We trained the model with 21 variables that scored a P value of less than .05 in the univariable analysis. The multivariable model determined 16 significant risk factors, and 6 of them were time-dependent. These included preoperative hemoglobin a1c level, preoperative creatinine, preoperative hematocrit, intraoperative hemoglobin, postoperative creatinine, and postoperative hemoglobin. Area under the receiver operating characteristic values were 0.906 and 0.868 for training and validation sets, respectively., Conclusions: Time-dependent perioperative variables in an isolated coronary artery bypass grafting cohort provided better predictive ability to a readmission model. This study was unique in the inclusion of time-dependent covariates in the predictive model for readmission after discharge after coronary artery bypass grafting., (Copyright © 2020 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Children with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority of these solid tumors highly express GD2. Dinutuximab, an anti-GD2 monoclonal antibody, significantly improved event-free survival in children with GD2 + NB post autologous stem cell transplantation and enhanced natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity. Thus, approaches to increase NK cell number and activity, improve persistence and trafficking, and enhance tumor targeting may further improve the clinical benefit of dinutuximab. N-803 is a superagonist of an interleukin-15 (IL-15) variant bound to an IL-15 receptor alpha Su-Fc fusion with enhanced biological activity., Methods: The anti-tumor combinatorial effects of N-803, dinutuximab and ex vivo expanded peripheral blood NK cells (exPBNK) were performed in vitro using cytoxicity assays against GD2 + OS, NB and GBM cells. Perforin and interferon (IFN)-γ levels were measured by ELISA assays. Multiple cytokines/chemokines/growth factors released were measured by multiplex assays. Human OS, GBM or NB xenografted NOD/SCID/IL2rγnull (NSG) mice were used to investigate the anti-tumor combinatorial effects in vivo., Results: N-803 increased the viability and proliferation of exPBNK. The increased viability and proliferation are associated with increased phosphorylation of Stat3, Stat5, AKT, p38MAPK and the expression of NK activating receptors. The combination of dinutuximab and N-803 significantly enhanced in vitro cytotoxicity of exPBNK with enhanced perforin and IFN-γ release against OS, GBM and NB. The combination of exPBNK+N-803+dinutuximab significantly reduced the secretion of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), platelet-derived growth factor-BB (PDGF-BB), and stem cell growth factor beta (SCGF-β) from OS or GBM tumor cells. Furthermore, OS or GBM significantly inhibited the secretion of regulated on activation, normal T cell expressed and presumably secreted (RANTES) and stromal cell-derived factor-1 alpha (SDF-1α) from exPBNK cells (p<0.001) but significantly enhanced monokine induced by gamma interferon (MIG) secretion from exPBNK cells (p<0.001). N-803 combined with dinutuximab and exPBNK cells significantly extended the survival of OS, GBM or NB xenografted NSG mice., Conclusions: Our results provide the rationale for the development of a clinical trial of N-803 in combination with dinutuximab and ex vivo exPBNK cells in patients with recurrent or metastatic GD2 + solid tumors., Competing Interests: Competing interests: MC serves as a consultant for Jazz, Omeros and Novartis and a Speakers Bureau for Jazz, Servier, Amgen, Sanofi and Sobi. DL reports personal fees and other from Kiadis Pharma, CytoSen Therapeutics, Courier Therapeutics, and Caribou Biosciences outside the submitted work; In addition, DL has a patent broadly related to NK cell therapy of cancer with royalties paid to Kiadis Pharma. PS-S is a majority shareholder of ImmunityBio, Inc. related to N-803. JTS is an employee of NantKwest, an affiliated company to ImmunityBio, Inc related to N-803. Other co-authors declare they have no conflict of interest., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Both completeness of revascularization and multiple arterial grafts (multiple arterial coronary artery bypass grafting) have been associated with increased midterm survival after coronary artery bypass grafting. The purpose of this study was to evaluate the relative impact of completeness of revascularization and multiple arterial coronary artery bypass grafting on midterm survival after coronary artery bypass grafting., Methods: A retrospective review of 17,411 isolated, primary coronary artery bypass grafting operations from January 2002 to June 2016 at a US academic institution was performed. Patients were divided into groups based on complete or incomplete revascularization and number of arterial grafts. Inverse probability of treatment weighting based on the generalized propensity score was performed to minimize imbalance in preoperative characteristics. Between-group differences in outcomes were assessed using multivariable logistic and Cox regression analyses, incorporating the propensity score weights., Results: Patients undergoing multiple arterial coronary artery bypass grafting in this study were younger, had fewer comorbid conditions, and had lower incidence of left main stenosis compared with patients undergoing single-arterial coronary artery bypass grafting. Short-term perioperative outcomes were similar between groups once propensity score weighting was used to minimize between-group differences in preoperative variables. Median follow-up in the entire population was 630 days, but was 1366 days in the cohort with data available from the Social Security Death Index. Multiple arterial coronary artery bypass grafting was protective for midterm survival compared with single arterial coronary artery bypass grafting, regardless of complete or incomplete revascularization or strategy (multiple arterial complete revascularization vs single-arterial complete revascularization: hazard ratio, 0.82; 95% confidence interval, 0.69-0.97; P = .02; multiple arterial incomplete revascularization vs single-arterial incomplete revascularization: hazard ratio, 0.70; 95% confidence interval, 0.53-0.90; P = .007)., Conclusions: After controlling for preoperative comorbidities, multiple arterial coronary artery bypass grafting provides a modest midterm survival benefit over single-arterial coronary artery bypass grafting irrespective of completeness of revascularization, suggesting that when forced to choose, surgeons may elect to pursue multiple arterial conduits., (Copyright © 2019 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Pulmonary complications often cause morbidity and mortality in pediatric allogeneic hematopoietic stem cell transplant (HSCT) recipients. While detection of infection and initiation of appropriate antimicrobial therapy improves survival, present techniques oftentimes do not detect infections in bronchoalveolar lavage (BAL) samples because of pretreatment with antimicrobial therapies and the need for a priori knowledge of likely viral pathogens, decreasing the yield of BAL., Objective: We evaluated whether RNA-based massively parallel sequencing (MPS) would improve detection of infections in BAL fluid in pediatric allogeneic HSCT recipients., Results: Nine patients underwent 10 BAL (1 patient underwent 2 BAL) and had sufficient BAL fluid for inclusion in this study. Clinical microbiological testing identified infections in 7 patients, and MPS identified infections in 5 patients, although some of these detected organisms were not detected by clinical testing. Results were fully concordant in 5 patients, fully discordant in 3 patients, and partially discordant in 2 patients. Bacterial, viral, and fungal infections were detected via both techniques., Conclusion: This suggests that MPS in conjunction with routine clinical testing increases the yield of detection of infectious organisms in the BAL fluid., (© 2021 S. Karger AG, Basel.)

Background: The prognosis of patients with relapsed or progressive B cell (CD20 + ) non-Hodgkin's lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemoradiotherapy resistance. Novel therapeutic strategies are urgently needed. N-820 is a fusion protein of N-803 (formerly known as ALT-803) to four single-chains of rituximab. This agent has tri-specific binding activity to CD20 and enhanced antibody-dependent cell-mediated cytotoxicity., Methods: We investigated the anti-tumor combinatorial effects of N-820 with ex vivo expanded peripheral blood natural killer (exPBNK) cells against rituximab-sensitive and rituximab-resistant CD20 + BL in vitro using cytoxicity assays and in vivo using human BL xenografted NOD/SCID/IL2rγnull (NSG) mice. We also investigated the cytokines/chemokines/growth factors released using ELISA and multiplex assay. Gene expression changes were examined using real-time PCR arrays., Results: N-820 significantly enhanced the expression of NK activating receptors (p<0.001) and the proliferation of exPBNK cells with enhanced Ki67 expression and Stat5 phosphorylation (p<0.001). N-820 significantly enhanced the secretion of cytokines, chemokines, and growth factors including GM-CSF, RANTES, MIP-1B (p<0.001) from exPBNK cells as compared with the combination of rituximab+N-803. Importantly, N-820 significantly enhanced in vitro cytotoxicity (p<0.001) of exPBNK with enhanced granzyme B and IFN-γ release (p<0.001) against BL. Gene expression profiles in exPBNK stimulated by N-820+Raji-2R showed enhanced transcription of CXCL9 , CXCL1 , CSF2 , CSF3 , GZMB, and IFNG . Moreover, N-820 combined with exPBNK significantly inhibited rituximab-resistant BL growth (p<0.05) and extended the survival (p<0.05) of BL xenografted NSG mice., Conclusions: Our results provide the rationale for the development of a clinical trial of N-820 alone or in combination with endogenous or ex vivo expanded NK cells in patients with CD20 + B-NHL failing prior rituximab containing chemoimmunotherapy regimens., Competing Interests: Competing interests: DL reports personal fees and other from Kiadis Pharma, Courier Therapeutics, and Caribou Biosciences outside the submitted work; In addition, DL has a patent broadly related to NK cell therapy of cancer with royalties paid to Kiadis Pharma. PS-S is a majority shareholder of ImmunityBio, Inc. and Altor BioScience, LLC, related to N-820. JTS is an employee of NantKwest, an affiliated company to ImmunityBio, Inc and Altor BioScience, LLC, related to N-820. Othe co-authors declare they have no conflict of interest., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Ewing sarcoma (ES) is a malignant pediatric bone and soft tissue tumor. Patients with metastatic ES have a dismal outcome which has not been improved in decades. The major challenge in the treatment of metastatic ES is the lack of specific targets and rational combinatorial therapy. We recently found that protein phosphatase 1 regulatory subunit 1A (PPP1R1A) is specifically highly expressed in ES and promotes tumor growth and metastasis in ES. In the current investigation, we show that PPP1R1A regulates ES cell cycle progression in G1/S phase by down-regulating cell cycle inhibitors p21 Cip1 and p27 Kip1 , which leads to retinoblastoma (Rb) protein hyperphosphorylation. In addition, we show that PPP1R1A promotes normal transcription of histone genes during cell cycle progression. Importantly, we demonstrate a synergistic/additive effect of the combinatorial therapy of PPP1R1A and insulin-like growth factor 1 receptor (IGF-1R) inhibition on decreasing ES cell proliferation and migration in vitro and limiting xenograft tumor growth and metastasis in vivo . Taken together, our findings suggest a role of PPP1R1A as an ES specific cell cycle modulator and that simultaneous targeting of PPP1R1A and IGF-1R pathways is a promising specific and effective strategy to treat both primary and metastatic ES., Competing Interests: CONFLICTS OF INTEREST S.L.L. declares a conflicts of interest as a member of the advisory board for Salarius Pharmaceuticals, and is also a listed inventor on United States Patent No. US 7939253B2, “Methods and compositions for the diagnosis and treatment of Ewing’s sarcoma”. S.L.L. and W.L. are also listed inventors on United States Patent No. US 8557532, “Diagnosis and treatment of drug-resistant Ewing’s sarcoma.”, (Copyright: © 2020 Luo et al.)

Objectives: Primary pulmonary vein stenosis (PPVS) is increasingly diagnosed in children with no prior pulmonary vein intervention history, and management is challenging. We describe characteristics of patients who underwent surgical repair of PPVS at our center, and examine factors associated with treatment failures., Methods: A retrospective review of all patients who underwent surgical intervention for PPVS (2002-2016) was completed. Patients who had undergone prior cardiac surgery involving the pulmonary veins or atrial switch were excluded. Regression analyses were performed to examine characteristics, PPVS features, including severity score, and surgical details associated with treatment failures., Results: Thirty-four children underwent initial surgical intervention for PPVS. Median age was 8.9 months (interquartile range, 5.9-18.4 months). Most patients (n = 31; 91%) had unilateral pulmonary vein involvement and the median PPVS severity score was 3.5 (interquartile range, 3-5). On competing risk analysis, 1 year following surgical repair, 9% of patients had died, 14% had undergone reintervention, and 77% were alive without reintervention; at 5 years the numbers were 9%, 30%, and 61%, respectively. Factors associated with mortality included bilateral disease and PPVS severity score >6. Bilateral disease and PPVS severity score >5 were associated with reintervention risk., Conclusions: Multidisciplinary management strategy is required for PPVS. Despite satisfactory early repair, patients continue to be at risk for recurrence and subsequent mortality, especially those with extensive primary involvement. The disappointing results underscore the need for multi-institutional collaborations to better understand this complex disease, establish management and follow-up protocols, and explore investigational treatment modalities that could modify the unfavorable outcome of this uncommon and challenging disease., (Copyright © 2019 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Objective: Quality metrics and reimbursement models focus on 30-day readmission rates after coronary artery bypass grafting (CABG). Certain preoperative variables are associated with higher rates of readmission. The purpose of this study was to determine whether STS Predicted Risk of Mortality (PROM) scores predict 30-day readmission following CABG., Methods: A retrospective review of all patients undergoing isolated CABG between 2002 and 2017 at a US academic institution was performed. Logistic regression analysis was used to determine the association between PROM and 30-day readmission, and the area under the receiver-operator curve (ROC) was calculated to estimate predictive accuracy., Results: During the study period, 21,719 patients underwent CABG and 2,023 (9.2%) were readmitted within 30 days. Readmitted patients were sicker with higher rates of comorbid conditions and higher STS PROM scores (1.03% vs 1.42%, GMR 1.33, CI 1.27-1.38, p < 0.0001). Median time to readmission was 8 days (IQR 4-15) with length of stay 5 days (4-6). By PROM quintile, higher PROM scores were associated with increased odds of readmission. PROM-adjusted 30-day mortality was higher in the readmitted group (1.04% vs 0.21%, OR 4.53, CI 2.67-7.69, p < 0.001), and mid-term survival was worse as well. PROM alone was a modest predictor of readmission (area under ROC 0.59, CI 0.57-0.60) compared to insurance status (0.55, 0.53-0.56), ejection fraction (0.52, 0.50-0.54), and history of heart failure (0.51, 0.50-0.52)., Conclusion: STS PROM scores are associated with increased risk of readmission following CABG.

Successful repair of complex thoracoabdominal aortic aneurysms requires careful surgical planning based on anatomic and patient considerations. Not only are surgical considerations key, but also post-operative care, regardless of surgical approach, can dramatically impact both short- and long-term outcomes. While open repair has been the gold standard for decades, the technical challenges associated with operative repair, a specialty approach requisite for good outcomes, and the unique challenges in the post-operative care of these patients have given providers pause when considering operative intervention. The relatively recent development of elegant endovascular and hybrid approaches to this problem has shown improved short-term morbidity and reasonable durability. Here, we discuss these three techniques for correction of complex thoracoabdominal aortic aneurysms to provide some guidance for optimization of outcomes based on individual patient anatomy and comorbid conditions.

Background: Valve-sparing root replacement (VSRR) is an attractive option in type A aortic dissection (TAAD) repair for a young patient with normal cusp anatomy, but conventional root replacement using a composite valved-conduit (ROOT) remains the gold standard in this emergent clinical setting. We examine the long-term safety and durability of the David V VSRR compared with ROOT in TAAD repair., Methods: From March 2004 to April 2017, 136 patients underwent repair of acute TAAD using either ROOT (n = 77; 56.6%) or VSRR (n = 59; 43.4%). Annual echocardiograms were performed for follow-up in VSRR patients. Univariable regression, Kaplan-Meier, and competing risk analyses were performed., Results: Preoperative characteristics were similar between groups, except that VSRR patients were younger (mean age 43.5 ± 11.4 years VSRR vs 50.4 ± 3.0 years ROOT; P = .001). Both groups had similar rates of preoperative malperfusion or shock (29.3% VSRR vs 37.0% ROOT; P = .35) and ≥3+ aortic insufficiency (63% VSRR vs 76.8% ROOT). Thirty-day mortality in the VSRR group was 2/59 (3.4%) and 11/77 in the ROOT group (14.3%; P < .001). All-cause survival at 9 years was 92% (VSRR) and 59% (ROOT; P = .002). The incidence of aortic reintervention was similar between groups (20%-23% at 5 years; P = .81). At 9 years of follow-up, 5/52 (9.6%) VSRR patients had ≥2+ aortic insufficiency, and 1 patient required valve reintervention., Conclusions: In highly-selected patients, the David V VSRR provides a safe repair of acute TAAD with concomitant root pathology and valve insufficiency. In our center, the incidence of valve-related reintervention at long-term follow-up is low after emergent repair., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Protein phosphatase inhibitors are often considered as tumor promoters. Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) is a potent protein phosphatase 1 (PP1) inhibitor; however, its role in tumor development is largely undefined. Here we characterize, for the first time, the functions of PPP1R1A in Ewing sarcoma (ES) pathogenesis. We found that PPP1R1A is one of the top ranked target genes of EWS/FLI, the master regulator of ES, and is upregulated by EWS/FLI via a GGAA microsatellite enhancer element. Depletion of PPP1R1A resulted in a significant decrease in oncogenic transformation and cell migration in vitro as well as xenograft tumor growth and metastasis in an orthotopic mouse model. RNA-sequencing and functional annotation analyses revealed that PPP1R1A regulates genes associated with various cellular functions including cell junction, adhesion and neurogenesis. Interestingly, we found a significant overlap of PPP1R1A-regulated gene set with that of ZEB2 and EWS, which regulates metastasis and neuronal differentiation in ES, respectively. Further studies for characterization of the molecular mechanisms revealed that activation of PPP1R1A by PKA phosphorylation at Thr35, and subsequent PP1 binding and inhibition, was required for PPP1R1A-mediated tumorigenesis and metastasis, likely by increasing the phosphorylation levels of various PP1 substrates. Furthermore, we found that a PKA inhibitor impaired ES cell proliferation, tumor growth and metastasis, which was rescued by the constitutively active PPP1R1A. Together, these results offered new insights into the role and mechanism of PPP1R1A in tumor development and identified an important kinase and phosphatase pathway, PKA/PPP1R1A/PP1, in ES pathogenesis. Our findings strongly suggest a potential therapeutic value of inhibition of the PKA/PPP1R1A/PP1 pathway in the treatment of primary and metastatic ES.

Background: The Adult Congenital Heart Association (ACHA) is dedicated to supporting patients with congenital heart disease. To guide patients to qualified providers and programs, it maintains a publicly accessible directory of dedicated adult congenital cardiac programs. We analyzed the directory in 2006 and 2015, aiming to evaluate the growth of the directory as a whole and to evaluate the growth of individual programs within the directory. We also hope this raises awareness of the growing opportunities that exist in adult congenital cardiology and cardiac surgery., Methods: Data in the directory are self-reported. Only data from US programs were collected and analyzed., Results: By the end of 2015, compared to 2006, there were more programs reporting to the directory in more states (107 programs across 42 states vs 57 programs across 33 states), with higher overall clinical volume (591 vs 164 half-day clinics per week, 96,611 vs 34,446 patient visits). On average, each program was busier (5 vs 2 half-day clinics per week per program). Over the time period, the number of reported annual operations performed nearly doubled (4,346 operations by 210 surgeons vs 2,461 operations by 125 surgeons). Access to ancillary services including specific clinical diagnostic and therapeutic services also expanded., Conclusion: Between 2006 and 2015, the clinical directory and the individual programs have grown. Current directory data may provide benchmarks for staffing and services for newly emerging and existing programs. Verifying the accuracy of the information and inclusion of all programs will be important in the future.

Background: Adult patients with CHD often require complex operations, and indications for particular aspects of the operation are sometimes unclear. The aims of our study were as follows: to characterise concomitant procedures performed during adult congenital cardiac surgery, and to better define the risk involved with performing concomitant procedures during a single operation., Methods: We retrospectively studied 458 adult congenital cardiac surgical patients. Major procedures were characterised as aortic, mitral, pulmonary, tricuspid, septal defect, single ventricle, transplant, and others. We constructed logistic regression models to assess the risk for mortality, major adverse event, and prolonged length of stay., Results: A total of 362 operations involved a single major procedure, whereas 96 involved concomitant procedures. Performing concomitant procedures increased mortality (7.3 versus 2.5%), major adverse events (21.8 versus 14.9%), and prolonged length of stay (29.2 versus 17.1%). The added risks of concomitant procedures on mortality, major adverse event, and prolonged length of stay were 2.9 (95% CI 1.0-8.5, p=0.05), 1.9 (95% CI 1.1-3.3, p=0.02), and 2.4 (95% CI 1.4-4.1, p=0.003), respectively. There were 200 patients with conotruncal anomalies who underwent pulmonary valve surgery. In this subset, the added risks of concomitant procedures in addition to pulmonary valve surgery on mortality, major adverse events, and prolonged length of stay were 6.6 (95% CI 1.2-37.3, p=0.03), 2.8 (95% CI 1.2-6.6, p=0.03), and 3.3 (95% CI 1.5-7.4, p=0.005), respectively., Conclusion: Concomitant procedures performed during adult congenital heart surgery increase risk. Awareness of this risk may improve surgical decision making and outcomes.

Objective: Hybrid coronary revascularization (HCR) combines minimally invasive left internal mammary artery (LIMA)-left anterior descending artery (LAD) bypass with percutaneous intervention of non-LAD vessels. The purpose of this study was to compare outcomes of HCR to conventional coronary artery bypass graft (CABG) surgery with single internal mammary artery (SIMA) or bilateral internal mammary artery (BIMA) grafting., Methods: Between October 2003 and September 2013, 306 consecutive patients who underwent HCR were compared with 8254 patients who underwent CABG with SIMA (7381; 89.4%) or BIMA (873; 10.6%) at a US academic center. The primary outcome was a composite of 30-day death, myocardial infarction, and stroke (major cerebrovascular and cardiac event [MACCE]). In addition to multiple logistic and linear regression analysis, a propensity score-matched analysis was used to compare HCR with SIMA and with BIMA., Results: The Society of Thoracic Surgeons-predicted risk of mortality was 1.6% for HCR, 2.1% for SIMA, and 1.1% for BIMA (P < .001). Factors associated with HCR use were older age, lower body mass index, history of percutaneous coronary intervention, and 2-vessel disease. In propensity-matched groups, 30-day MACCE rates were comparable (3.3% for HCR vs 1.3% for BIMA [odds ratio (OR), 2.50; P = .12] and vs 3.6% for SIMA [OR, 1.00; P = 1.00]). In-hospital complications were lower after HCR versus SIMA or BIMA (OR, 0.59; P = .033 and OR, 0.55; P = .015, respectively), as was the need for blood transfusion (OR, 0.44; P < .001 and OR, 0.57; P < .001). HCR was associated with shorter hospital stay compared with SIMA (OR, 1.28; P = .038) or BIMA (OR, 1.40; P = .006). No survival difference between matched groups was found at midterm follow-up (HCR vs SIMA: hazard ratio [HR], 0.66; 95% confidence interval [CI], 0.32-1.38; P = .66; HCR vs BIMA: HR, 1.05; 95% CI, 0.48-2.29; P = .91)., Conclusions: HCR may represent a safe, less invasive alternative to conventional CABG in carefully selected patients, with similar short-term and midterm outcomes as CABG performed with either SIMA or BIMA grafting., (Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

An increasing number of patients with congenital heart disease are surviving into adulthood, and many will require repeat surgical intervention. Those patients with tetralogy of Fallot likely represent the largest group. Of the clinically important complications that affect adults with repaired tetralogy of Fallot, pulmonary regurgitation is the most common. Without intervention, pulmonary regurgitation results in dilation of the right ventricle and may lead to additional complications, such as biventricular dysfunction, arrhythmias, heart failure, and death. Pulmonary valve replacement (PVR) is a means to ameliorate these adverse sequelae and is the most common reoperation performed in adults with congenital heart disease. Although still controversial, much has been written on the indications for and timing of PVR. This article examines some of the less-reported issues surrounding PVR in repaired tetralogy of Fallot, including contemporary surgical outcomes, the role of the percutaneous and mechanical valve, and the role of concomitant aortic and tricuspid operations during PVR., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Osteosarcoma is the most common primary malignant bone tumor in children. Validated biological markers for disease prognosis available at diagnosis are lacking. No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma to the best of our knowledge. To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes. At more than 17% of the tested loci, samples obtained from patients who experienced disease relapse were more methylated than those from patients who did not have recurrence while patients who did not experience disease relapse had more DNA methylation at fewer than 1%. In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation with 6.6% of gene promoter loci being more methylated and 2% of promoter loci being less methylated in patients with disease relapse. A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and 5 y event-free survival (P-value = 1.7 × 10(-6)), with empirical annotation of this locus showing promoter characteristics. Our data indicate that DNA methylation information has the potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies.

Recipient CD4 T regulatory cells inhibit the acute T cell-mediated rejection of renal allografts in wild-type mice. The survival of single class II MHC-disparate H-2(bm12) renal allografts was tested in B6.CCR5(-/-) recipients, which have defects in T regulatory cell activities that constrain alloimmune responses. In contrast to wild-type C57BL/6 recipients, B6.CCR5(-/-) recipients rejected the bm12 renal allografts. However, donor-reactive CD8 T cells rather than CD4 T cells were the primary effector T cells mediating rejection. The CD8 T cells induced to bm12 allografts in CCR5-deficient recipients were reactive to peptides spanning the 3 aa difference in the I-A(bm12) versus I-A(b) β-chains presented by K(b) and D(b) class I MHC molecules. Allograft-primed CD8 T cells from CCR5-deficient allograft recipients were activated during culture either with proinflammatory cytokine-stimulated wild-type endothelial cells pulsed with the I-A(bm12) peptides or with proinflammatory cytokine-simulated bm12 endothelial cells, indicating their presentation of the I-A(bm12) β-chain peptide/class I MHC complexes. In addition to induction by bm12 renal allografts, the I-A(bm12) β-chain-reactive CD8 T cells were induced in CCR5-deficient, but not wild-type C57BL/6, mice by immunization with the peptides. These results reveal novel alloreactive CD8 T cell specificities in CCR5-deficient recipients of single class II MHC renal allografts that mediate rejection of the allografts., (Copyright © 2014 by The American Association of Immunologists, Inc.)

One's cellular immune repertoire is composed of lymphocytes in multiple stages of maturation - the dynamic product of their responses to antigenic challenges and the homeostatic contractions necessary to accommodate immune expansions within physiologic norms. Given that alloreactivity is predominantly a cross-reactive phenomenon that is stochastically distributed throughout the overall T-cell repertoire, one's allospecific repertoire is similarly made up of cells in a variety of differentiation states. As such, the continuous expansion and elimination of activated memory populations, producing a 'recollective homeostasis' of sorts, has the potential over time to alter the maturation state and effector composition of both ones protective and alloreactive T-cell repertoire. Importantly, a T cell's maturation state significantly influences its response to numerous immunomodulatory therapies used in organ transplantation, including depletional antibody induction. In this review, we discuss clinically utilized depletional induction strategies, how their use alters a transplant recipient's cellular immune repertoire, and how a recipient's repertoire influences the clinical effects of induction therapy., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Donor Ag-reactive CD4 and CD8 T cell production of IFN-gamma is a principal effector mechanism promoting tissue injury during allograft rejection. The CXCR3-binding chemokines CXCL9 and CXCL10 recruit donor-reactive T cells to the allograft, but their role during the priming of donor-reactive T cells to effector function is unknown. Using a murine model of MHC-mismatched cardiac transplantation, we investigated the influence of CXCL9 and CXCL10 during donor-reactive T cell priming. In allograft recipient spleens, CXCL9 and CXCL10 were expressed as early as 24 h posttransplant and increased with similar kinetics, concurrently with CXCR3 expression on T cells. CXCL9, but not CXCL10, expression required NK cell production of IFN-gamma. The absence of CXCL9 in donor allografts, recipients, or both significantly decreased the frequency of donor-reactive CD8 T cells producing IFN-gamma and increased the frequency of donor-reactive CD8 T cells producing IL-17A. In contrast, the absence of CXCL10 increased the frequency of IFN-gamma-producing CD8 T cells in a CXCL9-dependent manner. These data provide novel evidence that donor-reactive CD8 T cells use the CXCR3 chemokine axis as a costimulation pathway during priming to allografts where CXCL9 promotes the development of IFN-gamma-producing CD8 T cells, and CXCL10 antagonizes this skewing.

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2(bm12) cardiac allografts is restricted by CD4(+)CD25(+) regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2(bm12) cardiac allografts. In contrast to the long-term survival of B6.H-2(bm12) allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5(-/-) recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-gamma and IL-4 were markedly increased in spleens of B6.CCR5(-/-) versus wild-type recipients. Wild-type and B6.CCR5(-/-) mice had equivalent numbers of splenic FoxP3(+) Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro. However, diminished numbers of FoxP3(+) Tregs infiltrated B6.H-2(bm12) allografts in B6.CCR5(-/-) recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4(+)CD25(+) Tregs to CCR5(-/-) recipients restored long-term survival of B6.H-2(bm12) cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.

Chemokines, including monokine induced by interferon-gamma (Mig/CXCL9), are produced both in allografts and during the direct T-cell infiltration that mediates graft rejection. Neither the specific production nor contribution of allograft donor versus recipient Mig in allograft rejection is currently known. C57BL/6 mice with a targeted deletion in the Mig gene were used as both skin allograft donors and recipients in a class II major histocompatibility complex-mismatched graft model to test the requirement for donor- versus recipient-derived Mig for acute rejection. B6.Mig(-/-) allografts had a 10-day prolonged survival in B6.H-2(bm12) recipients when compared with wild-type C57BL/6 allograft donors, and B6.H-2(bm12) skin allografts had a 5-day prolonged survival in B6.Mig(-/-) versus wild-type recipients. Transplantation of B6.Mig(-/-) skin grafts onto B6.H-2(bm12).Mig(-/-) recipients resulted in further prolonged allograft survival with more than 30% of the grafts surviving longer than 60 days. Prolonged allograft survival was also associated with delayed cellular infiltration into grafts but not with altered T-cell proliferative responses to donor stimulators. Immunohistochemical staining of allograft sections indicated that Mig is produced by both donor- and recipient-derived sources, but Mig from each of these sources appeared in different areas of the allograft tissue. These results therefore demonstrate the synergy of donor- and recipient-derived Mig in promoting T-cell infiltration into allografts.

Background: Current immunosuppression regimens are toxic to transplant recipients and, in many cases, acute rejection episodes occur because of escape of donor-reactive lymphocytes from the immunosuppression. T cells are the mediators of acute, cell-mediated graft damage and are hypothesized to use the CXCR3 chemokine axis for migration into the allograft. This study investigates the effect of CXCR3 blockade using a nonpeptide, small molecule inhibitor, AMG1237845, in murine cardiac allograft survival., Methods: C57BL/6 (H-2) mice received vascularized cardiac allografts from A/J (H-2) donors and were treated with the CXCR3 antagonist. Histologic and flow cytometric analyses were used to measure infiltration of leukocytes, and quantitative reverse-transcriptase polymerase chain reaction and interferon-gamma ELISPOT assays were used to measure donor-specific reactivity., Results: CXCR3 antagonism modestly prolonged allograft survival compared with vehicle treatment, but at time-matched intervals posttransplant, neutrophil, CD8, and CD4 T cell infiltration was indistinguishable. Although proliferation of donor-reactive naïve T cells was unaffected by CXCR3 antagonism, the frequency of interferon-gamma-producing cells in the recipient spleen was significantly reduced by AMG1237845 treatment. CXCR3 blockade for 30 days synergized with short-term, low-dose anti-CD154 monoclonal antibodies to prolong survival past 50 days in 75% of grafts and past 80 days in 25% of the cases., Conclusions: These results indicate that in synergy with co-stimulation blockade, CXCR3 is a viable therapeutic target to prevent acute graft rejection.

CD4 T cell-dependent mechanisms promoting allograft rejection include expression of inflammatory functions within the graft and the provision of help for donor-reactive CD8 T cell and Ab responses. These studies tested CD4 T cell-mediated rejection of MHC-mismatched cardiac allografts in the absence of both CD8 T and B lymphocytes. Whereas wild-type C57BL/6 recipients depleted of CD8 T cells rejected A/J cardiac grafts within 10 days, allografts were not rejected in B cell-deficient B6.muMT(-/-) recipients depleted of CD8 T cells. Isolated wild-type C57BL/6 and B6.muMT(-/-) CD4 T cells had nearly equivalent in vivo alloreactive proliferative responses. CD4 T cell numbers in B6.muMT(-/-) spleens were 10% of that in wild-type mice but were only slightly decreased in peripheral lymph nodes. CD8 T cell depletion did not abrogate B6.muMT(-/-) mice rejection of A/J skin allografts and this rejection rendered these recipients able to reject A/J cardiac allografts. Redirection of the alloimmune response to the lymph nodes by splenectomy conferred the ability of B6.muMT(-/-) CD4 T cells to reject cardiac allografts. These results indicate that the low number of splenic CD4 T cells in B6.muMT(-/-) mice underlies the inability to reject cardiac allografts and this inability is overcome by diverting the CD4 T cell response to the peripheral lymph nodes.

A key event during T cell-mediated rejection of allografts is the trafficking of donor antigen-primed effector T cells from the lymphoid tissue to the graft. This trafficking is mediated in part by chemokine produced in the graftengaging receptors on the T cells and other graftinfiltrating leukocytes. The presence of specific sets of chemokines and chemokine receptors is detectable in rejecting allografts. In animal models, allograft rejection is delayed when chemokine-chemokine receptor function is absent or antagonized but cellular infiltration and graft survival eventually occur, suggesting that T cells and other leukocytes use several trafficking mechanisms during rejection. The use of chemokines as footprints of rejection may be of considerable value as noninvasive biomarkers in transplantation.