Faculty Bibliography

The humanized IgG1κ monoclonal antibody siplizumab and its rat parent monoclonal IgG2b antibody BTI-322 are directed against the CD2 antigen. Siplizumab is species-specific, reacting with human and chimpanzee cells but not with cells from any other species, including other non-human primates. Because siplizumab treatment has recently shown great potential in clinical transplantation, we now present the results of our previous pharmacokinetic, pharmacodynamic and safety studies of both antibodies. Fourteen chimpanzees received 1-3 doses of 0.143 to 5.0 mg/kg iv The effects were followed with flow cytometry on peripheral lymphocytes and staining of lymph nodes. Side effects were recorded. Serum antibody concentrations were followed. Across the doses, a rapid, transient depletion of CD2, CD3, CD4 and CD8 lymphocytes and NK cells was observed for both antibodies. Immune reconstitution was more rapid for BTI-322 compared to siplizumab. Paracortical lymph node T cell depletion was moderate, estimated at 45% with doses of >0.6 mg/kg. Restoration of lymph node architecture was seen after two weeks to two months for all animals. All four subjects receiving BTI-322 experienced AEs on the first dosing day, while the eight subjects dosed with siplizumab experienced few mild, transient AEs. Infusion with siplizumab and BTI-322 resulted in rapid depletion of CD2⁺ cells in circulation and tissue. Siplizumab had a longer t_1/2 and fewer AEs compared to BTI-322.

BACKGROUND:Cytomegalovirus (CMV) infection is a serious complication in immunosuppressed patients, specifically transplant recipients. Here, we describe the development and use of an assay to monitor the incidence and treatment of CMV viremia in a Cynomolgus macaque model of bone marrow transplantation (BMT) for tolerance induction. We address the correlation between the course of viremia and immune reconstitution. METHODS:Twenty-one animals received a nonmyeloablative conditioning regimen. Seven received cyclosporine A for 28 days and 14 received rapamycin. A CMV polymerase chain reaction assay was developed and run twice per week to monitor viremia. Nineteen recipients were CMV seropositive before BMT. Immune reconstitution was monitored through flow cytometry and CMV viremia was tracked via quantitative polymerase chain reaction. RESULTS:Recipients developed CMV viremia during the first month post-BMT. Two animals developed uncontrollable CMV disease. CMV reactivation occurred earlier in cyclosporine A-treated animals compared with those receiving rapamycin. Post-BMT, T-cell counts remained significantly lower compared with pretransplant levels until CMV reactivation, at which point they increased during the viremic phase and approached pretransplant levels 3 months post-BMT. Management of CMV required treatment before viremia reached 10 000 copies/mL; otherwise clinical symptoms were observed. High doses of ganciclovir resolved the viremia, which could subsequently be controlled with valganciclovir. CONCLUSIONS:We developed an assay to monitor CMV in Cynomolgus macaques. CMV reactivation occurred in 100% of seropositive animals in this model. Rapamycin delayed CMV reactivation and ganciclovir treatment was effective at high doses. As in humans, CD8 T cells proliferated during CMV viremia.

BACKGROUND:Although short-term outcomes for liver transplantation have improved, patient and graft survival are limited by infection, cancer, and other complications of immunosuppression. Rapid induction of tolerance after liver transplantation would decrease these complications, improving survival and quality of life. Tolerance to kidneys, but not thoracic organs or islets, has been achieved in nonhuman primates and humans through the induction of transient donor chimerism. Since the liver is considered to be tolerogenic, we tested the hypothesis that the renal transplant transient chimerism protocol would induce liver tolerance. METHODS:Seven cynomolgus macaques received immune conditioning followed by simultaneous donor bone marrow and liver transplantation. The more extensive liver surgery required minor adaptations of the kidney protocol to decrease complications. All immunosuppression was discontinued on postoperative day (POD) 28. Peripheral blood chimerism, recipient immune reconstitution, liver function tests, and graft survival were determined. RESULTS:The level and duration of chimerism in liver recipients were comparable to those previously reported in renal transplant recipients. However, unlike in the kidney model, the liver was rejected soon after immunosuppression withdrawal. Rejection was associated with proliferation of recipient CD8 T effector cells in the periphery and liver, increased serum interleukin (IL)-6 and IL-2, but peripheral regulatory T cell (Treg) numbers did not increase. Antidonor antibody was also detected. CONCLUSIONS:These data show the transient chimerism protocol does not induce tolerance to livers, likely due to greater CD8 T cell responses than in the kidney model. Successful tolerance induction may depend on greater control or deletion of CD8 T cells in this model.

Patients awaiting lung transplantation face high wait-list mortality, as injury precludes the use of most donor lungs. Although ex vivo lung perfusion (EVLP) is able to recover marginal quality donor lungs, extension of normothermic support beyond 6 h has been challenging. Here we demonstrate that acutely injured human lungs declined for transplantation, including a lung that failed to recover on EVLP, can be recovered by cross-circulation of whole blood between explanted human lungs and a Yorkshire swine. This xenogeneic platform provided explanted human lungs a supportive, physiologic milieu and systemic regulation that resulted in functional and histological recovery after 24 h of normothermic support. Our findings suggest that cross-circulation can serve as a complementary approach to clinical EVLP to recover injured donor lungs that could not otherwise be utilized for transplantation, as well as a translational research platform for immunomodulation and advanced organ bioengineering.

PURPOSE OF REVIEW:To describe the most recent progress towards tolerance in xenotransplantation. RECENT FINDINGS:Mixed chimerism and thymic transplantation have been used to promote tolerance in xenotransplantation models. Intra-bone bone marrow transplantation is a recent advance for mixed chimerism, which promotes longer lasting chimerism and early graft function of subsequent organ transplantation. The hybrid thymus, an advancement to the vascularized thymokidney and vascularized thymic lobe, is being developed to allow for both donor and recipient T-cell selection in the chimeric thymus, encouraging tolerance to self and donor while maintaining appropriate immune function. Regulatory T cells show promise to promote tolerance by suppressing effector T cells and by supporting mixed chimerism. Monoclonal antibodies such as anti-CD2 may promote tolerance through suppression of CD2+ effector and memory T cells whereas Tregs, which express lower numbers of CD2, are relatively spared and might be used to promote tolerance. SUMMARY:These findings contribute major advances to tolerance in xenotransplantation. A combination of many of these mechanisms will likely be needed to have long-term tolerance maintained without the use of immunosuppression.

BACKGROUND AND AIMS:Ex vivo surgery may provide a chance at R0 resection for conventionally unresectable tumors. However, long-term outcomes have not been well documented. In this study, we analyze our 11-year outcomes to define its role. STUDY DESIGN:We retrospectively analyzed 46 consecutive patients who underwent ex vivo surgery at our institution 2008-2019. RESULTS:The types of tumors were: carcinoma (n = 20), sarcoma (n = 20) and benign to low grade tumor (n = 6). The type of ex vivo surgery was chosen based on tumor location and vascular involvement. The most commonly performed procedure was ex vivo hepatectomy (n = 18), followed by ex vivo resection and intestinal autotransplantation (n = 12), ex vivo Whipple procedure and liver autotransplantation (n = 8) and multivisceral ex vivo procedure (n = 7). Twenty-three patients (50%) are currently alive with median follow-up of 4.0-years (11 months-11.8 years). The overall survival was 70%/59%/52%, at 1-/3-/5-years, respectively. Patient survival for benign to low grade tumors, sarcoma, and carcinoma was 100%/100%/100%, 65%/60%/50%, and 65%/45%/40%, at 1-/3-/5-years, respectively. Ninety-one percent patients had R0 resection, and 57% had no recurrence to date with median follow-up of 3.1-years. Two patients (4.3%) died within 30 days due to sepsis and gastroduodenal artety (GDA) stump blowout. Two additional patients died between 30 and 90 days due to sepsis. Perioperative mortality in the last 23 consecutive cases was limited to 1 patient who died of sepsis between 30 and 90 days. CONCLUSIONS:For a selected group of patients with conventionally unresectable tumors, ex vivo surgery can offer effective surgical removal with a reasonably low perioperative mortality at experienced centers.

We present a case of COVID-19 hepatitis in a living donor liver allograft recipient whose donor subsequently tested positive for COVID-19. The patient is a female infant with biliary atresia (failed Kasai procedure). She recovered well, with improving liver function tests for 4 days. On post-operative day (POD) 4 the patient developed respiratory distress and fever. COVID-19 testing (polymerase chain reaction) was positive. Liver function tests increased approximately 5-fold. Liver biopsy showed moderate acute hepatitis with prominent clusters of apoptotic hepatocytes and associated cellular debris. Lobular lymphohistiocytic inflammation was noted. Typical portal features of mild to moderate acute cellular rejection were also noted.

Human intestinal transplantation often results in long-term mixed chimerism of donor and recipient blood in transplant patients. We followed the phenotypes of chimeric peripheral blood cells in 21 patients receiving intestinal allografts over 5 years. Donor lymphocyte phenotypes suggested a contribution of hematopoietic stem and progenitor cells (HSPCs) from the graft. Surprisingly, we detected donor-derived HSPCs in intestinal mucosa, Peyer's patches, mesenteric lymph nodes, and liver. Human gut HSPCs are phenotypically similar to bone marrow HSPCs and have multilineage differentiation potential in vitro and in vivo. Analysis of circulating post-transplant donor T cells suggests that they undergo selection in recipient lymphoid organs to acquire immune tolerance. Our longitudinal study of human HSPCs carried in intestinal allografts demonstrates their turnover kinetics and gradual replacement of donor-derived HSPCs from a circulating pool. Thus, we have demonstrated the existence of functioning HSPCs in human intestines with implications for promoting tolerance in transplant recipients.

OBJECTIVE:There are multiple approaches to manage the clinical complications of portal hypertension (PHTN) to treat/prevent spontaneous hemorrhage by mitigating thrombocytopenia. No single approach is ideal for all patients given the heterogeneity of this population. Our goal was to determine whether partial splenic embolization (PSE) was safe and effective in the pediatric population. METHODS:This is a retrospective review of our single-center experience for all patients ages 0 to 21 who underwent PSE between January 2010 and August 2017. The embolized splenic volume targeted was 60% to 70%. RESULTS:Twenty-six patients underwent PSE due to thrombocytopenia and/or recurrent variceal bleeding. Patients ranged in age from 18 months to 20 years (mean 13.1 years). The median platelet count before PSE was 53.0 (×10/L). The platelet count improved after PSE with values >100,000 in 21 patients (80.8%). Children with prior esophageal varices showed improvement after PSE with only 9 (34.6%) requiring further endoscopic therapy. After PSE, patients developed transient abdominal pain, distention, fever, and perisplenic fluid collections. Serious complications such as splenic abscess, splenic rupture, bleeding, pancreatic infarction, opportunistic infection, or death were not observed. One patient experienced thrombotic complications after PSE and was later diagnosed with myelodysplastic syndrome. CONCLUSIONS:PSE is a safe and effective alternative in the management of pediatric PHTN in select populations. PSE may be a favorable alternative to splenectomy and portal systemic shunting because it preserves functional spleen mass and avoids postprocedure accelerated liver disease or encephalopathy.