Jane Burgess

Donor-derived CMV specific T-cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation

We investigated the use of adoptively transferred donor-derived cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) as immune reconstitution postallogeneic transplant in a phase 2 study. Fifty patients were infused with a single dose of 2 × 10(7)cells/m(2) after day 28 post-transplant. Twenty-six patients reactivated CMV posttransplant (only 5 post-CTL infusion) and 9 required therapy with ganciclovir or foscarnet (only 1 post-CTL infusion). There was 1 case of fatal CMV disease, attributable to high levels of antithymocyte globulin at the time of T cell infusion. We compared the patients in the phase 2 study with a group of contemporaneous controls also treated at the trial centers. There was no increase in acute or chronic graft-versus-host disease attributable to CTL infusion; overall and progression-free survival were similar in both groups. There was a reduction in the percentage of patients who required CMV directed antiviral therapy (17% vs 36%, P = .01) and in the total number of treatment days in the cohort receiving CTL (3.4 days vs 8.9 days, P = .03) without a reduction in CMV reactivation rates. We postulate that adoptively transferred cells are able to expand in response to viral antigen, limit viral replication, and prevent progression to tissue infection. This study was registered on the Australian Clinical Trial Registry as #ACTRN12605000213640 and #ACTRN12607000224426.

Pancreatic islet xenograft tolerance after short-term costimulation blockade is associated with increased CD4+ T cell apoptosis but not immune deviation.

BACKGROUND Our purpose was to determine if short-term inhibition of the CD40/CD40L and CD28/B7 costimulatory pathways was capable of inducing specific unresponsiveness to pancreatic islet xenografts and to ascertain the mechanism of tolerance induction. METHODS Diabetic B6AF1 mice were transplanted with Wistar or DA rat islets and were treated short term with CTLA4-Fc and anti-CD40L mAb (MR1). RESULTS Coadministration of CTLA4-Fc with MR1, resulted in indefinite rat islet xenograft survival in mice. Tolerance was species but not strain specific as long-term surviving recipients rejected third party BALB/c islet allografts but accepted a second rat islet xenograft from the same or different donor strain. Tolerance induction was associated with a large leukocyte infiltrate that did not exhibit features of immune deviation as intragraft T cell-specific cytokine gene expression was globally reduced. In particular, interleukin-4 gene expression was markedly suppressed. There was a complete inhibition of anti-donor IgG, IgG1, and IgM antibody in the serum of CTLA4-Fc/MR1- treated animals. Tolerance induction was associated with increased CD4+ T cell apoptosis as there was an increased proportion of annexin-V staining and Fas expressing CD4+ T cells and a decrease in CD4+ T cell Bcl-2 expression in the grafts and draining lymph nodes of CTLA4-Fc/MR1-treated recipients. CONCLUSION Combined costimulatory blockade was capable of producing tolerance to pancreatic islet xenografts. The induction of this tolerant state was associated with increased T cell apoptosis, whereas the maintenance phase of tolerance was associated with the accumulation of a large number of inactive lymphocytes within the graft.

Genetic and functional evaluation of the level of inbreeding of the Westran pig: a herd with potential for use in xenotransplantation

Abstract:  Background:  The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full‐sib matings for nine generations.

Requirement of MyD88 for macrophage-mediated islet xenograft rejection after adoptive transfer.

Background. Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. Methods. In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88−/− and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. Results. Both wild-type C57BL/6 and MyD88−/− mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88−/− recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88−/− mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. Conclusion. This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.

Early Cyst Growth Is Associated with the Increased Nuclear Expression of Cyclin D1/Rb Protein in an Autosomal-Recessive Polycystic Kidney Disease Rat Model

Background: In this study we hypothesised that proliferation, and the increased expression of G1-phase cyclins (D1, E) and phosphorylated retinoblastoma protein (p-Rb) is restricted to the early period of synchronized cyst growth in autosomal-recessive polycystic kidney disease (ARPKD). Methods: Lewis polycystic kidney disease (lpk) rats (model of ARPKD; postnatal weeks 1, 3, 6, 12 and 24; n = 6 each) as well as human juvenile cystic renal disease tissue (n = 2) were examined. Results: Between weeks 1 and 3, the percentage cyst area increased 6-fold in lpk rats, followed by a more progressive rise (1.5-fold increase) until week 24. The number of Ki-67-, cyclin D1- and p-Rb-positive cells increased in lpk rats and peaked at week 3, declining thereafter. By serial sections, cysts co-expressed Ki-67, cyclin D1 and p-Rb. The expression of cyclin E was variable, and peaked at week 24. In human tissue, small cysts had a higher expression of p-Rb. Conclusion: Proliferation and the increased nuclear expression of cyclin D1 and p-Rb coincide with the early phase of cyst growth in rats and humans, suggesting that there might be a therapeutic window in which cyclin-dependent kinase inhibitors are most effective in preventing kidney enlargement in ARPKD.

Subcapsular fetal pig pancreas fragment transplantation provides normal blood glucose control in a preclinical model of diabetes.

Objective. Identifying a limitless source of &bgr;-cells that survive transplantation into a neovascularised site and provide normal blood glucose control remains an important goal in the development of pancreatic islet xenotransplantation. It was our hypothesis that fetal porcine pancreas fragments could achieve these objectives, and this was tested in a large preclinical animal model. Research Design and Methods. Inbred “Westran Pig” fetal porcine pancreas fragments were transplanted beneath the splenic capsule into syngeneic Westran Pig recipients without immunosuppression, and 3 months later, a total native pancreatectomy was performed to demonstrate function. Results. Histologic analysis showed appropriate development of islet-like structures up to and beyond 120 days after transplantation. After native pancreatectomy, recipients survived more than 100 days without exogenous insulin and with normal glucose homeostasis as assessed by normal glucose tolerance tests, K values, and normal glucagon secretion. Conclusions. This study confirms that fetal pig islet tissue has the potential to mature and function normally in a neovascularised site, hence, avoiding the innate immune destruction that occurs when islet tissue is exposed directly to the circulation.

Therapeutic role of sirolimus in non-transplant kidney disease

Sirolimus is a member of a novel class of immunosuppressant drug that potently suppresses T cell proliferation and expansion by inhibition of the Target of Rapamycin Complex 1 (TORC1) protein kinase. Sirolimus also has anti-proliferative effects on intrinsic cells of the kidney, and increasing evidence suggests that it may have a therapeutic role in non-transplant renal diseases. In the normal kidney, sirolimus is considered to be non-nephrotoxic. In the diseased kidney, sirolimus may be beneficial or detrimental, depending on the type of renal injury. In polycystic kidney disease, TORC1 activation mediates renal tubular epithelial cell (TEC) proliferation and cyst growth in animals, and Phase III clinical trials are underway to determine the effect of sirolimus in attenuating disease progression in humans. In contrast, in acute kidney injury, sirolimus transiently impairs proximal TEC regeneration and delays renal recovery. In animal models of lupus nephritis and diabetic kidney disease, sirolimus prevents disease progression. However, the efficacy of sirolimus in human glomerulonephritis as well as in diabetic chronic kidney disease remains unclear, as it paradoxically exacerbates renal dysfunction when the baseline glomerular filtration rate is low (< 40 ml/min/1.73 m(2)) and there is heavy proteinuria (> 300 mg/day). This may, in part, be due to inhibition of compensatory glomerular capillary repair through the suppression of endothelial cell proliferation and angiogenic growth factor production by podocytes. Therefore, at present, polycystic kidney disease is the most promising therapeutic application for sirolimus in non-transplant renal diseases, and further studies are needed to clarify its role in other situations.

Pre-clinical model of composite foetal pig pancreas fragment/renal xenotransplantation to treat renal failure and diabetes

Hawthorne WJ, Simond DM, Stokes R, Patel AT, Walters S, Burgess J, O’Connell PJ. Pre‐clinical model of composite foetal pig pancreas fragment/renal xenotransplantation to treat renal failure and diabetes. Xenotransplantation 2011; 18: 390–399.

Long-term control of recurrent or refractory viral infections after allogeneic HSCT with third-party virus-specific T cells

Donor-derived adoptive T-cell therapy is a safe and effective treatment of viral infection posttransplant, but it is limited by donor serostatus and availability and by its personalized nature. Off-the-shelf, third-party virus-specific T cells (VSTs) appear promising, but the long-term safety and durability of responses have yet to be established. We conducted a prospective study of 30 allogeneic hemopoietic stem cell transplant (HSCT) patients with persistent or recurrent cytomegalovirus (CMV) (n = 28), Epstein-Barr virus (n = 1), or adenovirus (n = 1) after standard therapy. Patients were treated with infusions of partially HLA-matched, third-party, ex vivo-expanded VSTs (total = 50 infusions) at a median of 75 days post-HSCT (range, 37 to 349 days). Safety, viral dynamics, and immune recovery were monitored for 12 months. Infusions were safe and well tolerated. Acute graft versus host disease occurred in 2 patients, despite a median HLA match between VSTs and the recipient of 2 of 6 antigens. At 12 months, the cumulative incidence of overall response was 93%. Virological control was durable in the majority of patients; the reintroduction of antiviral therapy after the final infusion occurred in 5 patients. CMV-specific T-cell immunity rose significantly and coincided with a rise in CD8+ terminal effector cells. PD-1 expression was elevated on CD8+ lymphocytes before the administration of third-party T cells and remained elevated at the time of viral control. Third-party VSTs show prolonged benefit, with virological control achieved in association with the recovery of CD8+ effector T cells possibly facilitated by VST infusion. This trial was registered at www.clinicaltrials.gov as #NCT02779439 and www.anzctr.org.au as #ACTRN12613000603718.

Adjuvant Peptide Pulsed Dendritic Cell Vaccination in Addition to T Cell Adoptive Immunotherapy for Cytomegalovirus Infection in Allogeneic Hematopoietic Stem Cell Transplantation Recipients

Adoptive cellular immunotherapy has been shown to be effective in the management of cytomegalovirus (CMV) reactivation and disease. Whether adjuvant dendritic cell (DC) vaccination will provide additional benefit in prophylaxis or treatment of CMV in hematoietic cell transplantation (HSCT) recipients is unknown. In this study, we administered prophylactic CMV-peptide specific T cell infusions, followed by 2 doses of intradermal CMV peptide-pulsed DC vaccine, to 4 HSCT recipients. There were no immediate adverse events associated with T cell infusion or DC vaccinations. One of the 4 patients developed grade III acute gut graft-versus-host disease. Immune reconstitution against CMV was detected in all 4 patients. Patients receiving CMV peptide-specific T cells and DC vaccination had peak immune reconstitution at least 10 days after the second DC vaccination. In summary, combining DC vaccine with T cell infusion appears feasible, although further study is required to ascertain its safety and efficacy in augmenting the effects of infusing donor-derived CMV-specific T cells.