P. T. Coates

Review of Epstein–Barr virus and post-transplant lymphoproliferative disorder post-solid organ transplantation (Review Article)

SUMMARY:  Post‐transplant lymphoproliferative disorder (PTLD) following solid organ transplantation is an important form of post‐transplant malignancy. PTLD is typically associated with Epstein–Barr virus (EBV) and occurs in the setting of profound immunosuppression resulting in a deficiency of EBV‐specific cytotoxic T lymphocytes (CTL). Predisposing factors include EBV mismatch between donor and recipient, use of immunosuppression especially T‐cell depletive therapies and genetic predisposition of recipients. The standard approach has been to reduce immunosuppression but is often insufficient to induce tumour regression. Further understanding of the immunobiology of PTLD has resulted in improved monitoring techniques (including EBV viral load determined by polymerase chain reaction) and newer treatment options. Recent work has highlighted a potential role for dendritic cells in both the pathogenesis and treatment of PTLD. Current treatment modalities include adoptive immunotherapy using ex vivo generated autologous EBV‐specific CTL or allogeneic CTL, cytokine therapies, antiviral agents, and more recently, rituximab and dendritic‐cell based therapies. This review focuses on the developments and progress in the pathogenesis, diagnosis and treatment of PTLD.

Insulin-Like growth factor-II (IGF-II) prevents proinflammatory cytokine-induced apoptosis and significantly improves islet survival after transplantation.

Background The early loss of functional islet mass (50–70%) due to apoptosis after clinical transplantation contributes to islet allograft failure. Insulin-like growth factor (IGF)-II is an antiapoptotic protein that is highly expressed in &bgr;-cells during development but rapidly decreases in postnatal life. Methods We used an adenoviral (Ad) vector to overexpress IGF-II in isolated rat islets and investigated its antiapoptotic action against exogenous cytokines interleukin-1&bgr;– and interferon-&ggr;–induced islet cell death in vitro. Using an immunocompromised marginal mass islet transplant model, the ability of Ad-IGF-II–transduced rat islets to restore euglycemia in nonobese diabetic/severe combined immunodeficient diabetic recipients was assessed. Results Ad-IGF-II transduction did not affect islet viability or function. Ad-IGF-II cytokine-treated islets exhibited decreased cell death (40%±2.8%) versus Ad-GFP and untransduced control islets (63.2%±2.5% and 53.6%±2.3%, respectively). Ad-IGF-II overexpression during cytokine treatment resulted in a marked reduction in terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive apoptotic cells (8.3%±1.4%) versus Ad-GFP control (41%±4.2%) and untransduced control islets (46.5%±6.2%). Western blot analysis confirmed that IGF-II inhibits apoptosis via activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. Transplantation of IGF-II overexpressing islets under the kidney capsule of diabetic mice restored euglycemia in 77.8% of recipients compared with 18.2% and 47.5% of Ad-GFP and untransduced control islet recipients, respectively (P<0.05, log-rank [Mantel–Cox] test). Conclusions Antiapoptotic IGF-II decreases apoptosis in vitro and significantly improved islet transplant outcomes in vivo. Antiapoptotic gene transfer is a potentially powerful tool to improve islet survival after transplantation.

Antigen-encoding bone marrow terminates islet-directed memory CD8+ T-cell responses to alleviate islet transplant rejection

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease, and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by “conventional” therapies, memory T cell–mediated attack is a substantial challenge in islet transplantation, and this will extend to application of personalized approaches using stem cell–derived replacement β-cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement β-cells. Here, we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions, inactivates established memory CD8+ T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8+ memory T cell–mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell–mediated gene therapy effectively terminates antigen-specific memory T-cell responses, and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and, importantly, the clinical application of personalized β-cell replacement therapies using patient-derived stem cells.

Ablation of pathogenic memory T-cell responses by bone marrow-mediated gene therapy under immune-preserving conditions

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.Changes in microbiome, mucosal immunity and intestinal integrity have been associated with the onset of Type 1 Diabetes (T1D) in children. Toll-like Receptors (TLR) have been associated all three factors. The role of TLR and their effects on microbiome in autoimmunity were studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. Physical parameters of the intestines were compared; ileal weight was reduced in NOD.Tlr1-/-mice. Similarly, by histology, these mice had reduced villus length and width. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA), in two cohorts, 18 months apart. Analysis of caecal 16S sequences clearly resolved the mouse lines and there were significant differences in beta diversity between the strains, with individual bacterial species contributing greatly to the differences in the microbiota of individual TLR-deficient strains. To test the relationship between microbiome and T1D, all strains were re-derived onto the parental NOD/Lt line and the incidence of T1D re-assessed within two generations. All rederived lines expressed an incidence of disease similar to that of the parental line. TLR deficiencies are associated with changes in microbiome; changes of microbiome are associated with T1D; the effects of TLR deficiencies on T1D appear to be mediated by their effects on gut flora.Intestinal TCRb+CD4-CD8b-CD8a+ (CD8aa) IELs alleviate T cell induced colitis and have been suggested to play a role in virus infection and cancer. Their thymic development has been elucidated to some extent, as IEL precursors (IELp) are known to be CD4-CD8-CD5+TCRb+, but is not yet fully understood. Within the thymus, mature IELp were identified based on their expression of CD122 and MHC class I. Two major phenotypic subsets exist within this mature thymic IELp population: a PD1+Tbet- population that preferentially expresses a4b7, and a PD1-Tbet+ population with preferential CD103 expression. These two populations were also distinct in their Valpha repertoire. The PD1+a4b7+ population contains clones that are strongly self-reactive as judged by Nur77GFP and their dramatic increase in Bim deficient mice, while the PD1-Tbet+ population did not show these characteristics. Both gave rise to CD8aa IELs upon adoptive transfer into RAG-/- recipients. However intrathymic labeling revealed that PD1+a4b7+ IELp were the major thymic emigrating population, and emigration was S1P1-dependent. In contrast, PD1-Tbet+ IELp expressed CXCR3, were retained, and accumulated in the thymus with age. Preliminary immunofluorescence data furthermore indicate differential thymic cortico-medullary localization of the IELp subtypes. These experiments more precisely define the behavior of IEL precursors.

ADENOVIRUS-MEDIATED TRANSDUCTION OF ISOLATED PANCREATIC ISLETS USING INSULIN-LIKE GROWTH FACTOR-II TO PROMOTE ISLET SURVIVAL POST-TRANSPLANTATION: 2000

A. Hughes1, A.J. Kupke2, C.J. Drogemuller1, C.F. Jessup1, D.M. Mohanasundaram1, C.R. Milner3, G.R. Russ4, P.T. Coates4 1Renal And Transplantation Immunobiology Laboratory, Hanson Institute, Adelaide/AUSTRALIA, 2Islet Transplantation Facility, The Queen Elizabeth Hospital, Adelaide/SA/AUSTRALIA, 3Renal And Transplantation Immunobiology Laboratory, Hanson Institute, Adelaide/SA/AUSTRALIA, 4Central Northern Adelaide Renal And Transplantation Service, Royal Adelaide Hospital, Adelaide/ AUSTRALIA