Boosting beta-cell function for treatment of type 1 diabetes

In type 1 diabetic (T1D) patients the pancreatic insulin-producing beta cells are selective destroyed by the immune system, leading to an impaired glucose metabolism. The steep increase in the incidence of this chronic autoimmune pathology especially in young children in the last decades raises serious concerns. To date, insulin therapy is considered the gold standard for the treatment of T1D. Nevertheless, limitations persist, such as the frequent episodes of hypoglycemia, and the chronic micro- and macrovascular complications. Islet cell transplantation offers an alternative treatment for T1D patients, specifically for those with hypoglycemic unawareness following insulin administration. Despite the improved outcome of islet cell transplantation over the last few years, drawbacks remain, such as a limited supply of cadaveric donors, the necessity of several donors for a single transplantation, and (immediate) graft failure through metabolic pressure, continued autoimmunity, alloimmunity, high concentrations of immunosuppressive drugs, and oxidative stress caused by hypoxia or due to cytokine exposure. In this PhD thesis, we propose that in the longterm, tolerogenic antigen-based and beta-cell replacement/regenerative approaches could be promising in both blocking the autoimmunity to prevent continued beta-cell destruction and boosting beta-cell function and revascularization to restore sustainable insulin regulation.

Contrasting with alloimmune responses, autoimmune disease recurrence is difficult to control with standard immunosuppression and therefore poses a challenging obstacle for a successful longterm islet cell transplantation outcome. In chapter 4, we used a combination therapy consisting of a short induction therapy of low doses of anti-CD3 mAbs combined with the intragastric delivery of genetically-modified L. lactis-based vaccines producing human proinsulin along with pro-tolerogenic IL10 to reestablish tolerance to islet antigens in longstanding diabetic NOD islet recipients. We first demonstrate that an early regimen (starting 5 days before islet implantation) with low-dose anti-CD3 combined or not with L. lactis-based antigen-specific therapy induced poor graft acceptance in around 20% of NOD islet recipients. However, when therapeutic regimen started at the time of islet implantation, the overall graft acceptance was around 56% in L. lactis-based therapy-treated NOD islet recipients. Next, we observed that diabetes duration before islet implantation impacts on the rate of success. The efficiency to prevent autoimmune diabetes recurrence markedly decreased in anti-CD3-treated mice with diabetes duration, while L. lactis-based combination therapy-treated mice were much less influenced by this parameter. The efficiency of glycemic control and the rate of graft acceptance in relation to the timing of diabetes diagnosis is in part related to the protection of residual endogenous islets in the pancreas as reflected by the insulin content of the native pancreas at different time points after diabetes diagnosis. Further, we found that NOD islet recipients treated with anti-CD3 alone or combined with the L. lactis-based vaccine had Foxp3+ Tregs present in the lymph nodes draining the islet implantation site (i.e. kidney) but this was not different between mono- and combination therapy and independent of diabetes duration. Based on these observations, we concluded that a mild lymphodepletion with low-dose anti-CD3 mAbs in combination with an antigen-specific approach using genetically-modified L. lactis can by-pass autoimmune diabetes recurrence in 56% of syngeneic islets transplanted in longstanding diabetic NOD mice. Moreover, we found that islet cell transplantation outcome will certainly depend on the time of islet implantation in relation to T-cell-targeting strategy and on the time of islet implantation after diabetes diagnosis. Integration of these antigen-specific technologies to enhance engraftment and combat graft destruction may help to advance the therapeutic efficacy and availability of islet cell transplantation in selected patient populations.

In the first days following transplantation, islets lack an adequate vascular network, leading to severe hypoxia and cell death. It is believed that this is one of the major causes for the poor performance of islet grafts longterm. Thus, in chapter 5 we used a novel class of progenitor cells MAPC® and co-transplanted these with mouse islets to improve islet engraftment. First, we demonstrated that human MAPC produce high amounts of angiogenic growth factors, including VEGF, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the CAM assay. Next, we showed for the first time that human MAPC co-transplanted as composite pellets with mouse islets can improve islet graft function in a syngeneic marginal mass islet cell transplantation model as measured by the initial glycemic control, diabetes reversal rate, glucose tolerance, and serum C-peptide concentration compared with transplantation of islets alone. Moreover, we found that grafts composed of islet-human MAPC composites had an improved revascularization process. We found a significant improvement in the development of a new islet capillary network in mice where islets were co-transplanted with human MAPC. Indeed, higher numbers of capillary-like structures with a lining of endothelial cells were found on the periphery and in the intra-islet space of the islet-human MAPC composites at week 5 post-transplantation, suggesting that host-derived vessels are directly feeding transplanted islets and that close proximity and even direct contact between the transplanted pancreatic islets and human MAPC is of critical importance for the improved glucose control, diabetes reversal rate and increased revascularization. The present data propose that co-transplantation of mouse pancreatic islets with human MAPC, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularization and subsequently improve islet graft function.

As a general conclusion, the data presented in this PhD thesis show promising therapeutic approaches to improve islet transplant outcomes. Our work also indicates that these approaches should act on different levels to overcome the many hurdles of beta-cell replacement in the modility of islet cell transplantation. We showed that genetically-modified L. lactis-based antigen-specific vaccines combined with a short course of low doses of anti-CD3 mAbs can avoid autoimmmune diabetes recurrence depending on the time of islet implantation in relation to T-cell-targeting strategy and on the time of islet implantation after diabetes diagnosis. We also showed that the use of human MAPC in islet cell transplantation can improve islet graft morphology and function by transplantation of islets-human MAPC composites, possibly via the promotion of graft revascularization mediated by human MAPC. In the near future, we hope it will be possible to reach longterm islet transplant outcomes by implementing and combining our approaches avoiding not only the immunological issues of islet cell transplantation but also improving islet engraftment.