Objective
We aim to further our previous work engineering an islet graft for the skin site by including immune modulating components such that our graft promotes not only islet function, but also immunological tolerance. We will create a vascularized, immune privileged site under the skin, exploiting an important advantage of one of the materials that we have been using in our most recent work. That material is based on methacrylic acid (MAA) and without additional biological components (growth factors or cells), it induces the formation of blood vessels. This is instead of the scar that typically surrounds most implanted materials, and which isolates the implant (say a microcapsule) from the host and prevents integration of islet and host vasculatures. The material is a mixture of poly(methacrylic acid) with a common biomaterial (polyethylene glycol, PEG) and is to be used as an injectable gel to create a vascular bed under the skin to enable successful islet transplantation. The MAA-PEG hydrogel is initially liquid but gels once injected under the skin; it degrades within a week to enable integration and engraftment of transplanted islets. Interestingly, exposure of DCs to the MAA hydrogel appears to enhance the ability of DCs to promote the desired regulatory T cell (Treg) formation, a key feature of immune tolerance.
To circumvent the problem of immune suppression, we will use the MAA-PEG gel to deliver both islets and dendritic cells (DC) which are released locally as the gel degrades. DC and especially the tolerance-inducing vitamin D3 treated DC (VD-DC) are expected to result in what is termed “infectious tolerance”. This is long-term immune acceptance achieved by triggering an infection-like immune response which recapitulates the regulatory mechanisms used to protect the infected individual’s own cells, but here are used in the context of islet transplantation.
We propose to use the MAA-PEG gel and VD-DC to modulate the adaptive immune response (Aim 1) and yield an immune privileged, vascularized, under-the-skin (subcutaneous) islet graft, sufficient to restore normal glucose levels in chemically induced diabetic mice (Aim 2). Islets will be transplanted from one mouse strain (BALB/c) into another strain (C57Bl/6), an allograft. We use mouse islets in current studies but anticipate that we will use human pancreatic precursor cells in future studies. We will demonstrate that colocalization of the allogeneic islets and the tolerogenic DC within the degradable MAA-PEG gel will take advantage of the resulting vascularized, immune modulated environment to promote islet survival without the systemic toxicity and side effects seen with systemic immunotherapies and chronic immunosuppression.
A comparison with immune isolation (i.e. alginate microencapsulation) will demonstrate that immune modulation is a viable strategy for promoting islet graft survival, without a chronically implanted protective material. We presume that integrating the vasculature of the islet with that of the recipient will benefit islet function and reduce the number of islets needed to be transplanted. While microencapsulation is a means of eliminating the need for immune suppression, we note that the capsule wall is a barrier to engraftment and integration of the islet vasculature with the implant site– a critical issue for this method. Instead, by showing that we can promote tolerance, we establish a platform which, in the future, could be used to beneficially impact auto-immune causes of diabetes.
The goal is to enable minimally invasive, islet transplantation under the skin in a retrievable, vascularized “device-less”, physiologically integrated implant site, without the need for systemic immune suppression. This is an important step towards, the widespread clinical implementation of this strategy for better control of blood glucose levels and reduced complications.
Background Rationale
The invasiveness of islet transplantation (typically into the liver) and resulting immune responses result in a significant loss of transplanted islets. With JDRF support, we have shown that the skin, once vascularized is an attractive, minimally invasive site for islet transplantation. At this alternative site, host vessels are linked to the beta-cells to promote survival, proper glucose-sensing and insulin secretion. Furthermore, the graft can be accessed with ease. The focus now is on controlling the immune response to islets in this site. Microencapsulation is one approach, but the encapsulated islets are isolated from the vasculature as well as the immune system and there is the problem of the foreign body reaction. We propose that there is an opportunity to generate a state of immunological tolerance in this new hitherto unstudied transplant site (i.e., the vascularized skin).
We focus here on using an inherently vascularizing methacrylic acid based injectable hydrogel (MAA-PEG) to deliver islets together with antigen-primed tolerogenic dendritic cells (DCs) that we expect will induce immune tolerance to the graft. Most importantly, the MAA-PEG gel generates blood vessels (and nerves) without growth factors or cells. MAA creates this regenerative environment by modulating the behaviour of responding cells. The potential to re-innervate islets may prove useful in the vascularized skin site, although this is not a focus here. MAA-PEG benefits islet engraftment not only through an effect on vascularization, but also by generating a beneficial, regenerative microenvironment – one that enhances the recruitment of endogenous DCs and the generation of regulatory T cells.
By adding DCs to this system, we expect to build on the MAA-PEG regenerative environment to promote immunological tolerance of islet allografts. This is expected to mitigate allogeneic immune rejection while avoiding the need for long-term, systemic immune suppression; nonetheless short term (one-week) therapy is expected to still be needed to dampen the peri-operative host response. We use vitamin D3 treated dendritic cells (VD-DC) as our source of tolerogenic DCs. The added VD-DC are released from the gel as the material degrades and are expected to trick the host immune system into considering the transplanted islets as host cells, exploiting mechanisms akin to what occurs after an infection ("infectious" tolerance), creating a locally privileged environment within the vascularized islet graft.
VD-DC interact with responding host immune cells in a way which recapitulates the inherent mechanisms that protect host cells from the hosts own immune system – in particular deleting destructive T cells and recruiting antigen-specific regulatory T cells. These regulatory T cells are crucial for locally protecting the graft, ensuring that the normally destructive immune response against this graft is diminished. Hence, we aim to "re-train" the host immune system to accept transplanted islet cells in order to promote long-term tolerance of the graft. One can imagine a similar impact on autoimmunity in follow-on studies.
We expect the resulting vascularized, immune privileged islet graft to be unencumbered by the systemic toxicity associated with immune suppression or the foreign body response of immuno-isolation, yet able to restore normal glucose levels in diabetic mice. This approach works with the immune system rather than fighting against it.
Description of Project
Widespread cell therapy for those living with diabetes is becoming more of a clinical reality. However, current sites of islet transplantation present “hostile” environments for islets. With the support of JDRF, we showed that we can create a vascular bed or injection site under the skin, sufficient to support insulin producing cells and restore normal glucose levels in diabetic rodents. Without such intervention, there are too few blood vessels to support islet viability and function. Insulin-secreting beta cells depend on connection with host vasculature for long term survival, accurate sensing of blood glucose and proper secretion of insulin. The skin also has the clinical advantage of being more accessible and being considered “safer” for patients; critically, the transplanted cells are potentially retrievable.
We are focused here on using a vascular regenerating material based on methacrylic acid (MAA) which does not require additional biological components (growth factors or cells) to induce the formation of blood vessels. The recruited blood vessels are sufficient to support an islet graft under the skin even when the material degrades within a week. The MAA material is used as an injectable gel: it is initially liquid but gels once injected. To circumvent the problem of immune suppression, we will use the MAA-PEG gel to deliver both islets and tolerogenic dendritic cells, the latter induced by treatment with the biologically active form of vitamin D (vitamin D3). The resulting VD-DC are expected to create long-term immune acceptance by a recapitulation of the inherent regulatory mechanisms which normally protect host cells from their own immune system, but here are used to "trick" the immune system to instead accept an islet transplant.
We will use the MAA-PEG gel and VD-DC to modulate the adaptive immune response (Aim 1) and yield an immune privileged, vascularized, under-the-skin islet graft, sufficient to restore normal glucose levels in chemically induced diabetic mice (Aim 2). Interestingly, exposure of DCs to the MAA hydrogel appears to enhance the ability of DCs to promote the desired regulatory T cell (Treg) response. Regulatory T cells are crucial for both locally protecting the graft (when VD-DC are co-delivered with islets) and ensuring that the host immune response against this graft is diminished (after these VD-DC migrate to secondary lymph organs). We aim to "re-train" the host immune system to accept target antigens (in this case allogeneic islet antigens) in order to promote long-term tolerance of the graft. VD-DC treated animals are expected to show evidence of immunological memory demonstrating successful “re-training”. Furthermore, we will compare the efficacy of this approach to the microencapsulation approach, where islets are only physically protected from the immune system; we expect microencapsulation to be less effective because it prevents integration with the host vasculature and does not exploit the immune system for a functional benefit.
The goal of this work is to enable minimally invasive islet transplantation under the skin in a retrievable, vascularized, physiologically integrated implant site, without the need for systemic immune suppression. The therapeutic success of skin implants requires the need to promote islet vascularization, such as by using MAA-PEG, while simultaneously mitigating the adaptive host response. These are important steps towards, the widespread clinical implementation of this strategy for better control of blood glucose levels with reduced secondary complications.
We focus on rodent islets in this project, but we expect to use stem cell derived human cells in future studies. Excitingly, strategies for converting pluripotent stem cells into mature beta cells are emerging, perhaps eliminating the need for donor islets and making widespread use of engineered islet transplants clinically feasible.
Anticipated Outcome
We will demonstrate that the vascular regenerating MAA-PEG gel, together with the added dendritic cells, is an effective means of transplanting pancreatic islets in a minimally invasive manner to a vascularized, under-the-skin (subcutaneous) site. The primary outcome is showing that this combination is sufficient to restore normal glucose levels without the need for long-term immunosuppression. Furthermore, we will compare the efficacy of this approach to that of microencapsulation which we expect to be less effective because of the capsule membrane which generates a detrimental foreign body response, precludes integration of the vasculature inside the islet with that of the host and which does not present the opportunity for inducing tolerance.
In Aim 1, we focus on the immune modulatory ability of the delivered dendritic cells to induce a more tolerogenic response/microenvironment (particularly a greater regulatory T cell response) compared to an islet transplant under the skin, but without dendritic cells. In aim 2, we expect to see fewer islets needed to return diabetic mice to normal glucose levels (compared to microencapsulation) and a delay in the return to hyperglycemia with effective immune modulation by the tolerance inducing DC.
Tolerance inducing dendritic cells are expected to present donor islet antigens in an anti-inflammatory context to protect the transplanted islets from the immune response through their effect on transplant-specific regulatory T cells. These regulatory T cells are expected to work with the VD-DC to further polarize responding host cells to a regulatory state, resulting in an amplified regulatory T cell and anti-inflammatory DC response. Hence, we expect to see a higher ratio of regulatory T cells to (cytotoxic) effector T cells and more anti-inflammatory DCs (both delivered DC and those recruited from the host) in the graft and draining lymph nodes than for the transplants without additional cells.
Since MAA-PEG has a beneficial effect on dendritic cells, in addition to its vascular regenerating properties, we should see some of this Treg effect with MAA-PEG without delivered dendritic cells, but this should be less than the combination of both MAA-PEG and delivered DC. We expect the short-term presence of the degradable MAA-PEG will mean that any suppressive tolerogenic response seen without added DC will diminish as the material degrades. We expect the response to MAA-PEG alone to be insufficient to prevent immune-mediated killing of islet grafts – the rationale for including VD-DC.
Dendritic cell treated animals are expected to show evidence of immunological memory. T cells from DC treated, recipient animals are expected to have a reduced response when exposed to islet donor antigens relative to when these cells are exposed to antigens from a third-party animal. When given a second transplant, we expect that islets from the same strain as the original donor will survive but that third-party islets will invoke a strong host response and be destroyed soon after transplantation – evidence that the immune system is tolerant only to the original islet graft. We do not expect this impact with microencapsulation or without the tolerogenic dendritic cells.
Without the vascularizing material, the islets will not survive because the skin is not a suitable site for islet transplantation without vascularization, such as that induced by the MAA-PEG. Because islets can now be deployed under the skin, and without pre-vascularization, we expect that strategies developed to enable islet engraftment without immune suppression, become feasible. Here we use tolerogenic dendritic cells to modulate the immune response with a view to inducing tolerance, but MAA-PEG is a platform for other approaches as well.
Relevance to T1D
The creation of an immune privileged, vascularized, under-the skin injection site for islets will benefit those who live with diabetes and suffer the long-term consequences of the limited control of blood glucose associated with multiple daily insulin injections. There are ~3 million people with type 1 diabetes in United States and Canada, with many more diagnosed each year. Worldwide there are >10 times as many living with this disease, driving significant concerns about how developing countries will be able to cope with the costs of such chronic diseases and their degenerative consequences.
Islet transplantation is a promising approach to treatment with a view to minimizing the degenerative complications that affect those who live with diabetes and lifelong insulin therapy. Excitingly, strategies for converting pluripotent stem cells into mature beta cells are emerging, perhaps eliminating the need for donor islets.
However, both the abdominal cavity and the liver present “hostile” environments for islets. As a result, excess islets are needed for a return to normal glucose levels placing increased pressure on the limited amount of donor tissue. To address this issue, we created a vascularized injection site under the skin, enabling the use of the skin as the site for delivery of islets or stem cell derived beta cells. The skin site is less hostile towards islets and has the clinical advantages of being more accessible and being considered “safer” for patients; critically, the transplanted cells are potentially retrievable. In the absence of our approach, the skin has relatively few blood vessels and thus islets are starved for oxygen and fail to secrete enough insulin. Our past work with JDRF support shows that islets will survive and function well with appropriate vascularization.
In this proposal, we build on the vascularized skin site to create an immune privileged transplant site, by locally modulating the immune response through the pro-tolerogenic properties of our vascularizing MAA-PEG material together with the additional immune cells (tolerogenic dendritic cells) that we deliver along with pancreatic islets. The therapeutic success of skin implants depends on the simultaneous re-vascularization of delivered islets and mitigation of the adaptive host response. We expect to enable acceptance of the grafted tissue without long term immune suppression, while better yet, modulating it to a pro-tolerogenic state.
Through vascularizing the skin, the MAA-PEG gel becomes a platform for the systematic study of islets and the impact of immune modulation in a site that is less hostile and more accessible than the peritoneal cavity. Achieving this recognizes the critical importance of linking the vasculature to insulin secreting beta-cells for proper glucose-sensing and insulin secretion. Furthermore, it recognizes that immune modulation is site-specific: what may be effective for transplants in the liver or kidney may not be appropriate for those under the skin. We aim to "re-train" the host regulatory immune system to accept transplant antigens in order to promote long-term tolerance of an islet graft. Although outside the scope of this project, the same approach can be translated to the autoimmune case, in which autoantigens are targeted.
Enabling modulation of human disease, is a critical next step. Using a biomaterial without biological components, at least for the purpose of islet vascularization and skin delivery simplifies this path. A vascularized injection site under the skin created using MAA-PEG and tolerogenic dendritic cells obviates the concerns associated with using the peritoneal cavity or the liver and systemic immune suppression. While patient-derived tolerogenic DC have been deemed safe in several clinical contexts including diabetes, integrating the material, insulin secreting cells (human islet or PP cells) and human DC will require follow-on investigation in humanized mice and primates.