Objective
We aim to improve our existing subcutaneous islet transplant platform to improve results in immune competent models using clinically relevant methods, and incorporate immunomodulation so that implants are protected from immune rejection.
This project has three primary objectives:
1. Understand the local environment provided by neutrophil depletion (via Ly6G antibody) that can enable islet survival at the vascularizing subcutaneous site.
a. We have found that mitigating neutrophils at early times is necessary for subcutaneously implanted islets to survive. We want to understand how islet survival is promoted by neutrophil depletion so that we can achieve similar effects with a better method.
2. Develop a new protocol to replace anti-Ly6G with FDA approved drugs that achieves the environment outlined by (1) and similarly prolongs islet graft survival.
a. Anti-Ly6G has shown promising results but cannot be used in humans; Ly6G is a protein only found in mice. Moreover, neutrophil depletion is not desirable in humans since neutrophils are useful cells (in most other contexts). Here we want to generate a protocol that lets us continue research into our MAA-PEG subcutaneous islet transplant platform.
3. Prolong islet graft survival without immune suppression using immune tolerance-promoting tolerogenic dendritic cells.
a. We hope to re-train the host immune system to “tolerate” the foreign implanted islet cells so that we do not have to use immune suppression. Tolerogenic dendritic cells have the potential to achieve this re-training but it has not yet been possible to use these cells with islets in the subcutaneous space. Here we will test how to best incorporate these cells into a vascularizing subcutaneous islet graft so that they can provide a therapeutic effect and prolong islet survival.
The overall goal is to yield a vascularized subcutaneous islet graft that regulates blood glucose (reverses diabetes) and is accepted by the host immune system. Here we study how to combine a vascularizing transplant vehicle with agents to mitigate the early inflammatory immune response and the later organ rejecting immune response. The results of these experiments and the development of such a therapy would be an important step towards the widespread clinical implementation of cell replacement as an improved treatment option for T1D.
Background Rationale
Islet transplantation and the subcutaneous space:
Pancreatic islet transplantation is a promising regenerative, insulin injection-free therapy for type 1 diabetes (T1D). However, it is not possible to transplant islets to their native site in the pancreas, and there is no perfect alternative transplant site. Islets need connections to a dense network of blood vessels in order to properly function and survive (sense blood glucose and secrete the proper amount of insulin).
Our research group and others have shown that the subcutaneous space (under the skin) is promising transplant site for islets. This site can be easily accessed (surgery is minimally invasive and grafts can be easily retrieved), is large enough to accommodate the necessary number of cells, and is considered safe for patients. However, blood vessels have to be recruited to the site for islets to survive. The subcutaneous site also has a unique inflammatory (neutrophil) response (that can destroy implants) because of its role as a barrier organ; this “neutrophil response” typically protects cuts from infection, for example. Moreover, all islet transplants including those in the subcutaneous space must be protected from immune rejection and autoimmunity. Immunosuppression provides some protection, but secondary complications (toxicity) and insufficient protection make this option less than ideal.
MAA biomaterials for islet transplantation:
During my PhD training I showed that islets subcutaneously injected in an inherently vascularizing methacrylic acid (MAA)-based hydrogel (“MAA-PEG”) survived in the subcutaneous space and reversed diabetes in immune compromised mice. The Sefton lab had previously shown that methacrylic acid (MAA)-based biomaterials including this hydrogel recruit numerous blood vessels in the subcutaneous space. This is in part because MAA shifts the behavior of cells investigating the implant towards a state that regenerates biological tissue. My work showed that MAA-PEG caused the host’s own cells to locally generate a (blood vessel rich) environment in the days after implantation that allowed islets to survive and engraft for months, even though MAA-PEG disappears in roughly a week.
In more recent experiments I showed that MAA-PEG also allows us to subcutaneously transplant islets in more relevant immune competent mice, but only when we dampened the above noted inflammatory response with a neutrophil depleting antibody. The neutrophil depletion approach permitted further experimentation, but depleting the neutrophils is not an option in the clinic. We propose that there is an opportunity to develop a clinically relevant protocol to dampen this early inflammatory response and make the subcutaneous space more accessible to researchers and clinicians as an islet transplant site.
Immunomodulation and tolerogenic dendritic cells:
In this work I will add immunomodulating tolerogenic dendritic cells as a means of protecting the islet implant from rejection at later times, instead of using immunosuppression. These tolerogenic cells are naturally responsible for regulating immune responses and preventing the immune system from attacking healthy tissue. We aim to use these cells to “re-train” the host immune system to recognize transplanted islets as healthy tissue (instead of suppressing or avoiding the immune response); thus, the implant will generate a regulatory immune response that will counteract transplant rejection. We focus on these cells over other immunomodulating options (like delivery of protective regulatory T cells (Tregs)) because of the natural role of tolerogenic dendritic cells as orchestrators of immune responses. We have already seen a benefit when combining tolerogenic dendritic cells and MAA-PEG in a mouse model of autoimmune diabetes (e.g., more Tregs at the implant and in the draining lymph nodes). Here we will build on these findings and evaluate how immunomodulation by tolerogenic dendritic cells and the MAA-based hydrogel will influence rejection of allogeneic (non-self) islets at the vascularized subcutaneous site.
Description of Project
Islet transplantation (beta cell replacement) is a promising regenerative, insulin injection-free therapy for type 1 diabetes (T1D). However, challenges around chosen transplantation site (and method of transplantation) and attack from the immune system (inflammation, autoimmunity and conventional transplant rejection) still prevent widespread use of this therapy.
I aim to further develop a cell replacement therapy for type 1 diabetes (T1D) that can overcome these challenges using a regenerative hydrogel “delivery platform.” This hydrogel is a synthetic hydrogel blend of poly(methacrylic acid) (MAA) and poly(ethylene glycol) (PEG) (“MAA-PEG”) that inherently causes blood vessels, nerves and other tissues to grow and regenerate. I have shown that islets injected subcutaneously (under the skin) in MAA-PEG survive and reverse diabetes in mice because of the regenerative properties of MAA. The subcutaneous space is a promising transplant site that is less hostile than alternatives like the liver. However, without vascularization by a component like MAA it does not have enough blood vessels needed to keep transplanted cells alive. In more clinically relevant models I have recently found that islets injected subcutaneously in MAA-PEG can only survive and reverse diabetes if we also dampen inflammation at the time of surgery. In those experiments we used a neutrophil-depleting antibody (“anti-Ly6G”) to promote cell survival. Unfortunately this method won’t work in humans; Ly6G is only found in mice, and neutrophil depletion is not clinically desirable. Moreover, islets that engrafted with anti-Ly6G and MAA-PEG were still eventually rejected by the host immune response.
In this project I will study how neutrophil depletion enabled islet survival (Aim 1), devise a strategy to achieve the same effects with FDA-approved, clinically relevant drugs or approaches (Aim 2) and then combine these methods with immunomodulating tolerogenic dendritic cells to prevent rejection (Aim 3). We have already tested these tolerogenic dendritic cells with MAA-PEG in an autoimmune model and found that combining MAA with the immunomodulating cells led to greater therapeutic outcomes (a delay in onset of autoimmune diabetes compared to vehicle controls, and the generation of a regulatory immune response). We are interested in immunomodulation because it “treats” the host immune system instead of suppressing or avoiding it; immunosuppression can provide some benefit (delayed rejection) but is associated with secondary complications (organ toxicitiy) and is not sufficient in the long run. We expect that the combination of the regenerative MAA material, the FDA-approved anti-inflammatory drugs and tolerogenic dendritic cells will protect islets from early or late rejection (at least from weeks to months, in initial experiments). Our goal is to build a subcutaneous, immune privileged insulin secreting organ under the skin that reverses diabetes in mice and avoids rejection.
Anticipated Outcome
We will show that allogeneic (foreign) islets can be subcutaneously injected in a MAA-PEG hydrogel in immune competent mice, and that added tolerogenic dendritic cells will start a regulatory immune response that protects islets from immune rejection without immune suppression.
As part of this work we will develop a clinically relevant protocol that could be used by any subcutaneous islet / beta cell method to improve early transplant outcomes. We have recently found that neutrophils destroy islet grafts at early times at the subcutaneous site; depleting neutrophils improves survival significantly. Our findings with neutrophil depletion have been replicated by others in the JDRF research group, highlighting their relevance to subcutaneous transplantation. However, neutrophil depletion is not a viable clinical option. In aims 1 and 2 of this project I will better understand the effects of neutrophil depletion and try to replicate them using FDA-approved, clinically relevant drugs. We expect to find high levels of inflammatory proteins involved in the recruitment, activation, and function of neutrophils. Further, we expect that inhibiting these proteins (individually or inhibiting multiple targets at once) will sufficiently disrupt the local neutrophil response to enable islet survival. Without mitigation of this early response or without vascularization by the MAA component we do not expect to see an impact on blood glucose levels for more than 1-2 days following transplantation. We will mimic the useful effects of neutrophil depletion on islet transplantation success while maintaining the healthy benefits of host neutrophils.
In aim 3 we expect that adding tolerogenic dendritic cells will further improve islet survival by triggering an immune response that protects the graft from immune rejection. We have already combined tolerogenic dendritic cells and MAA-PEG (without islets) in a mouse model of autoimmune diabetes and saw that the combination led to greater therapeutic effects (more useful regulatory immune cells throughout the host, and a delay in diabetes onset compared to delivery in a control hydrogel). Here we expect that these effects will be maintained in the C57BL/6 mouse model and work together with the anti-inflammatory protocol from aim 2 to trigger an enhanced “infection-like” regulatory immune response. This infectious tolerance will grow and counteract typical organ rejection; we will see this at earlier times by higher levels of Tregs and fewer cytotoxic T cells at the graft and peripheral immune tissues (lymph nodes). We anticipate that these effects on immune responses will manifest as prolonged islet survival and reversal of diabetes for months or longer. Controls without the tolerogenic dendritic cells may show some longer survival (as we have seen with just MAA and neutrophil depletion) but we do not expect these implants to be protected from chronic rejection.
Relevance to T1D
My project to develop a clinically feasible method to subcutaneously transplant islets and prevent rejection through immunomodulation would be a significant step towards a cell replacement therapy that would benefit those living with diabetes.
Islet transplantation (i.e., beta cell replacement) is a promising approach that can restore intrinsic blood glucose control and provide insulin injection independence. This treatment would prevent the degenerative complications associated with lifelong insulin therapy. Promising advances in stem cell-derived beta cells (including first in human trials) offer to overcome limitations in the availability of cells for transplantation. Despite these advances, methods to deliver these cells are still limited, in part by transplant site; implants often fail within 5-10 years.
In this project I will further develop a means of transplanting islets to the promising subcutaneous (under the skin) site using a vascularizing hydrogel “platform” (“MAA-PEG”) as a therapy for T1D. Currently in the clinic, islets are transplanted to the hostile liver site, leading to significantly early cell loses and precluding long term graft survival. On the other hand, the subcutaneous space is less hostile, more easily accessed (to retrieve, monitor, or modify grafts in place) and generally safer for patients than sites like the liver (there are fewer expected complications). The subcutaneous space does not normally have the blood vessels needed to support islets so it has not typically been available as a transplant site for other researchers or clinicians. Together with the Sefton lab I have already shown that islets injected into the subcutaneous space in MAA-PEG survive and reverse diabetes in mice.
However, in recent work we found that islets can only survive with MAA at the subcutaneous site if we dampen inflammation (i.e., deplete neutrophils) around the time of surgery. Neutrophils hinder graft survival but are also important in maintaining health and preventing infection (among other uses) and so depletion is not a clinical option. In this project I will develop an alternative, clinically appropriate protocol to dampen early subcutaneous inflammation (neutrophils) that will re-enable access to the vascularizing subcutaneous space as a site for islet transplantation. This protocol will be of interest to the diabetes research community; our findings on neutrophil depletion have already benefitted the experiments of other JDRF research groups.
I will further build on the MAA-PEG transplant platform to show that added tolerogenic dendritic cells can prolong islet survival by preventing rejection from the host immune system. Tolerogenic dendritic cells are regulatory immune cells that can “re-train” other immune cells and provide an infection-like protective effect, “infectious tolerance.” These tolerogenic dendritic cells are already being tested in the clinic as a therapy for autoimmune diabetes. Our work here will be the first use of these cells to generate a tolerogenic response to prevent immune attack and rejection of a vascularizing subcutaneous islet graft. The results of these experiments combining tolerogenic dendritic cells, islets, MAA-PEG and anti-inflammatory mitigation at the subcutaneous site will provide a pre-clinical basis for the development of an immunosuppression-free, subcutaneous islet transplantation therapy for diabetes.
My project will benefit regenerative therapies for diabetes through the continued development of the MAA-PEG hydrogel platform. My belief is that a form of MAA-PEG could be used with existing practices to make a substantive clinical impact in the short run. This project will also further highlight MAA-PEG as a useful tool for pre-clinical diabetes research; others may find the simplicity of MAA-PEG useful in developing their technologies (e.g., testing new forms of stem beta cells through MAA-PEG transplant experiments). The results of our experiments will also provide useful references for future development of regenerative therapeutics for diabetes.