Objective
As presented within the Summary section, our laboratory has pioneered the development, and use, of antigen (Ag)-specific poly(lactide-co-glycolide) (PLGA) nanoparticle-based Tolerogenic Immune Modifying Particle (TIMP) therapy for the restoration of immunological self-tolerance. To date the majority of the work completed has focused on the ability of Ag-specific TIMP treatment to increase CD4+ regulatory T cells, while decreasing inflammatory CD4+ T cells. In complex autoimmune diseases, such as T1D, other T cell populations, i.e., CD8+ T cells, have been shown to be present within the pancreas of both people living with T1D and NOD mice. Therefore, it is hypothesized that CD8+ T cells play a role in both the initiation and progression of T1D. The first objective of the present grant proposal is to determine the ability of Ag-specific TIMP treatment to regulate a CD8+ T cell-induced mouse model of T1D. Our preliminary data presented within the grant proposal show that treatment of mice with TIMPs containing peptides that either the disease-inducing CD8+ T cells respond to, or a peptide derived from the same protein from which the CD8+ T cells are specific for, is able to significantly modulate the CD8+ T cell-induced responses in vivo. Therefore, we will determine how Ag-specific TIMP treatment modulates both CD8+ T cells and CD4+ T cells depending on the specific peptide (either specific for the CD8+ T cells or specific for the CD4+ T cells) changes the number, location, and phenotype of these disease-inducing T cells over time. Given our ability to modulate the T cells responses over time, we will also utilize the present experimental model system and Ag-specific TIMP treatment as a tool to assess the progression of T cell specificities present within the pancreas during various stages of disease. The second objective of the present grant proposal is to determine if the initial short term CD8+ T cell-induced inflammation within the pancreas is sufficient to activate T cells that will continue and propagate further disease progression. In doing so, we will address mechanisms by which people living with T1D may experience an early relapse-remitting course of disease following the initial diagnosis, and if Ag-specific therapies, such as Ag-specific TIMP treatment, directly or indirectly inhibit these mechanisms.
Background Rationale
To date we have addressed the functional activity of antigen (Ag)-specific poly(lactide-co-glycolide) (PLGA) nanoparticle-based Tolerogenic Immune Modifying Particle (TIMP) treatment in multiple autoimmune disease models. We have been able to identify the overall functionality of TIMP treatment with regard to CD4+ T cell function. However, given the complexity of the autoimmune response responsible during autoimmune disease, exemplified by the progression of T1D, the presently proposed studies are designed to further define the following key questions. 1) What is the phenotype of the Ag-specific CD4+ T cells and CD8+ T cells induced follow treatment with TIMPs containing CD4+ T cell versus CD8+ T cell epitopes? 2) Are these T cells located only within the spleen, or do the cells migrate to the pancreas and pancreatic lymph nodes to regulate tolerance within the target tissue? 3) Which regulatory protein(s) are required for the induction/maintenance of tolerance? 4) Are TIMP-induced regulatory CD4+ T cells found to be spatially located near and in contact with the CD8+ T cells and/or dendritic cells? 5) Is the secondary dysglycemia due to a loss of CD4+ T cell control of the inflammatory CD8+ T cells, or retention within the spleen? 6) Alternatively, is epitope spreading responsible for the eventual occurrence of secondary dysglycemia? 7) Can the re-dosing of mice with the initial Ag-specific TIMP allow for the maintenance of tolerance over time? 8) And lastly, can co-treatment with TIMPs containing the spread epitope diabetogenic proteins inhibit the secondary dysglycemia?
Description of Project
Aberrant immune reactions against self or environmental antigens (Ags) are the main drivers for autoimmune and allergic diseases respectively, and both autoimmune and allergic diseases are increasing in number both within the United States and worldwide. For autoimmune diseases, and more specifically, type 1 diabetes (T1D), the current standard of care involves administration of therapeutics targeting disease symptoms or broadly suppressing the inflammatory immune response leaving patients vulnerable to opportunistic infections and cancers. Presently, there are no available therapies for people living with T1D that modulate the activity of the primary driver of disease development and progression, i.e., self-reactive immune cells. Initiation and progression of T1D is likely due to activation of T cells specific for multiple β-islet cell expressed diabetogenic proteins. To test this, we have previously tested the ability of poly(lactide-co-glycolide) (PLGA) nanoparticle-based Tolerogenic Immune Modifying Particle (TIMP) containing diabetogenic proteins to inhibit the development of T1D in the non-obese diabetic (NOD) mouse model of T1D. While treatment of NOD mice with TIMPs containing a single protein did not inhibit disease, treatment of mice with TIMPs containing three proteins (TIMP-T1D) did result in a significant decrease in T1D development. The present grant proposal will address key therapeutic design and mechanism of action questions for the explicit purpose of restoring self-tolerance within the immune system of people living with T1D. To do so, our research group has developed a TIMP platform constituting an off-the-shelf treatment for induction of tolerance to specific Ag(s) to inhibit undesired immune responses, while not affecting the protective elements of the immune response. We have pioneered an approach in which intravenous administration of TIMPs loaded with disease-associated proteins/peptides induces tolerance to the protein/peptide contained within the TIMP. As we have recently published that TIMP treatment is both safe and induced Ag-specific tolerance in a Phase 1/2a celiac disease clinical trial, Ag-specific tolerance induced by nanoparticles encapsulating multiple diabetogenic proteins is a promising approach to T1D treatment. The overall hypothesis that Ag-specific TIMP induced CD4+ Tregs from the same autoantigen control autoreactive CD8+ T cells indirectly via CTLA-4 and PD-1 thereby removing APC help will be tested.
Anticipated Outcome
We anticipate that antigen (Ag)-specific poly(lactide-co-glycolide) (PLGA) nanoparticle-based Tolerogenic Immune Modifying Particle (TIMP) treatment will induce a stable population of Ag-specific regulatory T cells. While we have generated sufficient data to support this hypothesis, the cellular function in the context of a CD8+ T cell-mediated autoimmune disease has not been characterized to date. While the present experimental model is very robust and the initiation of disease is rapid, the preliminary data generated to date are compelling. We presently hypothesize that prophylactic treatment with Ag-specific TIMPs will completely block the initiation of disease, while the therapeutic treatment scheme shows that T cells of various specificities have been activated during the initial stage of disease. Therefore, if our preliminary data are correct, then the later treatment of mice with TIMPs containing the proteins/peptides that T cells activated during the first phase of disease respond to will block the later secondary disease. Together, these data will support the conclusion that both blockade of disease-associated target tissue inflammation and induction of Ag-specific tolerance to the ongoing autoimmune response are sufficient for the restoration of immune tolerance.
Relevance to T1D
Our laboratory has pioneered the development of antigen (Ag)-specific therapies. Autoimmune diseases, such as multiple sclerosis (MS), psoriasis, rheumatoid arthritis, and type 1 diabetes, are a prevalent cause of human morbidity and mortality may affect up to 1 in 10 people within the population. In these disease states, a failure in immune regulation of autoreactive CD4+ T cells results in T cell–mediated destruction of self-tissues. Critically, the pre-clinical mouse model findings have recently been successfully transferred into Phase I/IIa clinical trials for the treatment of celiac disease. These published data show that treatment with TIMPs is both safe and specifically inhibited the induction of inflammatory cytokine secretion by CD4+ T cells post gluten challenge. While in other studies outside of the present grant proposal are focused on the cellular mechanisms by which Ag-specific TIMP treatment induces a significant increase in the number of Ag-specific regulatory CD4+ T cells, the present proposal focuses on the ability of Ag-specific TIMP treatment to modulate CD8+ T cells. Besides the relevant data that will be obtained with successful completion of the proposed studies for clinical translation for patients living with T1D, the present model system may allow for increased understanding of the progression of T1D. Successful completion of the presently proposed studies is hypothesized to support the conclusion that activated CD8+ T cell within the disease-associated target tissue may induce β-islet cell quiescence and eventual cell death. It is this β-islet cell death that would allow for the presentation of β-islet cell Ags by antigen-presenting cells within the pancreas resulting in the activation of additional Ag-specific T cells, thereby resulting in the progression of disease. Data from others point to the presence of inflammatory cytokines within the pancreas and β-islets inducing a decrease in insulin production by β-islet cells. If correct, the present model system may also be used to identify druggable pathways for the development of co-therapies to be used in combination with Ag-specific TIMP therapy to target both β-islet cells directly, as well as inducing Ag-specific tolerance within the immune system.