Objective
The ultimate objective of the present project is to modulate human -cells to make them more resistant and less “visible” to the immune system. Increased expression of HLA Class I and antigen presentation to the immune system are key steps for immune-induced -cell killing, but -cells attempt to defend themselves by expressing molecules such as PDL1 that block the attack by the invading immune cells. Of concern, both HLA Class I and PDL1 are regulated at least in part by similar molecules and intracellular signals, rendering it difficult to inhibit HLA Class I without also inhibiting the protective PDL1 molecule. The Eizirik group has shown that this is actually possible – by inhibiting one transcription factor, STAT2, and then exposing human -cells to interferon- they observed up-regulation of the protective PDL1 in parallel to down-regulation of HLA Class I. Departing from this proof of principle, Eizirik and the biotech company InSphero will join forces to develop a platform to identify novel agents that dissociate HLA Class I expression from PDL1 expression and then apply the platform to screen, test and ultimately validate new therapeutic agents to protect β-cells in T1D. They will utilize two complementary human pancreatic islet models: 1. Islet-like cells differentiated from inducible pluripotent cells (iPSC), a model of “early development β-cells” relevant for the early stages of the autoimmune process, already in use by the Eizirik group; and 2. Human islet microtissues (hIsMT), a robust and reproducible model of adult human islets developed by InSphero via gentle enzymatic dissociation and scaffold-free reaggregation of isolated human islets. The project will pursue three aims:
1. In Aim 1 key methodologies will be established at InSphero with the help of the Eizirik group, and previous results generated by the Eizirik group in iPSC-derived islet-like cells will be reproduced in hIsMTs at InSphero;
2. In Aim 2, two already identified agents (i.e., STAT2 knockdown and metformin) plus novel agents (identified by promoter studies and in silico drug mining) with the potential to inhibit IFN-induced HLA Class I up-regulation while enabling up-regulation of PDL1 in human β-cells will be tested in both islet models;
3. In Aim 3, the hIsMT platform will be adapted to be screening-compatible and utilized to identify novel agents involved in HLA Class I and PDL1 dissociation, with a subset to be subsequently validated in both systems. This platform will be open to utilization by Pharma for larger screenings in search of novel therapies for T1D.
Background Rationale
Stress pathways triggered within β-cells initiate and/or accelerate autoimmune β-cell destruction and multiple layers of evidence implicate interferon (IFN) signaling as a key component of T1D pathophysiology. The equilibrium between HLA Class I and PDL1 expression is of particular interest - β-cells in T1D typically overexpress HLA Class I, increasing presentation of islet antigens to the immune system, while IFN- and IFN- induce the expression of PDL1 in β-cells, protecting them at least in part, and temporarily, against immune attack. Of concern, inhibition of targets such as JAK or TYK2, that prevent IFN signalling and are currently under investigation as a treatment for T1D and other autoimmune diseases, leads to inhibition of both HLA Class I expression and PDL1. Against this background, the identification of agents that inhibit HLA Class I up-regulation while enabling up-regulation of PDL1 in β-cells would constitute a valuable new class of T1D therapeutics. We will utilize two complementary human model systems to achieve this goal: 1. Islet-like cells differentiated from inducible pluripotent cells (iPSC), a model of “early development β-cells” relevant for the early stages of the autoimmune process, already in use by the Eizirik group; and 2. Human islet microtissues (hIsMT), generated via gentle enzymatic dissociation and scaffold-free reaggregation of isolated human islets which provide a robust and reproducible model of adult human islets and are developed by InSphero.
Description of Project
Type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing pancreatic -cells. Therapeutic targeting of the immune system alone is not sufficient to prevent disease, emphasizing the need for complementary approaches to directly protect the β-cells, prevent their death and decrease stimulation of the immune system. The early steps of T1D involve local release of pro-inflammatory mediators (cytokines) from immune cells that infiltrate the pancreatic islets where -cells are found. Two of these mediators – key ones at different stages of the disease – are interferon-α (IFN-α; active at the early stages of T1D) and interferon- (IFN-, active at the later stages of T1D). Exposure of human pancreatic islets to IFNs induces three hallmarks of T1D, namely endoplasmic reticulum stress (a form of stress that contributes to -cell death), release of chemokines (proteins that attract cells of the immune system that will harm the -cells) and up-regulation of HLA Class I (which facilitates presentation of -cell autoantigens to the immune system, particularly to the cytotoxic CD8+ T-cells that do most of the pancreatic -cell killing). On the other hand, IFNs also induce expression of protective molecules, particularly PDL1 that functions as an inhibitor of the CD8+ T-cells expressing its receptor, PD1. PDL1-PD1 also protects cancer cells against immune assault, and the discovery that inhibitors of PDL1-PD1 have therapeutic value for some advanced cancers led to the Nobel prize in Medicine of 2018. A fraction of individuals affected by cancer and treated with these inhibitors develop endocrine autoimmune diseases, including T1D, confirming that PDL1 has indeed a role in protecting -cells against the immune system. The molecular mechanisms regulating HLA Class I and PDL1 expression in human β-cells remain to be clarified and the identification of agents that inhibit HLA Class I up-regulation while enabling up-regulation of PDL1 in β-cells would constitute a valuable new class of T1D therapeutics.
To achieve this objective, we will first develop a platform to identify novel agents that dissociate HLA Class I expression from PDL1 expression and then apply the platform to screen, test and ultimately validate new therapeutic agents to protect β-cells in T1D. We will utilize two complementary human pancreatic islet models: 1. Islet-like cells differentiated from inducible pluripotent cells (iPSC), a model of “early development β-cells” relevant for the early stages of the autoimmune process, already in use by the Eizirik group; and 2. Human islet microtissues (hIsMT), a robust and reproducible model of adult human islets developed by InSphero via gentle enzymatic dissociation and scaffold-free reaggregation of isolated human islets. The project will pursue three aims:
1. In Aim 1 key methodologies will be established at InSphero with the help of the Eizirik group, and previous results generated by the Eizirik group in iPSC-derived islet-like cells will be reproduced in hIsMTs at InSphero;
2. In Aim 2, two already identified agents (i.e., inhibitors of the transcription factor STAT2 and metformin) plus novel agents (identified by promoter studies and in silico drug mining) with the potential to inhibit IFN-induced HLA Class I up-regulation while enabling up-regulation of PDL1 in human β-cells will be tested in both islet models;
3. In Aim 3, the hIsMT platform will be adapted to be screening-compatible and utilized to identify novel agents involved in HLA Class I and PDL1 dissociation, with a subset to be subsequently validated in both systems. This platform will be open to utilization by Pharma for larger screenings in search of novel therapies for T1D.
Anticipated Outcome
We expect that the presently proposed project will generate the following anticipated outcomes:
1. The development of a platform to identify novel agents that dissociate HLA Class I expression from PDL1 expression.
2. The application of this novel platform to screen, test and ultimately validate at least 3 new therapeutic agents with the potential to protect β-cells in T1D. By protecting -cells and by inhibiting the deleterious “dialogue” between -cells and the infiltrating immune cells, these novel agents will introduce a new paradigm for the treatment and/or prevention of T1D, namely boosting -cell defenses instead of the usual approach of targeting the immune system only.
3. Upgrade and make the platform accessible to Pharma companies interested in developing novel approaches to treat T1D (see for instance the letter of support by Novo Nordisk), which will hopefully stimulate additional Pharma companies to join the field and, working together with academic researchers, accelerate the advancement of a cure for T1D.
Relevance to T1D
Pioneering research by the applicants introduced a paradigm shift in the field, indicating that the “battle” leading to β-cell loss in diabetes is fought, to a large extent, inside the β-cells, and leads to a “dialogue” with the invading immune cells that amplifies the immune assault. A key component of this dialogue is the presentation of -cell neoantigens in the context of HLA Class I to the immune system. Most of the research efforts for the discovery of novel treatments for T1D has focused on the immune system, and innovative therapies that protect -cells and block this deleterious -cell-immune system dialogue remain to be discovered. The presently proposed approach, focusing on the mechanisms that regulate both human pancreatic -cells up-regulation of HLA Class I and consequent presentation of autoantigens to the immune system, and of protective molecules such as PDL1 will clarify key steps in the “misguided dialog” between -cells and the immune system that eventually culminates in T1D and thus opens the door for the development of novel therapies for the disease.
This proposal is a very good fit for the present call, which aims to accelerate the development of therapies to prevent, slow, halt or reverse the progression of T1D through the advancement of high-impact research carried out by two highly complementary teams. Indeed, a successful completion of this collaborative project will allow the development of a platform to identify novel agents that dissociate HLA Class I expression from PDL1 expression and to apply the platform to screen, test and ultimately validate at least 3 new therapeutic agents with the potential to protect β-cells in T1D. By protecting -cells and by inhibiting the deleterious “dialogue” between -cells and the infiltrating immune cells, these novel agents will introduce a new paradigm for the treatment and/or prevention of T1D, namely boosting -cell defenses instead of the usual approach of targeting the immune system only. Importantly, the platform to be developed by InSphero in collaboration with Eizirik will be of clear value to Pharma interested in developing novel approaches to treat T1D (see the letter of support by Novo Nordisk) and will hopefully stimulate new companies to join the field and, together with academic researchers, accelerate the advancement of a cure for T1D.