Objective
The goal of this application is to test the physiological relevance of programmed cell death-ligand 1 (PD-L1) protein cargo that is packaged by pancreatic beta cells into small membrane-bound nanoparticles called extracellular vesicles (EVs). EVs are released by cells and contain various molecules that can be transferred to or interact with other cells. We plan to test the idea that PD-L1 molecules may be increased by beta cells under certain stressful conditions to bind to and block immune cell attacks on beta cells. Experiments will also test the idea that these nanoparticles can be tested in blood to provide a window into changes that are happening at the level of the pancreatic beta cell in people at-risk for type 1 diabetes.
Background Rationale
Type 1 diabetes (T1D) is an autoimmune disease where the immune system mistakenly attacks the beta cells, the worker cells responsible for producing insulin in the pancreas. Proinflammatory cytokines, are small proteins that can act as disruptive forces creating stress in the beta cell working environment. However, some cytokine signals also work to suppress or calm down the immune response, which can help protect the stressed beta cells and potentially slow down the progression of the disease. These interactions between beta cells and the immune system that influence T1D development are incompletely understood. Extracellular vesicles (EVs) are small membrane-bound nanoparticles that are released by cells, including beta cells and can carry specific molecules that can influence immune responses and contribute to the autoimmune process in T1D. Stressed beta cells can undergo changes in their EV contents to attempt to protect themselves from the destruction caused by autoimmunity. These altered EVs may carry specific molecules that promote the protective mechanisms aimed at evading immune-mediated destruction. One such molecule that has been identified in other organ systems is PD-L1 (programmed death-ligand 1). PD-L1 and PD1 (programmed cell death protein 1) are key players in the immune checkpoint pathway which regulates immune responses and is also implicated in cancer. PD-L1 on the cell surface inactivates immune cells, allowing cancer cells to evade destruction by the immune system. FDA-approved immune checkpoint inhibitor treatments inhibit proteins such as PD‐L1, allowing for activation of an individual’s own immune system to combat cancer cells. Intriguingly, a known side effect of these drugs is beta cell autoimmunity and new onset T1D. PD-L1 is present on the beta cell surface and can protect beta cells from destruction by engaging with and inactivating surrounding immune cells. Furthermore, certain cytokines upregulate different forms of PD-L1 message arising from the same PD-L1 gene, encoding for molecules with distinct structures, with potentially different roles and function. However, the mechanisms by which the beta cells upregulate different PD-L1 message and protein and how this protein impacts the communication to surrounding immune cells is not known. Given the potential of EVs to play role in beta cell: immune regulation and intercellular communication in the islet microenvironment under pathophysiologic conditions, the goal of this project is to study how PD-L1 cargo is packaged into EVs and how they contribute to regulating the immune system. By gaining a deeper understanding of how EV PDL1 participates in the immune response in T1D, we can pave the way for using EVs as blood-based biomarkers of disease, and ultimately allow for new interventions to prevent autoimmune beta cell destruction.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing beta cells in the pancreas by the immune system. Proinflammatory cytokines, triggered by genetic or environmental factors, act as stress alarm signals that tell the immune system to attack the insulin-producing beta cells and lead to beta cell death. Extracellular vesicles (EVs) are tiny nanoparticles released by cells that contain various molecules, including proteins, lipids, and nucleic acids, that cells release to communicate to other tissue via differing content. Interestingly, cytokines can influence these messenger EVs by changing their content, potentially affecting EV functional properties, such as influences on immune responses. While most cytokines induce beta cell death, some cytokines also play a protective role in the beta cell's attempt to protect itself from autoimmune assault. To this end, cytokine-triggered increases in PD-L1 (programmed cell death-ligand 1) on the beta cell surface can protect beta cells from destruction by engaging with and inactivating surrounding immune cells. Consistent with this, therapies blocking PD-L1 can lead to autoimmune diseases, including T1D. However, the role of PD-L1 in beta cell EV has not been explored. Our preliminary findings show that EVs released by beta cells carry PD-L1 cargo on their surface, and specific types of inflammatory cytokine signaling induce multiple forms of PD-L1 in beta cells and their EVs. We also identified that EV PD-L1 can directly bind to immune cell receptors. Based on these observations we propose that inflammatory cytokine signaling alters the EV content to include increased PD-L1, which ultimately protects beta cells from destruction by inactivating infiltrating immune cells. The goals of this project are to 1) isolate and characterize molecular regulators of beta cell PD-L1 EV cargo and its ability to communicate and impact surrounding immune cells; 2) determine the relative contributions of different forms of PD-L1 in beta cells and EVs; and 3) identify changes of EV PD-L1 cargo in circulation during the natural history of T1D. This work will reveal novel mechanisms adopted by stressed beta cells in islet: immune cell communication to regulate beta cell survival during the T1D disease course and provide training in beta cell and EV biology to a promising young scientist.
Anticipated Outcome
We anticipate that stressed beta cells under proinflammatory conditions modeling the environment of early developing type 1 diabetes (T1D) will upregulate different forms of PD-L1 protein cargo into extracellular vesicles (EVs) released into the extracellular space and blood, and that the PD-L1 on the EV surface will directly engage with and inactivate T cells. Elevations in EV PD-L1 cargo will be detectable in blood in individuals at-risk for T1D. These findings will lay the groundwork for future studies using circulating EV PD-L1 to predict T1D and dissect differences in T1D clinical outcomes.
Relevance to T1D
Understanding mechanisms inhibiting immune cell activation in type 1 diabetes (T1D) may ultimately allow for the development of new interventions exploiting these mechanisms to block autoimmune beta cell destruction. Additionally, because extracellular vesicles (EVs) and their cargo are extremely stable in circulation, changes in EV cargo that occur as T1D develops could be useful as noninvasive “liquid biopsies” or biomarkers for active beta cell attack and T1D prediction. Furthermore, the concept of stressed beta cells utilizing EVs as a means of evading immune destruction holds promise for developing novel therapeutic approaches for T1D.