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6-Month Project Update
we have made significant progress on our project over the past months. The goal is to investigate whether PD-L1 expression on stem cell-derived beta-like cells (sBCs) can protect against recurring autoimmunity, providing a stable source of insulin-producing cells for cell replacement therapy. Previous research has shown that PD-L1 expression on sBCs can prevent allo and xenograft rejection, but its effectiveness in an autoimmune environment is still unknown. To address this, we have developed novel in vitro and in vivo models to study the interaction between immune T cells and pancreatic beta cells, supported by an R01 MPI grant with Dr. Ed Phelps. Our initial findings indicate that PD-L1 expression on sBCs can reduce the stimulation of autoreactive T cells, but not completely suppress them, suggesting the need for further investigation into whether PsBCs can fully protect against autoimmunity.
We have made progress on several fronts. First, we developed a dox-inducible system to control PD-L1 expression on sBCs, but it showed incomplete protection. To improve this, we tested different promoter strategies to achieve high and uniform PD-L1 expression, resulting in a new cell type, cPsBC, which also expresses firefly luciferase for easy quantification. Second, we established a protocol for cryopreserving sBCs, allowing us to create frozen banks of various genotypes for co-culture assays. Third, we advanced our in vivo models, using NOD HHD mice that express human HLA-A2 and found that while PsBCs showed some survival advantage, they were eventually lost, indicating PD-L1 alone is insufficient for complete protection. Finally, we have been refining our co-culture assays with different T cell populations to achieve reproducible results and reliable measurements of beta cell survival. We are focusing on human CD8 T cells with the 1E6 TCR and subpopulations from NOD HHD splenocytes. Our progress on these aims is laying the groundwork for future research into human autoimmunity and improving cell replacement therapies for Type 1 Diabetes.
Project Description
Patients suffering from Type 1 diabetes (T1D) lack functional insulin-producing pancreatic beta cells. The absence of beta cells is due to an autoimmune attack that results in their destruction. Transplantation of cadaveric beta cells represent a promising practical cure for T1D patients; however, donor shortage and immune rejection have hampered these efforts. Thus, generating an unlimited source of beta cells and protecting them from recurrent autoimmune destruction is crucial for making cell replacement therapy a reality for T1D patients.
In our lab we specialize in the large-scale generation of beta-like cells using directed stem cell differentiation, directly addressing the issue of donor tissue scarcity. However, beta-like cells are still susceptible to an immune attack. We have shown that stem cell derived beta-like cell (sBC) grafts transplanted in humanized autoimmune diabetes mice are readily rejected in a similar manner as is seen in T1D patients. For this reason, my project aims to perform genetic engineering in sBCs to protect them from an autoimmune attack. I propose to force the expression of immune check-point inhibitor PD-L1 in sBCs and then expose them to multiple autoimmune cells derived from T1D patients. Using this platform, I will determine if PD-L1 alone, or in combination with other genetic modifications, can fully protect sBCs from T1D patient’s immune cells.
Once I determine the genetic modifications required for successfully protecting sBCs, I will transplant sBCs into autoimmune diabetic humanized mice. The immune system of these mice has been trained to recognize human cells as self and won’t attack sBCs unless autoimmune diabetes occurs. This mouse model is one of the most accurate representations of human autoimmune diabetes. Using this innovative approach, I will study 1) the potential of genetically modified sBCs to reverse diabetes and 2) if genetically modified sBCs survive an autoimmune attack.
This project will tackle two critical aspects of T1D cell replacement therapy, namely the production of unlimited beta cells, and how to protect sBCs from recurrent autoimmunity. If successful, I anticipate that results from my studies will pave the way for a transformational impact in the field of cell-based replacement therapies to cure T1D.
