Objective
Our overall hypothesis is that deletion of TET2 from human induced pluripotent stem cell derived beta cells will render them resistant to immune mediated killing. Our objective is to create TET2-deficient human iPSC-derived beta cells and to test their resistance to inflammatory mediators and immune cell mediated killing.
Background Rationale
Beta cell replacement would enable reversal of T1D. However, long term immune suppression, that has been required for transplantation of islet allografts carries risks that render it unacceptable in most patients with T1D. Moreover, the transplanted islet allografts to not necessarily maintain insulin independence. A way to render islet cells resistant to immune mediated killing would enable them to survive long term without the need for continuous immune suppression.
There is increased expression of TET 2 in human beta cells in settings of immune stress. Knockout of Tet2 in mice makes the islets resistant to immune (cell) mediated killing and the effects of cytokines. Tet2 is important in responses to IFNg that is thought to be a critical mediator of beta cell killing in T1D.
Description of Project
Our overall hypothesis is that deletion of TET2 from human induced pluripotent stem cell derived beta cells will render them resistant to immune mediated killing. This proposal represents a collaboration between the New York. Stem Cell Foundation (NYSCF) and Dr. Kevan Herold's lab at Yale University. TET2 (Tet2) is a gene whose proteins play an important role in inflammatory responses. We found that Tet2 deficient mice were resistant to autoimmune diabetes and the effects of inflammatory mediators. We postulate that as in other model systems, loss of Tet2 renders cells resistant to cytokines that can kill beta cells, such as IFNg but also to cell mediated killing. In our preliminary studies we have found that human Embryonic Stem cell derived beta cells show reduced inflammatory responses when TET2 is deleted by CRISPR/Cas9. In this proposal, we plan to test the effects of deletion of TET2 in human induced pluripotent stem cell derived beta cells on their function and resistance to immune mediated killing. To avoid the heterogeneity of growth and differentiation of iPCs into beta cells we plan to utilize existing lines that have been derived from healthy controls (n=2) and from 3 patients with Type 1 diabetes. These lines are available in the laboratory at the NYSCF.and the expertise to use CRISPR/Cas9 to delete the TET2 gene is available. The expansion and differentiation of the iPSC derived beta cells will be done at the NYSCF with studies in vitro and in vivo in Dr. Herold's lab. These labs are in close proximity (by train) and have shared technologies for the proposed studies. If successful, these studies will establish a proof of concept for development of inflammation resistant beta cells that may be used in the future for islet replacement therapy.
Anticipated Outcome
We anticipate that the TET2 deficient beta cells will grow normally and respond normally to metabolic stimuli. We also anticipate that these cells will be resistant to killing by immune cells and cytokine. We expect that the responses to inflammatory mediators will not be absolute will require considerably higher levels of exogenous cytokines to induce cell killing. We also anticipate that the cells will be resistant to killing in vivo.
Relevance to T1D
Restoring normal metabolic control without the need for exogenous insulin requires replacement of lost beta cells. However, the need for continuous immune suppression to prevent the rejection of islet grafts carried significant risks and in addition, the survival of transplanted islets is not sufficient to render most recipients insulin independent. This proposal is testing in human cells, the proof of concept, first made in murine cells, that deleting TET2, a gene that is centrally involved in inflammatory responses, can render beta cells resistant to immune mediated killing. The proposal represents a collaboration between laboratories with expertise in stem cell biology and differentiation of beta cells (New York Stem Cell Foundation) and in autoimmunity of diabetes (Kevan Herold MD at Yale). if successful, these studies would identify a strategy that could be incorporated into beta cell replacement cells that are now in development at many laboratories. In the future, we hope that the sequential combination of an agent to silence the autoimmune responses (e.g. teplizumab) with the inflammation resistant beta cells will lead to long standing reversal of the disease with out the need for chronic immune suppression.