Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Shirwan, Haval

Committee Co-Chair (if applicable)

Yolcu, Esma

Committee Member

Bodduluri, Haribabu

Committee Member

Kosiewicz, Michele

Committee Member

Ratajczak, Mariusz

Author's Keywords

SA-FasL; islet; Tregs; biomaterials; scaffolds; microgels

Abstract

Signaling through Fas/FasL is critical to immune homeostasis and tolerance to self-antigens. SA-FasL is a chimeric protein of FasL and streptavidin. SA-FasL exists as oligomers with potent apoptotic function on Fas expressing immune cells and tightly binds to biotinylated surfaces. Islet grafts engineered to transiently display SA-FasL on their surface established tolerance in allogeneic recipients with a short course of rapamycin. We hypothesized that SA-FasL on the islet allograft will induce apoptosis in alloreactive T effector (Teff) cells and phagocytes clearing apoptotic bodies will produce tolerogenic molecules, such as TGF-β, that will lead to the generation and/or expansion of Treg cells at the induction phase. Treg cells will then home to allografts in response to inflammatory cues and will be maintained in the graft by alloantigens for long-term graft protection. In support of this hypothesis, we demonstrated reduced alloreactive T cells in the draining lymph nodes of SAFasL- engineered grafts as compared to controls. Depletion of phagocytes or blocking TGF-β peri- and immediate post-transplantation of allogeneic SA-FasLislet grafts abrogated tolerance as the grafts were rejected acutely. Systemic nature of the tolerance at the induction phase was shown by demonstrating that both un-engineered as well as SA-FasL-engineered islet grafts simultaneously transplanted under the contralateral kidney capsules survived indefinitely. SA-FasL-islets had characteristics of immune privilege as chemical destruction of the long-term graft followed by the transplantation of an un-engineered graft at the same location 4 days later resulted in the protection of the second set graft. We also tested whether tolerance to allografts requires physical presence of SA-FasL on the graft. To investigate this, we engineered biotinylated poly ethylene glycol (PEG) hydrogel or poly(lactic-co-glycolide) (PLGA) scaffolds with SA-FasL and co-transplanted with unmanipulated allogeneic islets under the kidney capsule or epididymal fat pad, respectively, resulted in indefinite ( > 200 days) islet survival. Flow cytometry revealed increased amounts of Tregs in the graft and draining lymph nodes in the PEG model. Mice rejected these grafts when Tregs were depleted. Taken together, the studies presented herein elucidate the mechanistic basis of SA-FasL-mediated tolerance and show that SA-FasL-engineered biomaterials are also effective in inducing tolerance.

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