Date of Award

1-1-2021

Embargo Period

8-14-2026

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Stephen Tomlinson

Second Advisor

Carl Atkinson

Third Advisor

Shikhar Mehrotra

Fourth Advisor

Christina Voelkel-Johnson

Fifth Advisor

Xue-Zhong Yu

Abstract

Immunosuppressants used in organ transplantation (Tx), including vascularized composite allograft (VCA) Tx, have had limited success controlling chronic rejection. They are also associated with multiple serious adverse effects. Therefore, there is an unmet need for therapies that maintain long-lived graft tolerance with minimal systemic effects. Donor brain death (DBD) induced injury and ischemia reperfusion injury (IRI) are major factors known to promote early graft inflammation, the severity of which is associated with poorer outcomes. Complement is activated and deposited within allografts following DBD and has been shown to exacerbate IRI, and we therefore explored two novel complement targeted strategies to reduce graft injury and promote tolerance. In our first approach we investigate the therapeutic potential of pretreating VCAs (hindlimb grafts) with CR2-Crry, a graft targeted fusion protein; Crry inhibits all complement pathways, while CR2 binds C3d, a complement activation product deposited on brain dead donor (BDD) grafts. VCAs were procured from BDD or living donor (LD) Balb/C mice, perfused in situ with CR2-Crry, then transplanted into C57BL/6 recipients after graft cold-storage. We first showed that CR2-Crry bound and persisted at significantly higher levels in grafts from BDD vs LD, both before and after Tx. Moreover, BDD VCAs showed more severe IRI and rejected earlier compared to LD VCAs. These effects were reversed with CR2-Crry pre-treatment of grafts. In our second approach, we investigated whether the same complement targeting strategy using CR2 can be utilized to direct Treg cells to graft deposited C3d. For this purpose, graft specific Tregs can be created by genetically engineering them to express allospecific chimeric antigen receptors (CAR). In our approach, however, we utilized CR2 as a CAR to target C3d, which is universally deposited in grafts regardless of donor-recipient alloantigens. To enforce a Treg phenotype, we also co-expressed FoxP3. Isolated mouse T-cells were genetically modified to express CR2-CAR-FoxP3 using retrovirus. Following several rounds of construct redesign to optimize expression and stability, we prepared CR2-CAR-FoxP3 Tregs that exhibited a stable and suppressive phenotype in vitro. These CAR Tregs, together with engineered controls cells were investigated in a cardiac transplant model and an experimental autoimmune encephalomyelitis model but failed to show a protective effect. Future studies should investigate the use of these cells in an adjuvant settin

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All rights reserved. Copyright is held by the author.

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Available for download on Friday, August 14, 2026

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