Immunomodulation with SA-PDL1 protein on pancreatic islets promotes indefinite graft survival in allogeneic recipients
Lalit Batra1, Pradeep Shrestha1, Hong Zhao1, Kyle B. Woodward1, Alper Togay1, Min Tan1, Orlando Grimany-Nuno1, Mohammad Tariq Malik1, María M. Coronel2, Andrés J. García2, Haval Shirwan1, Esma Yolcu1.
1Department of Microbiology and Immunology, University of Louisville, Louisville, KY, United States; 2Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, United States
Introduction: The broad application of allogeneic islets for the treatment of type 1 diabetes is curtailed by the adverse effects of chronic immunosuppression used to control rejection. PD-1/PD-L1 axis plays a critical role in regulating immune responses to self and foreign antigens. Tumor cells effectively evade the immune system by engaging PD-L1 expressed on their surface with PD-1 on the surface of activated immune cells. We herein tested whether the transient display of PD-L1 protein on the surface of allogeneic islets simulates cancer immune evasion for sustained survival and function in mice without chronic immunosuppression.
Materials and Methods: A synthetic chimeric gene consisting of the extracellular domain of PD-L1 protein and core streptavidin (SA) was expressed in insect cells. The chimeric protein, SA-PDL1, was purified and characterized for structure using western blots and function using in vitro T cell assays. BALB/c islets were isolated, surface modified with biotin, and decorated with SA-PDL1 protein (~200 ng/1000 islets) by taking advantage of high-affinity interaction between SA and biotin. Engineered islets were transplanted under the kidney capsule of chemically diabetic C57BL/6 mice with a short course of rapamycin (0.2 mg/kg daily for 15 doses). Graft survival and function were assessed with non-fasting blood glucose monitoring and IPGTT. Flow cytometry, qRT-PCR, and immunohistochemistry were performed to elucidate the mechanistic basis of SA-PDL1-mediated immunomodulation.
Results and Discussion: SA-PDL1 protein formed oligomers and facilitated the in vitro conversion of T conventional cells into CD4+FoxP3+ Treg cells as well as blocked the proliferation of alloreactive T cells. Islets were effectively modified with biotin and engineered with SA-PDL1 protein without a negative impact on their viability and function. SA-PDL1-engineered islet grafts under a transient course of rapamycin achieved sustained survival (~90%) and function in allogeneic recipients over a 100-day observation period. Graft survival was associated with increased levels of intragraft transcripts for anti-inflammatory and regulatory factors, including IDO-1, arginase-1, FoxP3, TGF-β, IL-10, and decreased levels of pro-inflammatory factors T-bet, IL-1b, TNF-α, and IFN-ϒ. Long-term graft recipients generated a proliferative T cell response to donor antigens at a similar magnitude to T cells of naïve animals, suggestive of the localized nature of immune protection. Long-term grafts also had a high number of peri-islet Treg cells and systemic depletion of this cell population resulted in prompt graft rejection.
Conclusion: The transient display of SA-PDL1 protein on the surface of islet grafts serves as a practical and effective means of achieving sustained survival with potential clinical implications.
This study was funded in part by NIH (grants R21EB020107, R01AI121281, U01AI132817 and NIH T32 HL134664) and KSTC (KSEF-2927-RDE-016).
We thank Drs. H. Waldmann of Oxford University and TV Brennan of Duke University for provding us with transgenic mice used in this study..
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