536 Engineering an In Vitro Platform for Characterization of Antigen Recognition for Encapsulated Islets
Tuesday November 17, 2015 from 15:30 to 16:30
Room 110

Anthony W. Frei, United States

PhD Student

Biomedical Engineering

University of Florida


Engineering an In Vitro Platform for Characterization of Antigen Recognition for Encapsulated Islets

Ethan Yang2,3, Anthony Frei1, Cherie Stabler1.

1J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, United States; 2Diabetes Research Institute, University of Miami, Miami, FL, United States; 3Biochemistry and Molecular Biology, University of Miami, Miami, FL, United States

Alginate has been extensively used as an encapsulation material for islet transplantation, with strong immunological protection exhibited in allograft rodent models. Despite this success, the mechanisms by which biomaterials block antigen recognition and impact graft outcomes remain poorly understood. While the encapsulating material can block direct antigen presentation, the potential for indirect immune activation through shed antigen is still present, and this smoldering response could contribute to graft failure in xenografts and large animal models. Herein, we sought to establish an in vitro platform capable of delineating the contribution of direct and indirect antigen recognition on subsequent immune activation. With a focus on alginate encapsulation, the capacity of alginate to block direct and indirect antigen presentation was evaluated, as well as the impact of immune cell activation on encapsulated cell viability and function. The potential of this platform to understand mechanisms of immune activation for alginate hydrogels, and to serve as a screening tool for new encapsulation approaches, is discussed.

For this study, ovalbumin (OVA) was selected as the model antigen and islets isolated from membrane-bound ovalbumin (mOVA) mice were used. mOVA Islets were encapsulated within 1.6% w/v Pronova UP-MVG (Novamatrix) and co-cultured with responders (splenocytes from OTI mice) to evaluate antigen specific activation via flow cytometry for proliferation (Cell Trace) and activation (Granzyme B) after 2, 3 and 5 days. The impact of activation on cell viability (live/dead confocal microscopy imaging) and function (GSIR) was also evaluated.

Co-culture of OTI splenocytes with unencapsulated mOVA islets resulted in significant proliferation and activation of CD8+ T cells after 2 days, with 80.6 ± 15.1% of the proliferating CD8+ viable T cells expressing granzyme B, demonstrating that islets serve as potent stimulators. Further, the resulting effector T cells imparted a substantial islet loss, with extensive fragmentation and decreased viability. Conversely, co-culture of alginate encapsulated mOVA islets with OTI splenocytes also resulted in extensive CD8+ T cell activation (81.7 ± 16.5%) comparable to free mOVA islets (p = 0.92). Remarkably, the impact of effector T cells in the surrounding milieu on islet viability was minimal. Encapsulated islets were found to be robust and viable, with no discernable difference to untreated controls, indicating that the alginate was able to protect the islets from effector T cell destruction during the time period evaluated. The specificity of CD8+ T cell activation was confirmed by co-culture of free or encapsulated B6 islets with OTI splenocytes, which resulted in no significant T cell activation. These results establish the antigen specific nature of the platform, as well as inertness of the alginate hydrogel.

In conclusion, this study outlines the development of an in vitro platform to study the effect of islet encapsulation on indirect single antigen activation. For the first time, we demonstrate the inability of standard alginate encapsulation to block indirect antigen activation, although this activation did not result in subsequent destruction of the encapsulated islet. With this promising platform, future work will further characterize the mechanisms of antigen release and activation within encapsulation, as well as explore manipulation of these immune responses toward desired tolerogenic pathways.

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