Poster CC12 - Elucidating the Impacts of Shape and Stiffness on CD8+ T cell Activation Using Particulate Artificial Antigen Presenting Cells

Introduction: Decades of encouraging yet incomplete responses from cytotoxic (CD8+) T cell-based cancer immunotherapies have invigorated recent efforts to develop new biotechnologies that can better modulate the behavior of immune cells, giving rise to artificial antigen presenting cells (aAPCs). These acellular platforms are modeled after natural antigen presenting cells, such as dendritic cells (DCs), that deliver signals to prime and activate T cells against tumor cells. Biomaterial-based aAPC constructs are an attractive choice for studying, tuning, and optimizing the mechanisms of T cell activation. While certain key aAPC parameters, such as ligand surface density, have been investigated and found to be important for aAPC/T-cell stimulation, the physical parameters of the particle have not been well investigated, yet are critical features of acellular encounters (1-3). Previous literature indicates that shape plays a major role in the effectiveness of particulate aAPC systems (2,4-5). There is also evidence that elasticity exerts influence over the proliferation and functionality of T cells in 2D and 3D aAPC systems (1,6-7) and in vivo particle pharmokinetics (7-9), but the impact on 3D particulate aAPC effectiveness in relation to the combined parameters of particle shape and stiffness has not yet been previously investigated. The objective of this study is to synthesize novel nano-aAPCs of variable shape and stiffness, to investigate their impacts on T cell activation and quality, ultimately with the hope of generating a more effective and durable anti-tumor T cell responses.

Materials and

Methods: To study CD8+ T cell responses, we have recently developed micro-scale aAPCs of tunable stiffness using a biocompatible Poly(ethylene Glycol) (PEG) base (7). To study the interplay between stiffness and shape, this platform was optimized for nanoscale production of ellipsoidal aAPCs. Briefly, nanoparticles of variable stiffness were synthesized through a single emulsion process (Fig. 1A), stretched in one dimension, and conjugated with two important signals for T cell activation, Signal 1 (MHC I loaded with Gp100 tumor antigen) and Signal 2 (anti-CD28) molecules. Transmission electron microscopy (TEM) was used to confirm nanoparticle spherical and ellipsoidal shapes (Fig. 1B). CD8+ T cell proliferation was quantified after 7-day incubation with nano-ellipsoidal PEG aAPCs of variable stiffness (Fig. 1C)

Results, Conclusions, and Discussions: The synthesis of nanoparticulate hydrogel aAPCs was characterized by confirming size, as well as spherical and ellipsoidal shape. Our platforms showed similar amounts of bound Signal 1 and Signal 2 proteins. TEM results and image analysis suggests that nanoparticles are similar in size, with the smallest dimesion being less than 500 nm. At the highest aAPC does, our softest (50 kPa) nano-ellipsoidal platform induced significantly more proliferation of CD8+ T cells, when compared to T cell cultures with aAPCs of higher stiffnesses.

We have shown the synthesis and optimization of biocompatible PEG nano-aAPCs of tunable stiffness and shape. In ongoing studies, we are fully characterizing aAPC properties, including polydispersity, stability, and protein conjugation. We are characterizing the impacts of aAPCs in the context of in vitro CD8+ T cell stimulations and in vivo clearance and activation. Ultimately, our novel and highly tunable nano-aAPC technology has the potential to not only bear translational anti-cancer relevance but can also shed light on aspects of basic T cell biology that are not otherwise easily studied.

Acknowledgements (Optional): I would like to thank the Biomedical Engineering Society for hosting this wonderful conference. I would also like to thank Johns Hopkins Medicine, Dr. Jonathan Schneck, Dr. Jordan Green, as well as the Schneck and Green lab members. This data is supported by the National Science Foundation Graduate Research Fellowship (DGE-1746891) and National Cancer Institute of the NIH (F31CA284859) .