Title: Multidimensional T Cell Mechanosensing
Description: T cells are key agents in the adaptive immune response, responsible for robust and selective protection of the body against foreign pathogens. T cells are activated through their interaction with antigen-presenting cells (APCs) via a dynamic cell-cell interface called the immune synapse (IS). Numerous studies in recent years have shown that T cell activation is a mechanoresponsive process. Modulation of substrate rigidity and topology are emerging as powerful tools for controlling T cell activation. However, the majority of systems used to investigate the IS have used substrates that lack the rigidities and topographical complexities inherent in the physiological T cell - APC interface. Circumventing these limitations, elastomer micropillar arrays can be fabricated with physiologically-relevant rigidities and provide a topographically-deformable activating substrate. In this thesis, we examine the mechanisms behind T cell mechanosensing in order to gain a more complete understanding of T cell activation.

More specifically, we take advantage of micropillar substrate properties to examine the IS in both 2D and 3D, seeking new insights into how the structural and mechanical features of the IS modulate T cell activity. We first investigate the traditional paradigm of T cell force generation at the 2D IS by seeking to characterize the temporal relationship between TCR signaling and force generation. We find that in both mouse naive and preactivated CD4+ T cells, TCR signaling is robust, dynamic, and localized to the pillar features. However, no temporal correlation is found between signaling and force generation. A potential reason for this lack of correlation is recent research showing that the physiological IS is a 3D interface that is topographically dynamic. This phenomenon complicates our interpretation of the 2D IS, as our micropillar system is protrusion-inducing substrate. In order to investigate the implications of topographical cues, we then characterize T cell activation in the 3D IS with respect to force generation and cytoskeletal development over time.

We demonstrate that preactivated CD4+ T cells exhibit a dynamic and robust penetration into micropillar arrays. In the 3D IS, actin polymerization is again not correlated with force generation, but we find that microtubules (MTs) have a critical role in 3D T cell mechanosensing. Namely, MT architecture is correlated with the spatial distribution of force generation in the 3D IS, the centralization of microtubule-organizing center (MTOC) to the 3D IS is a mechanosensitive process that is modulated by surface rigidity, and while MT polymerization is not necessary for force generation, it is critical for maintaining synaptic integrity over time. Together, this work reveals important aspects of the underlying dynamics of the T cell cytoskeleton in IS formation and maintenance. The conclusions will help advance the concept of mechanobiology in immunology, which may in turn be leveraged towards the development of biomaterials that enhance T cell manufacturing in adoptive cell therapy.