Spatial organization and force generation in the immunological synapse

The immunological synapse (IS) formed between T cells and antigen presenting cells is a key regulatory point of the immune system. This interface is highly dynamic, involving organization of receptor-ligand pairs into compartmentalized regions that are proposed to modulate signaling cascades and T cell activation. The focus of this thesis is to address the role of spatial organization, dynamics, and force generation within the IS and evaluate their functional impact on T cell activation.In T cell-APC pairs, spatial segregation of CD3 and CD28 clusters, which provide antigenic and costimulatory signals to the T cell, has been observed to enhance secretion of IL-2, a key cytokine indicative of T cell activation. However, it is not clear in these cellular experiments if the increased IL-2 is driven by the pattern itself or if pattern is a byproduct of reduced stimulation. We developed the use of multi-component surfaces, combining multiple rounds of micro-contact printing on a single substrate, to create high-density arrays of these signals that recapitulate key geometric features of physiological synapses. We demonstrate that naive T cells form stable synapses on these surfaces localization of CD28 signal to the periphery of the IS enhances IL-2 secretion by naive CD4+ T cells. This increased cytokine production correlated with NFkappaB translocation and required PKB/Akt signaling.Furthermore, we introduced a method for creating surfaces containing multiple, aligned regions of supported membrane, capturing the mobility of receptors on cellular surfaces at scales from micrometers to tens of nanometers T cell receptor (TCR) and LFA-1 are tethered to separate, closely juxtaposed regions of bilayer, capturing an important aspect of the natural organization observed between T cells and antigen presenting cells. The results present a novel platform for the study of spatial separation of receptors in the immunological synapse and its impact on T cell signals.Lastly, we characterized the mechanical force generated by T cells during immunological synapse formation on a soft micro-pillar array, and identified Pyk2 as a key molecule in the process. Using micro-contact printing technique, we demonstrate that Pyk2 activation was primarily localized to the anti-CD3 stimulation, and enhanced by spatially colocalized LFA-1-mediated integrin interaction. Pyk2 association with SFK and acto-myosin cytoskeleton is revealed. Activated Pyk2 recruits actin to the TCR activation sites, and Myosin IIA activity elevates activated Pyk2 at integrin interaction regions surrounding anti-CD3 stimulation sites. IL-2 secretion by naive CD4+ T cells is dependent on the rigidity of underlying activating substrate, revealing the functional impact of the mechanical force generation during synapse formation on down-stream T cell functions.