Live Cell and Superresolution Microscopy of Vesicular and Interleukin-2 Transport in Jurkat T Cells

T cells are essential to the immune system driving the adaptive immune response, where they coordinate and communicate with various immune cells through the secretion of pro-inflammatory cytokines, such as interleukin-2 (IL-2). A comprehensive understanding of spatio-temporal regulation of cytokine trafficking in T cells would reveal potential targets for the modulation of cytokine secretion, prompting the development of new therapeutic options. Therefore, through the incorporation of live cell imaging and superresolution microscopy, this dissertation investigates the mechanisms and motor proteins involved in the trafficking of intracellular IL-2 from T cells, using Jurkat T cells, as a model system. The first section explores the dynamics of acidic vesicles stained with a pH sensitive dye, Acridine Orange, following activation with CD3/CD28 Dynabeads. Live cell imaging revealed an increase in acidic vesicle mobility, which appeared to peak with 8 h activation, correlating with the maximum production and secretion of IL-2. The generation of a Jurkat cell line expressing IL-2 fused to EGFP demonstrated the utilization of acidic vesicles in the trafficking of IL-2. The second section investigates the role of actin-associated motor protein, Myosin-VI, on the transport of both acidic vesicles and IL-2. Small molecule inhibition and siRNA mediated knockdown of Myosin-VI increased the dynamics of acidic vesicles in 8 h activated Jurkat T cells, as well as the trafficking of IL-2. The involvement of Myosin-VI in the transport of both acidic vesicles and IL-2 was implicated, suggesting Myosin-VI as a potential therapeutic target for cytokine modulation. The third section of this dissertation unveils the distribution and organization of IL-2, particularly near the cell periphery following in vitro activation. Superresolution microscopy revealed not only an increase in intracellular IL-2 as a function of activation but also an increase in the prevalence of elongated IL-2 clusters along the cell periphery, indicating the packaging of IL-2 at the nanoscale. In conclusion, this dissertation reveals the involvement of acidic vesicles and Myosin-VI on intracellular IL-2 trafficking, as well as the clustering of IL-2 along the periphery of the cell following activation. These findings provide insight into the mechanisms and proteins involved in IL-2 transport within T cells, establishing the groundwork for the exploration of therapeutic targets for the modulation of cytokine trafficking and release.