BPS2026 – Activation-induced lysosomal tethering enhances TNFα vesicle trafficking in Jurkat T cells

TNFα is a key pro-inflammatory cytokine implicated in the development of multiple inflammatory and autoimmune diseases, where its dysregulation drives severe immune responses. While transcriptional and secretion profiles of TNFα have been well-documented and shown to follow defined temporal patterns, the intracellular trafficking events that bridge production and secretion remain poorly defined. We hypothesize that intracellular transport of TNFα is also temporally regulated, representing a potential regulatory checkpoint for downstream secretion. To investigate this, we combined super-resolution 3D imaging, volumetric analysis, and live-cell single particle tracking to map TNFα vesicle dynamics in activated Jurkat T cells. Our results show three major features of activation-dependent trafficking: first, progressive tethering of TNFα-containing vesicles to LAMP1+ vesicles; second, an increase in TNFα vesicle volume over the first 2 h, followed by a reduction after 4 h; and third, sustained increases in lysosome and TNFα vesicle dynamics over activation time. Together, these findings demonstrate that intracellular TNFα trafficking exhibits distinct temporal phases that provide additional insights to existing endpoint assays such as ELISA or western blotting. By resolving these intracellular dynamics, this work lays the foundation for targeting pre-secretion transport steps, such as tethering or vesicle trafficking, with small molecules, offering a cost-effective and broadly accessible alternative to antibody-based therapeutics.