Dynamic Micropatterning Reveals Spatial Dynamics of B Cell Receptor Signaling and Immune Synapse Formation

Abstract

B cell activation by foreign antigens, recognized by the B cell receptor (BCR), is the key prerequisite for cell differentiation for antibody production. B cells typically encounter antigens bound to the surface of antigen presenting cells (APCs), leading to the formation of the immunological synapse (IS). To gain a deeper understanding of lymphocyte activation, we developed a dynamic micropatterning technique that enables the modeling of IS formation with exceptional spatial and temporal control. Using this method, we can image B cells before and after BCR engagement, in both fixed and live samples. We compared the activation of different BCR proximal signaling proteins in activatory and non-activatory areas of the synapse and found that the activated signaling proteins exhibited distinct spatial distributions. While pCD79A was strongly localized in the antigen-tethered area, surprisingly, pPLC{gamma}2 was enriched in regions lacking BCR ligands. We also visualized the formation of the IS in living cells using enhanced-resolution microscopy in 3D. We identified different cell behaviors during this process, including the repurposing of pre-existing actin-based protrusions as ready-made building blocks for the IS -- a feature uniquely detectable with this highly controllable system. Extending our approach to include a co-stimulatory B cell ligand, ICAM-1, and T cell system using CD3 and CD28 antibodies as ligands, we demonstrate the broader applicability of this method. Overall, our results highlight the power of dynamic micropatterning in elucidating the rapid and dynamic earliest steps of the IS formation with high spatial and temporal precision.