Dynamic micropatterning uncovers spatial dynamics of B cell immune synapse formation.

In the process of mounting humoral immune responses, B cells typically engage antigens on the surface of antigen-presenting cells (APCs) via the immunological synapse (IS). To study this critical cell interaction structure, we developed a dynamic micropatterning technique that models IS formation with precise spatial and temporal control. This method enables imaging of B cells before and after B cell antigen receptor (BCR) engagement, in fixed and live samples. We analyzed BCR-proximal signaling in A20 D1.3 mouse B cells and found distinct spatial distribution preferences for different signaling proteins. Also, the size and shape of the initial non-activatory adhesion modulated the signaling outcome. We 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. Overall, dynamic micropatterning provides powerful insights into the rapid, early steps of IS formation with remarkable spatial and temporal resolution.