Kinetic segregation of CD45 drives BCR activation by both soluble and FDC-displayed antigen 4087

Description The molecular mechanism governing B-cell receptor (BCR) activation by antigen remains elusive. Recent findings suggest the antigen footprint plays a determining role in its activating capacity by exclusion of bulky phopshatases such as CD45 from the BCR signalosome. Here, we further characterized the physicochemical determinants of this antigen footprint. However, during physiological responses, B cells often encounter antigen retained by follicular dendritic cells (FDCs). To elucidate the mechanistic underpinnings of this mode of antigen engagement, we first defined antigen immune complex dimensions on the surface of FDCs in vivo. Using sparse colloidal lithograpy, we then generated model antigen surfaces mimicking antigen complexes on FDCs. Antigen on force sensors was patterned on islands of 80, 210, 300, or 600 nm in diameter surrounded by ICAM-1, or distributed homogeneously with ICAM-1 across the surface. Antigen-specific B cells interacted with and spread on all these surfaces, but generated a larger number of mechanical events (>6 pN) on patterned vs. homogeneous antigen, and a greater degree of SYK phosphorylation was observed on 210-600 nm patterns. CD45 was excluded from the patterns and pSYK decreased as a function of the radial distance from the pattern center. Extension of the antigen force sensor length to accommodate CD45 in the pattern abrogated this effect. This supports a kinetic segregation model for BCR activation by both soluble and FDC-retained antigen. Funding Sources Supported by the Danish National Research Foundation Centre of Excellence, CellPAT, the Center for Cellular Signal Patterns (DNRF135), and a Carlsberg Foundation Distinguished Fellowship (CF18-0446) to S.E.D.