Marginal zone B cells have higher basal BCR signaling that correlates with altered mobility and spatial organization of IgM-BCRs

Abstract

Abstract Marginal zone (MZ) B cells exist in a partially-activated ‘primed’ state but the molecular basis for this priming is not fully understood. We found that MZ B cells exhibit greater antigen-independent ‘tonic’ BCR signaling than naïve follicular (FO) B cells. Because BCR signaling output is dependent on BCR spatial organization and BCR-BCR interactions, we hypothesized that the increased tonic BCR signaling in MZ B cells is due to altered lateral mobility and nanoscale organization of BCRs. Single-particle tracking showed that surface IgM-BCRs on MZ B cells have higher diffusion coefficients and decreased confinement compared to IgM-BCRs on FO B cells. In contrast, the mobility and confinement of IgD-BCRs was similar on MZ and FO B cells. To assess BCR nanoscale organization, we used dSTORM and a novel graph-based clustering algorithm. This revealed that IgM-BCRs were more dispersed and less clustered on MZ B cells than on FO B cells, whereas IgD-BCR spatial organization was similar on the two cell populations. Importantly, 3-color STED imaging revealed that phospho-CD79 nanoclusters overlapped to a much greater extent with IgM-BCRs than with IgD-BCRs on FO B cells, and that this IgM-pCD79 overlap was even greater in MZ B cells. MZ B cells also exhibited greater pCD79 signaling in response to membrane-bound antigens than FO B cells. Thus, MZ B cells have greater tonic and antigen-dependent BCR signaling, which correlates with altered IgM-BCR mobility and organization