Intravital quantification of absolute cytoplasmic B cell calcium reveals dynamic signaling across B cell differentiation stages

Abstract

Development, function and maintenance of lymphocytes largely depend upon the cellular mobilization and storage of Ca2+ ions. In B lymphocytes, the absolute amount of calcium mobilized and retained after cell signaling remains unknown, athough it is a crucial part of their selection within germinal centers and differentiation into plasma cells. Here, we introduce the novel reporter mouse strain YellowCaB that expresses the genetically encoded calcium indicator TN-XXL in CD19+ lymphocytes. The construct consists of the electrondonor fluorophore eCFP and the acceptor citrine, linked by a calcium sensitive domain. Its conformation and therefore donor quenching is directly linked to cytosolic calcium concentrations. By combining intravital two-photon fluorescence lifetime microscopy with our numerical approach for phasor-based analysis, we are able to extract absolute cytoplasmic calcium concentrations in activated B cells for the first time in vivo. We show that calcium concentrations in B cells are highly dynamic and fluctuations persist in extrafollicular B cells with functional relevance.