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In vivo analysis of B cell receptor signaling in germinal centers : new insights into affinity maturation via intravital microscopy

  • Ulbricht, Carolin
Publication Date
Aug 27, 2018
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Our immune system provides us with a variety of effectors that account for life-long protection against pathogens. One aspect of this mechanism is the production of antibodies by long-lived plasma cells that derive from B cells of germinal centers within secondary lymphoid organs. Within germinal centers, B cells, T cells and follicular dendritic cells form stable contacts that are essential for a process called affinity maturation. These contacts lead to acquisition of antigen from follicular dendritic cells by B cells, cognate T cell-B cell interactions that ensure survival of B cell clones and finally to the recirculation of B cells within distinct zones of the germinal center. During recirculation, the membrane-bound form of immunoglobulin, the B cell receptor, becomes increasingly affine to the antigen thanks to accumulating mutations in the immunoglobulin genes. The B cell receptor is not only responsible for antigen uptake but is also able to transduce signals to inner parts of the cell. Thus, apart from its facilitating antigen uptake, affinity of the B cell receptor might play a pivotal role in signaling as well, a role of the germinal center B cell receptor that has long been up for discussion. We here show that B cell receptor signaling-related Calcium influx is active in a subset of germinal center B cells and that calcium concentration is increasing over time and with affinity of the receptor to the antigen. To monitor B cell receptor signaling in vivo we developed a novel transgenic reporter mouse strain termed YellowCaB that expresses the calcium sensitive Förster Resonance Energy Transfer sensor protein TN-XXL in CD19+ B cells. Employing intravital two-photon microscopy, we are the first group to be able to spatiotemporally resolve B cell receptor signaling in living organisms, while simultaneously monitoring cell-to-cell interactions and migration. This allows us to preserve the natural architecture of the tissue, what otherwise would not have been possible in culture systems. Furthermore, fluorescence lifetime imaging is offering us the possibility to relate calcium concentrations to cell fate decisions, as we could show that naive and antigenspecific B cells inside and outside germinal centers show distinct quenching of TN-XXL. The observation of colocalized B cells that coincided with calcium influx in one reaction partner led us to hypothesize that apart from follicular dendritic cells, B cells itself are a source of B cell receptor stimulation via complement receptors. Together with CD40L-signals from T follicular helper cells and signals stemming from Toll like receptor engagement, the B-cellular calcium level is tightly regulated to control the outcome of the germinal center reaction. Terminal differentiation might be a direct downstream result of these signals adding up. To support this, we could show that B cells are able to react to external stimuli several times via calcium influx without exhaustion. Taken together, we here present a new mode of action for B cells taking part in the germinal center reaction and feedbacking their own fate. Thus, B cell receptor signaling is an essential part of B-cell-, plasma cell- and memory B cell development.

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