B-cell antigen receptor expression governs signaling potential and protection from apoptosis
Kanutte Huse1,2, Cara Wogsland3, Hannah Polikowsky4, Kirsten Diggins4, Jillian Wise1,2, Erlend B. Smeland1,2, Jonathan Irish3,4, June Myklebust1,2
1Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway; 2Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 3Department of Pathology, Microbiology and Immunology, and 4Department of Cancer Biology, Vanderbilt University, Nashville, TN, United States
Mature B cells depend on expression of a signaling-capable B-cell antigen receptor (BCR) for survival. However, the exact role of antigen-induced and tonic BCR signaling in fate decisions like survival and differentiation during human germinal center (GC) reaction is not fully known. viSNE analysis of 27-dimensional mass cytometry data was used to identify and characterize B-cell subsets present throughout the germinal center reaction. Next, fluorescens phospho-flow was used to measure signaling responses induced by FITC-conjugated anti-IgM, enabling direct comparison of signaling responses in B-cell subsets according to stage, isotype, and per-cell expression of IgM. Before controlling for isotype and level of BCR stimulation, human GC B cells were observed to have weaker BCR signaling responses compared to naïve and memory B cells. This result was attributed to the observations that 1) BCR expression was significantly lower in GC B cells, and 2) BCR engagement (measured by per-cell binding of anti-IgM-FITC) was correlated with the degree of subsequent SYK, BLNK, PLCγ and SFK phosphorylation. After controlling for isotype and per-cell BCR expression level, potentiated BCR signaling was observed in GC B cells. Similarly, degree of caspase-3 cleavage in cells cultured in vitro was directly compared to BCR-expression level. Spontaneous apoptosis was inversely correlated with the level of BCR expression in GC B cells. These results indicate that BCR expression levels govern the strength of human GC BCR signaling and protection from apoptosis, suggesting an important role in the GC reaction.