Thesis Casper Marsman

Summary

During the human adaptive immune response, B cells can differentiate into either memory B cells, which recognize and quickly react to antigens upon reinfection, and antibody-secreting cells (ASCs), that secrete large quantities of antibodies that specifically bind and neutralize pathogens through cooperation with the innate immune system. The results presented in this thesis contribute to the field of human B cell differentiation research by providing optimized T cell dependent and T cell independent in vitro stimulation protocols that allow in-depth analysis of ASC differentiation, specifically for when patient material is limited. Furthermore, this thesis provides multiparameter flow cytometry protocols to analyze B cell differentiation membrane markers over time, together with intracellular (phospho)signaling proteins or transcription factors, allowing in-depth investigation into activation and differentiation of B cells. Next, we utilize these techniques to show that naïve B cells efficiently differentiate into ASCs upon termination of CD40L stimulation, by promoting BLIMP1 expression and inhibition of PAX5. Furthermore, we demonstrate how physiologically relevant oxygen pressures found in lymph nodes, 3% and 1% pO2 compared to 21% pO2 generally used in cell culture incubators, majorly contribute to ASC differentiation and IgG class-switch recombination. We show that spatiotemporal, environmental and metabolic factors are crucial regulators of human B cells and that these should all be considered during in vitro and in vivo B cell differentiation studies. The insights and techniques presented within this thesis will support future B cell related research, both on a fundamental and a clinical level.