Enabling high-dimensional analysis of the memory B cell antibody repertoire
Antibodies provide protection against myriad pathogens. Two lineages of B cells are capable of producing these antibodies. Long lived plasma cells secrete antibodies that directly intercept homologous pathogens. Memory B cells, recruited upon secondary exposure, secrete antibodies with altered specificities to better recognize heterologous targets. To enable the first repertoire-scale comparison of these two compartments (homologous vs. heterologous), we optimized culture conditions for PBMCs (peripheral blood mononuclear cells) with the the goal of using optimized conditions as sample input into PePSeq, a highly multiplexed serological assay. We utilized a combination of an interleukin, to proliferate B memory cells, and a TLR (toll like receptor) agonist, to stimulate antibody production. Our group compared supernatant collection methods (with and without pipette-mixing), varying concentrations of cell inputs, and how these factors influenced total IgG concentration over time. Microscopy was utilized to qualitatively measure cell culture health and antibody quantification was performed using a commercial human IgG ELISA kit. ). Based on the degree of death observed in cultures handled using pipette-mixing, only unmixed cultures were quantified for the remainder of the experiment. For all cell input amounts (1E4, 1E5, 1E6, and 1E7), similar amounts of total IgG were detected on days 7 and 9 (357- 478 ng/mL. In conclusion, memory B cells can be isolated from a commercial PBMC’s, using an IL-2 concentration of 1000 IU/mg; memory B cells can be stimulated to produce detectable antibodies starting at day 3 in in vitro culture; and all B cell cultures will accumulate similar concentrations of total IgG by day 7.
