Capturing the Functional Antibody Repertoire from Multiple B-cell Subsets Following Antigen Stimulation.

Abstract

Abstract Antigen stimulation of mature B-cells induces activated B-cells followed by differentiation to either memory B-cells or plasmablasts. Each subset has a unique phenotype and potentially unique immunoglobulin repertoire that can be mined for therapeutic antibody candidates. As new targets for therapeutic antibodies have become more challenging, primary B-cell technologies have arisen to meet this challenge, but, their use has typically been limited to one of the B-cell subsets. We sought to develop a platform that could be used generally to interrogate all of the B-cell subsets (i.e., activated, memory and plasmablasts) that are potential sources of useful antibodies. Using fluorescence activated cell sorting (FACS), we have identified and sorted antigen-specific activated and memory B-cells by staining for CD19, surface IgG, and fluorochrome-tagged antigen. Antigen-specific plasmablasts have been identified and sorted after encapsulation of cells in microdroplets, capture of secreted IgG, CD19/CD138 staining, and fluorochrome-tagged antigen staining. Additionally, hybridomas have been evaluated using either protocol. The sorted cells have been sequenced by next generation sequencing (NGS) of paired heavy and light chains. With this platform ~10,000–20,000 antigen specific B-cells from a population of a few million viable lymphocytes have been sorted in 1–2 hours. From the sorted population, 100 – 700 unique paired clonotypes have been identified using this platform. The platform enables the capture of immunoglobulin repertoires from multiple B-cell subsets that is comprehensive, flexible, and cost-effective.