Antigen-specific expansion and phenotyping of primary B cells
B cells are vital in leading the adaptive immune response against foreign antigens. Memory B cells (Bmem) allow for a quicker and more robust immune response upon re-encounter of the same or similar antigens. If these Bmem subpopulations can be harvested, they offer a source of plasma cells and antibodies which can be used in vaccine development and immunotherapy. However, the pool of Bmems reactive to a given antigen may be minuscule, rendering the experimental or clinical value effectively moot. In this project, we sought to develop a reliable methodology for the antigen-specific expansion of rare subsets of human Bmems ex vivo. To date, we have demonstrated that primary B cells derived from COVID-convalescent blood donations can be a) maintained in culture for up to one week; b) selectively expanded using procedures evolved from published literature and firsthand observations; and c) identified using fluorescent antibodies. Using streptavidin-coated silica microspheres conjugated to biotinylated CpG and an anti-kappa antibody, we were able to model the selective targeting of B cells expressing kappa light chains in the B Cell Receptor (BCR), as confirmed using flow cytometry. Furthermore, we were able to identify a subset of antigen-specific B cells derived directly from uncultured blood samples by substituting biotinylated viral peptides for the model antibody and applying a secondary fluorophore prior to sample acquisition. With further development of this model, our selective culturing system will enable us to isolate and compare B cell clonotypes of interest from donor samples that have been exposed to SARS-CoV-2, either artificially through vaccination or naturally through infection. Once optimized, this system will be readily applicable to the ex vivo generation of choice Bmems or plasma cells, facilitating in-depth study of the adaptive immune response to SARS-CoV-2 or in vitro production of therapeutic antibodies. It could also be adapted to address the needs of researchers in related fields, such as T cell immunology, and as such has potential value in clinical studies.
