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Over the years, various techniques have been utilized to study the function and phenotype of antigen-binding B cells in the primary repertoire following immunization, infection, and development of autoimmunity. Due to the low frequency of antigen-reactive B cells (<0.05% of lymphocytes) in the periphery, preliminary enrichment of cells is necessary to achieve sufficient numbers for statistically sound characterization, especially when downstream analytic platform use, e.g., CyTOF, is low throughput. We previously described a method to detect and enrich antigen-reactive B cells from peripheral blood and tissues using biotinylated antigens in conjunction with magnetic nanoparticles, preparative to a downstream analysis by ELISPOT and flow cytometry. While mass cytometry (CyTOF) enables high dimensional immunophenotyping of over 40 unique parameters on a single-cell level, its low throughput compared to flow cytometry and requirement for removal of metal contaminants, such as nanoparticles, made it particularly unsuitable for studies of rare cells in a mixed population. Here we describe a novel CyTOF-compatible approach for multiplexed enrichment of antigen-reactive B cells, e.g., insulin and tetanus toxoid, using cleavable magnetic nanoparticles. This method allows improved monitoring of the phenotype and function of antigen-reactive B cells during the development of disease or after immunization while minimizing the amount of sample and run times needed. [ABSTRACT FROM AUTHOR]

The ongoing COVID-19 pandemic has had devastating health impacts across the globe. The development of effective diagnostics and therapeutics will depend on the understanding of immune responses to natural infection and vaccination to the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While both B-cell immunity and T-cell immunity are generated in SARS-CoV-2-infected and vaccinated individuals, B-cell-secreted antibodies are known to neutralize SARS-CoV-2 virus and protect from the disease. Although interest in characterizing SARS-CoV-2-reactive B cells is great, the low frequency of antigen-binding B cells in human blood limits in-depth cellular profiling. To overcome this obstacle, we developed a magnetic bead-based approach to enrich SARS-CoV-2-reactive B cells prior to transcriptional and antibody repertoire analysis by single-cell RNA sequencing (scRNA-seq). Here, we describe isolation of SARS-CoV-2 antigen-binding B cells from two seropositive donors and comparison to nonspecific B cells from a seronegative donor. We demonstrate that SARS-CoV-2 antigen-binding B cells can be distinguished on the basis of transcriptional profile and antibody repertoire. Furthermore, SARS-CoV-2 antigen-binding B cells exhibit a gene expression pattern indicative of antigen experience and memory status. Combining scRNA-seq methods with magnetic enrichment enables the rapid characterization of SARS-CoV-2 antigen-binding B cells.

Over the years, various techniques have been utilized to study the function and phenotype of antigen-binding B cells in the primary repertoire following immunization, infection, and development of autoimmunity. Due to the low frequency of antigen-reactive B cells (

The ongoing COVID-19 pandemic has had devastating health impacts across the globe. The development of effective diagnostics and therapeutics will depend on the understanding of immune responses to natural infection and vaccination to the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While both B-cell immunity and T-cell immunity are generated in SARS-CoV-2-infected and vaccinated individuals, B-cell-secreted antibodies are known to neutralize SARS-CoV-2 virus and protect from the disease. Although interest in characterizing SARS-CoV-2-reactive B cells is great, the low frequency of antigen-binding B cells in human blood limits in-depth cellular profiling. To overcome this obstacle, we developed a magnetic bead-based approach to enrich SARS-CoV-2-reactive B cells prior to transcriptional and antibody repertoire analysis by single-cell RNA sequencing (scRNA-seq). Here, we describe isolation of SARS-CoV-2 antigen-binding B cells from two seropositive donors and comparison to nonspecific B cells from a seronegative donor. We demonstrate that SARS-CoV-2 antigen-binding B cells can be distinguished on the basis of transcriptional profile and antibody repertoire. Furthermore, SARS-CoV-2 antigen-binding B cells exhibit a gene expression pattern indicative of antigen experience and memory status. Combining scRNA-seq methods with magnetic enrichment enables the rapid characterization of SARS-CoV-2 antigen-binding B cells. [ABSTRACT FROM AUTHOR]