Leveraging optimized oligonucleotide-tagged antigen assemblies and single-cell sequencing for multiplexed
Richard Apps1, Jessie J Polanco1, Kelsey E Lowman2,3
1NIH Center for Human Immunology, Autoimmunity, and Inflammation (CHI), Bethesda, MD 20894, United States.
None:
Antigen-specific responses to complex antigens encompass a range of cell states and reactivities to an array of epitopes, reflective of the heterogeneity in immune responses. Single-cell sequencing has created new opportunities when combined with flow cytometry for profiling of immune repertoire and cell phenotype. Rare B cells with fine specificities can be precisely isolated using flow cytometry, and with oligonucleotide-tagged antigen assemblies enable the isolation of a pool of cells reactive to different antigens in parallel from biological samples. Single-cell sequencing can then detect these tags to allow mapping of individual B cell reactivities to specific antigens, in addition to simultaneous profiling of cellular phenotypes. We establish workflows for these approaches with antigen reactivity frequencies typical of human peripheral blood, by conducting all staining prior to sorting to minimize loss of rare antigen-specific cells. We identify antigen:streptavidin ratio as a priority for optimization when using novel antigens to generate oligonucleotide-tagged assemblies for B cell staining, which is demonstrated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigens. We develop a novel approach of combining dual labelling using separately oligonucleotide- and fluor-tagged assemblies, to increase staining sensitivity sufficiently for exploratory detection of dengue-reactive cells in naturally exposed individuals. Together these findings support parallel profiling of cells with differing antigen-specificities, conferring new opportunities to advance understanding beyond characterization of individual clonotypes to their role in the broader humoral response. This can be important in pathogen, vaccine, malignancy, and autoreactivity responses, where both antigen-reactive cells at low frequencies and interactions between different epitope responses can shape clinical outcomes.
