Phenotyping complex human B cell compartments using high parameter spectral flow cytometry and scRNA-seq with parallel CITE-seq analyses.

Abstract

Abstract The human B cell compartment is very complex, consisting of numerous B cell subsets. Classifying this heterogeneous population into discrete subsets based on surface phenotype alone remains a major challenge in the field of B cell biology. Our lab has discovered a novel mouse IgM +IgD low/−B cell population that induces Treg proliferation in a GITRL-dependent manner, making it a potentially significant therapeutic target for autoimmune diseases. But due to their rarity and lack of specific cell surface identifiers, a more definitive phenotype is required to track this B cell subset in humans. The objective of these studies is to characterize human IgD low/−B cells from peripheral blood using both high parameter spectral flow cytometry and scRNA-seq with parallel CITE-seq analyses. Spectral flow cytometry has the major advantage over its conventional predecessor by allowing for the construction of large parameter panels that are not limited by standard compensation. Here, we have adapted a standardized gating scheme (CD27 vs. IgD and CD24 vs. CD38) that captures all the major canonical human B cell subsets. Furthermore, we have used analysis plugins in FlowJo (Phenograph and FlowSOM) that predict multiple discrete B cell subpopulations within the IgD low/−phenotype. Additionally, we have analyzed human B cells (CD19 +IgM +IgD low/−) using scRNA-seq combined with CITE-seq. This approach allowed us to analyze both the cell surface protein expression of CD27, CD21, CD24, and CD38 combined with the transcriptome at the single-cell level. Our analysis has revealed four individual subclusters within this populationthat can be validated by flow cytometry, showcasing the power of combining these two cutting-edge analytical tools.