A pipeline to unravel complexity of the human peripheral blood B cell compartment through multiomic cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analyses

Classifying heterogeneous human B cells into discrete subsets based on surface phenotype alone remains a major challenge. We discovered a mouse splenic IgM+IgDlow/- B cell subset (BDL) that induces proliferation of CD4+Foxp3+ T regulatory cells, making them a therapeutic target for autoimmunity. Because they are rare and lack sufficient cell surface markers, a multiomic strategy is required to identify and track them. The lack of correlation between mRNA expression levels and cell surface expression is a drawback to a transcriptome-only scRNA-seq approach because 'novel' cell populations identified by surface protein transcripts may prove impossible to validate. Thus, a pipeline was developed using human peripheral blood (PB) IgM+IgDlow/- B cells. First, a standardized flow cytometry gating scheme (CD27 versus IgD and CD24 versus CD38) that captures all major human PB B cell subsets was utilized. Second, CD19+IgM+IgDlow/- PB B cells were tagged with the four markers using cellular indexing of transcriptomes and epitopes by sequencing. Biaxial plots similar to flow cytometry were used to identify naïve, transitional, and memory B cells, and then confirmed with scRNA-seq. These studies using a multiomic workflow allowed the identification of IgM+IgDlow/- B cells by cell surface expression that could subsequently be analyzed for unique transcriptional profiles. Overall design: We performed scRNA sequencing to characterize BDL, a rare B cell subset in the heterogeneous CD19+IgM+IgDlow/- B cell compartment within human peripheral blood. We combined this technology to CITE-sequencing to obtain measurement at the protein and transcriptome level and to multiplex samples in order to measure these data simultaneously across three deidentified human donor samples: (3) CD19+IgM+IgDlow/- B cells and (1) CD19+IgM+IgDhigh B cells (negative control). All samples were stained with a panel of ADT (CD27, CD21, CD24, CD38) which were used to recreate flow cytometric analyses at the single cell level.