Single cell transcriptomics of in vivo differentiating antibody-secreting cells reveals a divergent activated B cell program dependent on IRF4

Abstract

Abstract The generation of antibody-secreting cells (ASC) requires reprogramming the B cell transcriptome, epigenome, and metabolic potential to support the processes necessary to synthesize and secrete antibodies. These molecular changes have largely been studied at defined stages representing naive B cells, activated B cells, and ASC. To further refine when ASC fate decisions occur, we adoptively transferred CTV labeled B cells and monitored differentiation to both type I and II T cell independent antigens. At the peak of ASC responses, transferred cells were phenotyped by flow cytometry and isolated for single-cell RNA-seq. These data captured a continuum of responding B cells representing all stages of differentiation, with the largest heterogeneity observed among actB. Computationally ordering cells along differentiation trajectories, revealed divergent actB differentiation paths, with one branch leading to ASC formation that was dependent on IRF4. Moreover, cells that followed this trajectory downregulated the surface marker L-selectin and could be separated from cells that followed the alternative non-ASC differentiation branch. Indeed, isolation of actB by L-selectin status confirmed that L-selectin negative cells were ten times more likely to form ASC in culture. In summary these data provide insights into the cell division kinetics, molecular heterogeneity, and differentiation paths that lead to ASC formation.