Abstract A74: Loss of L-selectin distinguishes activated B cells destined to differentiate to plasma cells
Authors: Patterson Dillon G. , Scharer Christopher D. , Mi Tian , Price Madeline J. , Hicks Sakeenah L. , Boss Jeremy M. 
Published: March 2020

Abstract
Plasma cells engineered to produce and secrete de novo proteins have the potential to be used as biologic therapeutics for the treatment of cancer and other diseases. Genetic manipulation, expansion, and differentiation of naive B cells has been performed in vitro, and significant efforts have been made to understand and model the mechanisms that govern B cell fate decisions. Collectively, these studies indicated B-cell differentiation occurred across all cell divisions and that considerable cell fate heterogeneity exists between responding B cells within the same cell division. However, the differences in transcriptional programming that drive these differences and whether the same kinetics of cell division and differentiation are observed in vivo remain unknown, leaving a gap in our knowledge that may be critical for the efficacy of future therapeutic applications. To begin to address this, we employed an in vivo murine adoptive transfer system and monitored the kinetics of cell division and differentiation in response to lipopolysaccharide. We report that in vivo B cell differentiation requires a minimum of eight cell divisions, which corresponded to peak IRF4 expression and Blimp-1-mediated reprogramming. However, only a fraction of the cells that divided eight times differentiated, indicating that additional guidance cues ultimately decide cell fate decisions. To resolve B-cell differentiation at finer molecular resolution, we performed single-cell RNA sequencing. These data captured a continuum of LPS-responding B cells representing all stages of differentiation, with the majority of heterogeneity observed among activated B cells (actB). Computationally ordering cells along differentiation, or pseudotime, revealed divergent actB trajectories, with one branch leading to plasma cell formation that was dependent on IRF4. Cells along this branch upregulated genes associated with MYC activation and oxidative phosphorylation, both of which are important for achieving the metabolic and catabolic requirements to support plasma cell functions. Moreover, cells that followed this trajectory downregulated the surface marker L-selectin and could be separated from cells that followed the alternative non-plasma cell differentiation branch. Indeed, isolation of actB that had divided eight times and by L-selectin status confirmed that L-selectin-negative cells were ten times more likely to form plasma cells in culture. In summary, these data provide insight into the cell division kinetics of B-cell differentiation in vivo, highlight an IRF4-dependent bifurcation event that occurs early during actB reprogramming, and specify a strategy to identify actB that preferentially differentiate to plasma cells. Together, these data may be leveraged to improve desired immune outcomes by forcing cells down a path to a plasma cell or by identifying and expanding cells that are more prone to differentiate.
Citation Format: Dillon G. Patterson, Christopher D. Scharer, Tian Mi, Madeline J. Price, Sakeenah L. Hicks, Jeremy M. Boss. Loss of L-selectin distinguishes activated B cells destined to differentiate to plasma cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A74.