Integrative Single-Cell RNA-Seq and Single-Cell ATAC-Seq Analysis of Human Plasma Cell Differentiation

Abstract

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. We have shown that PC differentiation (PCD) can be reproduced in vitrofrom memory B cells (MBC). MBC differentiate into CD20 low/-CD38 - pre-plasmablasts (prePB), CD20 -CD38 +CD138 - plasmablasts (PB) and CD20 -CD38 +CD138 + PC. The molecular changes occurring during PCD are recapitulated in this in vitrodifferentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PCD. We combined single-cell (sc) RNA-seq and scATAC-seq to decipher the trajectories involved in PCD. scRNA-seq experiments on the four populations (MBC, prePB, PB and PC) generated during normal PCD revealed a highly specific transcriptomic profile for MBC and PC, and a strong heterogeneity of the prePB and PB. The prePB stage presented the most differentially expressed genes (DEG) with almost 2000 DEG showing that the most important changes take place during this stage. Epigenetic analysis using scATAC-seq technology showed that prePB, PB and PC stages were clearly separated from the MBC highlighting chromatin remodeling induced by B cell activation and differentiation. As observed on the transcriptomic level, the number of differentially accessible peaks was higher in prePB (4660) than in other stages (MBC: 641; PB: 44; PC: 105). Among genes that were commonly identified using scATAC-seq and RNA-seq, we identified several TF such as BATF and BATF3 in prePB. Interestingly, differentially accessible peaks characterizing the prePB stage were enriched in motifs of BATF3, FOS and BATF belonging to the TF AP-1 family and among these differentially accessible peaks, 38% of peaks presented BATF3 motif. Since BATF3 TF was previously identified operating in short impulse manner at prePB stage, BATF3 target genes may represent a key transcriptional node involved in PCD. The integration of the chromatin accessibility and transcriptomic data revealed a more mature population of prePB cells characterized by open chromatin in PC genes without significant expression suggesting that a population of prePB were already committed to become antibody-secreting cells. To focus on the understanding of the processes occurring during the transition from prePB to PB which remains largely unknown, we computationally clustered and ordered cells. Pseudotemporal analyses underlined maturation trajectories in prePB with early prePB characterized by downregulation of B cell markers ( CD19, CD22, CD83, CCR7, CCL17 and CCL22) and B cell TF (SPIB, PAX5, STAT5A, RUNX3 and BATF3) together with upregulation of PC markers, adhesion molecules and growth factor receptors ( CD27, CD38, SLAMF7, BCMA, ITGA4, IL-6R, IL-6ST and INSR). The transition from early prePB to more mature prePB is associated with downregulation of AICDA and PRC2 complex ( EZH2, EED). In the transitional prePB, a first wave of UPR activation is observed, associated with mTORC1 pathway activation. In these prePB, HSAP5, also called binding immunoglobulin protein (BiP) is overexpressed together with ERN1, leading to the activation of the Ire1 pathway, known to splice XBP-1 (sXBP-1) producing a highly active TF. Then, prePB overexpress EGR1, FOS, CXCR4 and TFRC. Mature prePB overexpress KLF2 that participate in bone marrow PC homing. Then, PB overexpress gene involved in conventional UPR and protein secretion associated to immunoglobulin gene expression. Altogether, these data underlined that prePB present already UPR priming through mTORC1 pathway activation to prepare for PC function. XBP1 driven UPR activation will be coordinated in PB related to antibody synthesis increase. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of prePB already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our PCD model provide a resource for the identification of molecular circuits that are crucial for early and mature PC biological function and survival. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PCD.