Stage-Specific Non-Coding RNA Expression Patterns during In Vitro Human B Cell Differentiation into Antibody Secreting Plasma Cells

Abstract

The differentiation of B cells into antibody secreting plasma cells (PCs) is governed by a strict regulatory network that results in expression of specific transcriptomes along the activation continuum. In vitro models yielding significant numbers of PCs phenotypically identical to the in vivo state enable investigation of pathways, metabolomes, and non-coding (ncRNAs) not previously identified. The objective of our study was to characterize ncRNA expression during human B cell activation and differentiation. To achieve this, we used an in vitro system and performed RNA-seq on resting and activated B cells and PCs. Characterization of coding gene transcripts, including immunoglobulin (Ig), validated our system and also demonstrated that memory B cells preferentially differentiated into PCs. Importantly, we identified more than 980 ncRNA transcripts that are differentially expressed across the stages of activation and differentiation, some of which are known to target transcription, proliferation, cytoskeletal, autophagy and proteasome pathways. Interestingly, ncRNAs located within Ig loci may be targeting both Ig and non-Ig-related transcripts. ncRNAs associated with B cell malignancies were also identified. Taken together, this system provides a platform to study the role of specific ncRNAs in B cell differentiation and altered expression of those ncRNAs involved in B cell malignancies.

Keywords: RNA-seq; human B lymphocytes; in vitro B cell differentiation; non-coding RNA; plasma cells.

Figure 1

Morphology, cytoplasmic immunoglobulin (cIg) expression and phenotype of B cells differentiated into in vitro plasma cells (IVPC)s. (A) Wright staining of day 0 (D0) B cells (left panel) and day 10 (D10) IVPCs (right panel). (B) cIg staining of D10 IVPCs. Left panel: D10 IVPCs expressing IgA (red), IgG (blue) and IgM (green). Right panel: D10 IVPCs expressing κ light chain (LC; green), λLC (red), and DAPI (blue). (C) SLAMF7 expression. Left panel: D0 cells and D10 IVPCs analyzed by flow cytometry for SLAMF7 expression. Isotype control (black), SLAMF7 D0 B cells (red), SLAMF7 D10 IVPCs (blue). Right panel: SLAMF7 expression by RNA-seq (p < 0.0001; D4 = day 4; n = 3 at each time point).

Figure 2

Characterization of differentially regulated RNA transcripts. (A) Protein-coding RNAs (excluding immunoglobulin (Ig) transcripts) account for the majority of the differentially expressed RNA transcripts. (B) Long non-coding RNAs (lncRNAs) represent 87.4% of the non-coding (ncRNAs) shown in (A), with the antisense type representing the majority of lncRNAs.

Figure 3

Hierarchical clustering of differentially expressed transcripts during differentiation from D0 resting B cells to D10 IVPCs. The top 100 differentially expressed non-Ig protein-coding transcripts based on fold change (FC) ≥ 2.0 and false discovery rate (FDR) ≤ 0.05 were analyzed by Morpheus software for hierarchical clustering using log2 transformed pseudo-counts and Pearson correlation. The color scale bar represents relative expression based on log2 transformed pseudo-counts.

Figure 4

Memory B cells are preferentially expanded in the differentiation process. The percent of unmutated (UM) Ig heavy chain variable region (IGHV) transcripts decreases with differentiation, indicating that cells with a mutated (M) IGHV are preferentially expanded in this in vitro system (n = 3; p < 0.0001).

Figure 5

Hierarchical clustering of ncRNAs during the differentiation to IVPCs. A total of 982 ncRNA transcripts were differentially expressed across the transition from D0 resting B cells, D4 plasmablasts and D10 IVPCs based on log2 FC ≥ 2.0 and FDR ≤ 0.05. Five distinctive expression clusters are observed. The color scale bar represents relative expression based on log2 transformed pseudo-counts.

Figure 6

Differentially expressed ncRNA transcripts located in Ig loci. Fourteen differentially expressed ncRNA transcripts are located within Ig loci on chromosomes 2, 14 and 22.

Figure 7

Representative ncRNAs upregulated at D4 (Cluster IV). ncRNAs upregulated at D4 and associated protein-coding transcripts reflect increased proliferation and cytoskeletal conformational changes. Red squares = protein-coding transcripts; black circles = ncRNAs.

Figure 8

Representative ncRNAs downregulated during B cell differentiation (Cluster V). ncRNAs downregulated during IVPC differentiation and associated protein-coding transcripts. Red squares = protein-coding transcripts; black circles = ncRNAs.