RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs.

Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform.

Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response.

As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4.

Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell.

Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not.

Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma.