Background/Purpose:

The high prevalence of citrullinated protein antibodies and improvement following rituximab anti-CD20 therapy both suggest that B cells are important in the pathogenesis of RA. Nonetheless, this has been difficult to study due of the rarity of citrullinated protein-specific B cells and the lack of tools to physically isolate them. We recently developed citrullinated alpha-enolase peptide tetramer technology to identify and capture autoreactive blood B cells in RA, and now report the first whole transcriptome analysis of these autoreactive B cells.

Methods:

A female CCP+/RF+ RA patient previously shown to produce IgG against the citrullinated alpha-enolase peptide CEP-1 (CKIHA-X-EIFDS-X-GNPTVEC, where X represents citrulline) donated 60 ml of blood for further investigation (U. of Minnesota IRB Code Number: 9712M00072). CEP-1 peptide-specific B cells were then captured from PBMC using anti-phycoerythrin Miltenyi beads and a combination of CEP-1 [peptide-streptavidin-phycoerythrin] and native alpha-enolase EP-1 (CKIHAREIFDSRGNPTVEC) [peptide-streptavidin-phycoerythrin-AF647] decoy tetramers, as previously described in Taylor JJ et al., J Exp Med. 2012 Oct 22;209(11):2065-77. Both autoreactive CEP-1 tetramer- and control EP-1 tetramer-bound B cell fractions were purified by FACSAria flow cytometry, differentially stained, mixed, and loaded into a Fluidigm C1 small-cell chip. The Fluidigm C1 autoprep system was used for single cell capture and RNA from each cell was converted to cDNA.  Barcoded cDNA libraries were sequenced on an Illumina Hi-Seq sequencer. Differential gene expression between autoreactive CEP-1 peptide-specific B cells and decoy tetramer-bound control B cells was analyzed using Omics office software embedded in Tibco Spotfire version 6.5.2. False discovery rate (FDR) was controlled at 0.05 using the Benjamini-Hochberg procedure, and gene lists were analyzed using Ingenuity Pathway Analysis (IPA) software to detect significantly over-represented canonical pathways.

Results:

We found 119 genes which were differentially expressed between the captured autoreactive CEP-1 peptide-specific B cells and decoy EP-1 peptide tetramer-specific sorted B cells which withstood FDR correction (q <0.05).  Of these, only one transcript was down-regulated in autoreactive cells (IFI44L), whereas all the other 118 transcripts were up-regulated.  Transcripts that were up-regulated in the autoreactive cells included genes involved in B cell receptor and calcium signaling (CALM2), B cell activation, differentiation and transmembrane signaling (CD53), and cell cycle progression (CDKN2D). Using Ingenuity Pathways Analysis, 38 of 115 mapped genes were found to be involved with cellular proliferation (p = 0.004). Other over-represented canonical pathways included protein ubiquitination and integrin signaling. 

Conclusion:

In an RA patient demonstrating serological reactivity to citrullinated alpha-enolase, a CEP-1 peptide tetramer allowed for the capture of autoreactive B cells that demonstrated an activated and proliferative gene expression signature. These results illustrate that novel analytic approaches can now be applied to isolated autoreactive RA B cells.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-rna-seq-analysis-of-citrullinated-alpha-enolase-peptide-specific-b-cells-in-ra/