Date: Monday, November 9, 2015
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: In-depth analysis of the molecular and antigenic properties of antibody secreting cells (ASC) is critical for our understanding of autoimmune diseases. This goal however has been hampered by technological barriers imposed by low-throughput technologies to interrogate the ASC at the single cell level. We describe here the incorporation of a high throughput methodology for linking the B cell receptor’s heavy and light chain variable region (VH and VL) for analyzing the plasmablast B cell population from a SLE patient. Conventional techniques for sequencing of genomic DNA or cDNA from single cells are limited by low efficiency and low cell throughput (<200–500 cells), whereas >2 x 106B cells per experiment can be analyzed by this single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires.
Methods: We use a flow focusing apparatus to encapsulate the single B cells in to the emulsion droplets containing the lysis buffer and oligo dT magnetic beads for capturing the mRNA, followed by an emulsion RT-PCR for generating the VH and VL linked products for next generation sequencing. Approximately 50,000 plasmablasts (CD19+IgD-CD27highCD38highCD138neg) from a SLE patient were flow sorted and VH and VL transcript were linked using emulsion RT-PCR. IgH, IgL and linked transcripts were sequenced via Illumina MiSeq.
Results: 20,000 different sequences representing over 2,200 different clonotypes were identified, thereby demonstrating a very polyclonal PB repertoire during Lupus flares. High concordance was shown with Illumina miseq data obtained from bulk PB obtained from a separate aliquot of the same blood draw. Both experiments also demonstrated the presence of substantial clonal expansions of the SLE-associated VH4-34 clones.
Conclusion: We have incorporated a high-throughput methodology of linking immunoglubulin heavy and light chain variable region (VH-VL) transcripts prior to amplification and repertoire analysis via NGS. The ability of this approach to combine deep sequencing with single cell antibody generation should greatly enhance our understanding of the antigenic triggers involved in the pathogenesis of SLE and other autoimmune diseases.
To cite this abstract in AMA style:Tomar D, Tipton C, Sanz I. Analysis of SLE Plasmablasts By High Throughput Pairing of the Immunoglobulin Heavy and Light Chain (VH-VL) [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/analysis-of-sle-plasmablasts-by-high-throughput-pairing-of-the-immunoglobulin-heavy-and-light-chain-vh-vl/. Accessed January 29, 2020.
« Back to 2015 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/analysis-of-sle-plasmablasts-by-high-throughput-pairing-of-the-immunoglobulin-heavy-and-light-chain-vh-vl/