Jo-1-Binding and Clonally-Expanded Memory B Cells Express Germline and Somatically-Mutated B Cell Receptors in Anti-tRNA Synthetase Syndrome Patients

Erin Wilfong, Alberto Cisneros, Jennifer Young-Glazer, Scott Smith, Leslie Crofford and Rachel Bonami, Vanderbilt University Medical Center, Nashville, TN

Meeting: ACR Convergence 2021

Keywords: B-Lymphocyte, genomics, interstitial lung disease, Myositis

Session Information

Date: Saturday, November 6, 2021

Title: B Cell Biology & Targets in Autoimmune & Inflammatory Disease Poster (0001–0010)

Session Type: Poster Session A

Session Time: 8:30AM-10:30AM

Background/Purpose: Anti-tRNA synthetase syndrome (ARS) is a severe systemic autoimmune disease associated with myositis, interstitial lung disease, rash, and arthritis. ARS is associated with autoantibodies towards the tRNA synthetases, the most common being anti-histidyl tRNA synthetase (Jo-1). We previously showed Jo-1-binding B cells (JoBCs) isolated from Jo-1 autoantibody+ (Jo-1 ARS) patients are skewed towards a non-class-switched B cell subset that has phenotypic features of autoreactive-prone memory B cells. Mutated JoBC B cell receptors (BCRs), which dictate antigen specificity, would imply a requirement for T cell selection, whereas germline BCRs would point to T cell-independent mechanisms of JoBC expansion. Our objective was to define BCR V gene usage and mutation among BCRs expressed by JoBCs and determine phenotypic and BCR repertoire features of highly expanded B cell clones in ARS patients.

Methods: We applied human hybridoma technology to identify Jo-1 autoantigen-binding B cells. Additionally, single-cell, tandem RNAseq/BCRseq and a modified Seurat and IMGT/HighVQUEST-based pipeline was used to identify and analyze expanded B cell clones isolated from Jo-1 ARS, non-Jo-1 ARS, and healthy control peripheral blood (n=5 donors per group). BCR gene structure and phenotypic attributes were determined.

Results: JoBC hybridoma clones expressed germline and mutated BCRs. VH4-34 and VH3-21 were identified among JoBC hybridomas as well as clonally expanded B cells in our single-cell dataset isolated from distinct donors. Clonally-expanded B cells were biased towards memory and plasmablast subsets in both Jo-1 ARS and non-Jo-1 ARS patients. The majority of Jo-1 ARS expanded clones were not class-switched, whereas IgG and IgA class-switched clones were more predominant in non-Jo-1 ARS.

Conclusion: These data suggest B cell clones can expand, enter memory, and differentiate into plasmablasts prior to class-switching, particularly in Jo-1 ARS patients. Our approaches to identify autoantigen-specific and clonally-expanded B cells could be used to track B cell perturbations as correlates of conventional or experimental immune therapy responsiveness. Improved monitoring of deleterious expansion of autoantigen-specific B cells could improve disease management in ARS patients and other rheumatic diseases. Germline VH4-34 cross-reacts with commensal bacteria, providing a potential mechanism for JoBC immune tolerance breach prior to somatic hypermutation and T cell selection.

Figure 1. Somatically-mutated as well as germline BCRs confer Jo_1 autoantigen recognition. A) Jo_1 reactivity of representative JoBC monoclonal hybridoma lines is shown by ELISA. B) % Somatic hypermutation (y-axis) and VH gene identity (x-axis) is shown. C) BCR structural model of JoBC hybridoma line 1E7 depicts amino acid mutations as sticks (e.g., arrows). VH CDR loops (red), VL CDR loops (blue)

Figure 2. Single-cell transcriptomics identifies distinct B cell subsets in peripheral blood. Single-cell RNAseq and BCRseq technology was used to profile purified B cells isolated from ~5×106 cryopreserved PBMCs per donor from Jo_1 ARS patients, Non-Jo_1 ARS patients, and healthy controls (n=5 donors per group). Seurat was used to determine A) RNAseq-based B cell clusters, B) BCR isotype, and C) specific gene expression profiles to define B cell cluster identities (expression of selected genes is shown). Memory B cell (blue circles) and plasmablast subsets (pink circles) are highlighted in panels A-B.

Figure 3. Clonally expanded, memory B cells isolated from Jo_1 ARS and non-Jo_1 ARS patients express both minimally and highly mutated BCRs, which are skewed towards non-class-switched isotypes in Jo_1 ARS patients. B cell subsets were identified as in Fig. 2; memory B cell (blue circles) and plasmablast subsets (pink circles) are highlighted. A) Integrated Seurat and IMGT/HighV-QUEST BCR gene analysis identified clonally expanded B cells (n≥3 clones per clonotype, teal) among Jo_1 ARS (left), non-Jo_1 ARS (middle), or healthy controls (right), n=5 participants per group. B-D) Data filtered on clonally-expanded B cells from Jo_1 ARS or Non-Jo_1 ARS patients show: B) BCR isotypes, C) BCR V gene usage, and D) BCR somatic hypermutation (depicted by heat scale).

Disclosures: E. Wilfong, Boehringer-Ingelheim, 1, 5; A. Cisneros, None; J. Young-Glazer, None; S. Smith, None; L. Crofford, Boehringer-Ingelheim, 5, UpToDate, 9; R. Bonami, Boehringer-Ingelheim, 5.

To cite this abstract in AMA style:

Wilfong E, Cisneros A, Young-Glazer J, Smith S, Crofford L, Bonami R. Jo-1-Binding and Clonally-Expanded Memory B Cells Express Germline and Somatically-Mutated B Cell Receptors in Anti-tRNA Synthetase Syndrome Patients [abstract]. Arthritis Rheumatol. 2021; 73 (suppl 9). https://acrabstracts.org/abstract/jo-1-binding-and-clonally-expanded-memory-b-cells-express-germline-and-somatically-mutated-b-cell-receptors-in-anti-trna-synthetase-syndrome-patients/. Accessed .

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