Background/Purpose: Anti-histidyl-tRNA synthetase syndrome (Jo-1 ARS) is defined by the presence of autoantibodies against histidyl tRNA synthetase (Jo-1). Clinically, Jo-1 ARS can involve multiple tissues including muscle, lung, skin, and joints. Previously, we identified that Jo-1 binding B cells show skewing towards specific phenotypic subsets in the peripheral blood of Jo-1 ARS patients, but the mechanisms that promote B cell recognition of Jo-1 autoantigen are unknown.

Methods: Five patients with active anti-Jo-1 ARS were identified from the Vanderbilt MYSTIC cohort (VUMC IRB 141415).  We isolated live CD19+ B cells from cryopreserved peripheral blood mononuclear cells (PBMCs) using fluorescence activated cytometry sorting.  Five-thousand cells were targeted for sequencing per sample.  Single cell RNA-Seq, V(D)J sequencing, and Cite-Seq were performed using the 10X Chromium platform (5’-RNA, V(D)J, and CITE-Seq) de-multiplexed and processed using the CellRanger pipeline and analyzed with Seurat v4.0.0.  V(D)J gene identities and % somatic hypermutation were determined for B cell receptors (BCR) using IMGT/HighV-QUEST.  Seurat was used to merge these outputs with RNA-seq and CITE-seq data to enable integrated analysis of BCRs with transcriptomic and phenotypic profiling. Clonally expanded BCRs were identified and recombinantly expressed as monoclonal antibodies.  Total IgG, Jo-1, LPS, dsDNA, and insulin binding were measured by ELISA.

Results: Five B cell subsets (transitional, naïve, activated, memory, plasmablast) were assigned based on isotype switching and gene expression (Fig. 1A-B).  We identified 21 expanded clonotypes (defined as n ³ 3 per donor) amongst the five donors (Fig. 1C).  Clonally expanded cells were CD27+ and CD21lo (Fig. 2) and were primarily IgM+ (non-class-switched) (Fig. 3A).  Somatic hypermutation was highly variable and ranged from germline to very high (25%) (Fig. 3B).  Recombinant expression of clonally expanded BCRs as IgG1 antibodies and ELISA binding studies revealed that 5/21 (24%) bound Jo-1.  All Jo-1 binding clones exhibited some binding of dsDNA, LPS, or insulin, confirming polyreactivity.  Multiple Jo-1 binding B cells expressed the VH4-34 heavy chain gene, which has been previously associated with autoimmunity.

Conclusion: Clonally expanded B cells in patients with Jo-1 ARS are enriched for anti-Jo1 B cells, which display an autoreactive-prone (CD21lo) memory phenotype.  Somatic hypermutation analysis shows that germline recognition of Jo-1 autoantigen is possible, and that Jo-1-binding B cells can undergo extensive somatic hypermutation. Future studies will be required to determine if somatic hypermutation is associated with affinity maturation of Jo-1-binding B cells, and presumably enhanced pathogenicity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/clonally-expanded-b-cells-in-anti-histidyl-trna-synthetase-syndrome-patients-exhibit-an-autoreactive-prone-memory-phenotype-and-bind-jo-1-autoantigen/