Jo-1 autoantigen-specific B cells are skewed towards distinct functional B cell subsets in anti-synthetase syndrome patients

Abstract

Background Anti-Jo-1 autoantibodies which recognize histidyl-tRNA synthetase identify patients with the rare rheumatologic disease, anti-histidyl-tRNA synthetase syndrome (Jo-1 ARS), a phenotypically distinct subset of idiopathic inflammatory myopathies (IIM). Jo-1-binding B cells (JBCs) are implicated in disease pathogenesis, yet they have not been studied directly. We therefore aimed to characterize JBCs to better understand how they expand and function in Jo-1 ARS. Methods We enrolled 10 IIM patients diagnosed with Jo-1 ARS, 4 patients with non-Jo-1 IIM, and 8 age- and sex-matched healthy controls. We phenotypically characterized peripheral blood mononuclear cells (PBMCs) ex vivo using flow cytometry to define the B cell subsets in which JBCs reside. We further tested their ability to differentiate into antibody-secreting cells following stimulation in vitro. Results The majority of JBCs were IgM+ (not class-switched). Compared to non-JBCs in the same donors, JBCs contained a higher percentage of autoimmune-prone CD21lo cells and were increased in the CD21lo IgM+ IgD− CD27+ memory subset relative to healthy donor B cells. Whereas non-JBCs were present in the anergic BND B cell subset, JBCs were nearly absent from this compartment. JBCs were detected among plasmablasts in some donors, but a reduced frequency of JBCs differentiated into CD38hi24− plasmablasts compared to non-JBCs present in the same wells following in vitro stimulation. Conclusions JBCs are enriched for autoimmune-prone CD21lo B cells, some of which exhibit a memory phenotype in the peripheral repertoire of Jo-1 ARS patients. JBCs undergo limited class switch and show reduced capacity to differentiate into antibody-secreting cells. This suggests complex B cell biology exists beyond class-switched cells that differentiate to secrete anti-Jo-1 autoantibody (i.e., what is captured through serum autoantibody studies). New Jo-1 ARS therapies should thus ideally target non-class-switched JBCs in addition to those that have undergone IgG class-switching to most effectively block cross-talk with autoreactive T cells.

High-Throughput Detection of Autoantigen-Specific B Cells Among Distinct Functional Subsets in Autoimmune Donors

“…As shown in Figure 2A , specific B cell subsets were purified using flow cytometry sorting. The focus of this manuscript is on B cells derived from autoimmune donors; we presented detailed immunophenotyping to compare B cell subset percentages between patients with several of the autoimmune diseases highlighted in this manuscript and healthy controls in separate manuscripts ( 36 , 41 ). Purified B cell subsets (e.g.…”

“…We previously identified ASBCs using fluorescently-labeled autoantigen ( 36 ). This detection method could however miss B cells that downregulate surface BCR because of chronic antigen stimulation and/or immune tolerance mechanisms such as anergy.…”

“…Flow cytometry detection of ASBCs can be challenging due to low frequency of target cells. However, we recently identified a readily detectable population of Jo-1-binding B cells in the peripheral blood of Jo-1 autoantibody-positive anti-histidyl tRNA synthetase syndrome patients using flow cytometry staining with labeled autoantigen ( 36 ). This created an opportunity to directly examine expansion of ASBCs following our in vitro stimulation protocol.…”

“…Data were analyzed using FlowJo software (Tree Star, Inc.). Jo-1-binding B cells were identified by flow cytometry using GST-tagged Jo-1 antigen we previously cloned and recombinantly expressed, followed by detection with anti-GST secondary antibody (Abcam), as previously described ( 36 ).…”

“…For example, whereas naïve B cells proliferate in response to BCR stimulation, anergic (B ND ) and CD21 lo B cells do not (30,31). B ND and CD21 lo subsets may serve as reservoirs for autoreactive B cells in several autoimmune diseases, including type 1 diabetes, Sjögren's syndrome, anti-histidyl tRNA synthetase syndrome, and systemic sclerosis (32)(33)(34)(35)(36). We sought to develop high-throughput stimulation and screening methods to identify ASBCs among total PBMCs using ELISA detection of BCRs secreted as antibody.…”