Background/Purpose: Neonatal lupus (NL) is an uncommon autoimmune disease classically manifest as permanent complete heart block and/or transient cutaneous lesions. By definition of NL, there is universal exposure to maternal autoantibodies reactive with the Ro/La ribonucleoprotein complex; however, only 2% of women with these antibodies will have an affected child.  Accordingly, this study was initiated to address the hypothesis that antigen agnostic approaches (i.e. those that are antigen-independent) will have utility for identifying important functional antibodies and their targets.

Methods: Peripheral blood mononuclear cells were obtained from 10 Anti-Ro (plus or minus)/La positive mothers (asymptomatic, pauci-symptomatic, SLE, or Sjogren’s Syndrome) of children with complete heart block (5 of whom died).  Circulating plasmablasts were single-cell sorted and their IgH and IgL chains tagged with cell-specific DNA barcodes before sequencing with Illumina Miseq.  Phylogenetic trees of the antibody repertoire were generated via binning the reads from each B cell based on the barcodes.  Antibodies representative of clonal families were recombinantly expressed.  Profiling of the recombinant antibodies was done by immunofluorescence (IF) using the HEp-2 cell line and ELISAs using native Ro60, recombinant Ro52 and recombinant La.

Results: From inspection of the phylogenetic usage of IgH and IgL in NL mothers, the approach captured a profile inclusive of highly mutated antibodies.  Twenty three expressed antibodies representing the repertoires of two mothers were further evaluated.  For the majority of monospecific immunoglobulin IgG (16/23), there was strong nuclear and/or cytoplasmic staining which correlated with reactivity on ELISA.  Several antibodies showed polyreactivity for multiple antigens by ELISA.  In one mother with mild dry eyes and identification of anti-Ro only after the diagnosis of heart block in her fetus, there was evidence of a convergent autoimmune-specific antibody HCDR3 signature.  Interestingly, while the antibodies showing evidence of this convergent signature were both positive by IF, they were discordant regarding Ro/La reactivity.

Conclusion: While prior studies used approaches involving preselecting Ro/La antigens to further identify pathogenic autoantibodies, this is the first to define candidates based on clonal families identified through mining the plasmablast antibody repertoires in mothers with the pathogenic antibodies by virtue of their having a child with NL. Monospecific antibodies, particularly the subset with convergent specific antibody signatures, provide a platform for identifying the “pathoepitope” leading to cardiac injury.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/profiling-circulating-plasmablasts-from-anti-ro-positive-mothers-of-children-with-congenital-heart-block-to-identify-antigenic-targets-conferring-pathogenicity/