Alloreactivity and autoreactivity converge to support B cell epitope targeting

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. Using single cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific (alloreactive) B cell response in kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We identified 14 distinct alloreactive B cell lineages, which spanned the rejected organ and blood and expressed high-affinity anti-donor HLA-specific B cell receptors – many of which were clonally linked to circulating DSA. The alloreactive B cell response was focused on exposed, solvent-accessible mismatched HLA residues, while also demonstrating extensive contacts with self-HLA residues. Consistent with structural evidence of self-recognition, measurable autoreactivity by donor-specific B cells was common and positively correlated with anti-donor affinity maturation. Thus, allo- and self-reactive signatures appeared to converge, suggesting that during AMR, the recognition of non-self and breaches of tolerance conspire to produce a pathogenic donor-specific adaptive response. Overall design: After tissue processing, B cells and antibody-secreting cells (ASCs) from a rejected transplanted kidney and the peripheral blood of that same patient were sort-purified for bulk B-cell receptor IgVH sequencing and generation of single HLA-reactive recombinant monoclonal antibodies (rmAbs). rmAbs were utilized for experiments testing fine specificity, affinity, and binding structures. Plasma was affinity-purified against the donor HLA-A*01:01 to produce affinity-purified plasma Abs (appAbs) which were analyzed via proteomics.