Functional significance of plasma cell infiltrates of human cardiac allograft vasculopathy

PROJECT SUMMARY Cardiac allograft vasculopathy (CAV) is presently the leading cause of morbidity and mortality following heart transplantation. While the pathophysiology of CAV is still unclear, converging lines of evidence point to a critical role of local immune responses in the graft tissue in this complication. In human CAV, B cell and antibody- producing plasma cell (PC) infiltrates are consistently observed in or around coronary arteries, yet these infiltrating cells are still poorly characterized. In particular, the antigen specificity and effector functions of locally produced antibodies are currently unknown. Understanding how these antibodies contribute to mechanisms of CAV would undoubtedly facilitate the development of new treatments. Here, we propose to use state-of-the-art immunoglobulin gene repertoire analysis, single-cell-RNA-seq combined with paired single-cell-IgH+L sequencing (BCR-seq) to obtain a comprehensive characterization of plasma cells infiltrating cardiac allografts during CAV. The functional properties and pathogenicity of individual antibodies produced in situ will also be evaluated using both in vitro cell-based assays and in vivo experimental transplantation models after generation of recombinant monoclonal antibodies from intragraft plasma cells. Aim 1. To characterize graft-infiltrating plasma cells in human CAV. Studies in aim 1 will combine IGHV repertoire and single-cell-RNA-seq analyses to determine the clonal composition and transcriptome profile of plasma cells found directly at the graft site during CAV. These experiments will also identify predominant clones expanded in situ. Using an expression-cloning platform, we will generate recombinant monoclonal antibodies from a large number of plasma cells expanded in the graft infiltrates and identify their specificity. Aim 2. To determine the function of antibodies produced by intragraft plasma cells. We will focus here on the ability of antibodies secreted in situ to form immune complexes (IC) and activate FcγR-expressing cells in the graft. Experiments in aim 2 will use a scRNA-seq approach combined with CITE-seq to comprehensively map all immune and non-immune cells expressing FcγR in the graft and therefore capable of responding to IC. We will then investigate whether stimulation of these cells through specific FcγR leads to the engagement of pro- inflammatory and pro-fibrotic pathways associated with CAV. Lastly, we will assess whether antibodies secreted in situ amid CAV can modulate the function of biologically active metabolites they react to (e.g., bilirubin). Aim 3. To assess the capacity of antibodies produced by intragraft plasma cells to promote CAV in vivo In aim 3, we will use FcγR humanized mice and a heterotopic heart transplantation model to assess the capacity of antibodies secreted by graft-infiltrating PC to contribute to CAV in vivo. Moreover, we will use a series of constitutive or conditional knockout strains to determine which cells and FcγR are implicated in the effect. We will particularly investigate the involvement of the neonatal Fc receptor FcRn expressed by graft cells as this receptor was recently involved in IC-mediated inflammatory reactions in autoimmune diseases.