Local antibody responses in human cardiac allograft vasculopathy

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PROJECT SUMMARY Cardiac allograft vasculopathy (CAV) is one of the leading causes of morbidity and mortality following heart transplantation. While the pathophysiology of CAV is still poorly defined, converging lines of evidence point to a critical role of local immune responses in the graft tissue in this complication. In humans, CAV is consistently associated with B cells and antibody-producing plasma cell infiltrates in or around coronary arteries. These infiltrating cells have been poorly studied. 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 therapeutic agents to treat this form of rejection. Here, we are proposing to use state-of-the-art IGHV repertoire analysis, single-cell-RNA-seq combined with CITE- seq and paired single-cell-IgH+L sequencing 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 intragraft plasma cell infiltrates in human CAV. Studies in aim 1 will combine IGHV repertoire and single-cellRNA-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 (mab) from a large number of plasma cells present in the graft infiltrates and identify their reactivity. Aim 2 To identify FcR-mediated mechanisms whereby antibodies produced in situ contribute to CAV. We will focus here on the ability of antibodies secreted in situ to form immune complexes (IC) and activate Fc receptor (FcR)-expressing cells in the graft. Experiments in aim 2 will use a scRNA-seq approach combined with CITE-seq to systematically map all immune and non-immune cells expressing FcR in the graft and therefore capable of responding to IC. We will then investigate whether stimulation of these cells through specific FcR leads to the engagement of pro-inflammatory and pro-fibrotic pathways associated with CAV. Lastly we will look for evidence that a comparable process occurs in vivo in the context of CAV. Aim 3. To determine FcR-dependent mechanisms whereby antibodies promote vasculopathy in vivo. In aim 3, we will use a mouse aortic allotransplantation model to assess the capacity of antibodies secreted by graft-infiltrating PC to contribute to transplant vasculopathy in vivo. Moreover, we will use a series of constitutive or conditional knockout strains to determine which FcR are implicated in the effect and identify cells expressing these individual receptors. We will particularly investigate the involvement of the neonatal Fc receptor FcRn expressed by graft endothelial cells and smooth muscle cells.