Dominant intragraft plasma cells targeting bilirubin implicate local heme catabolism in human cardiac allograft vasculopathy

Cardiac allograft vasculopathy (CAV) is invariably associated with immune infiltrates around affected coronary arteries, including antibody-producing plasma cells (PC). We used single-cell-RNA-sequencing and IGHV profiling to characterize these infiltrating PC. We then generated 37 recombinant monoclonal antibodies (mAb) using rearranged immunoglobulin gene sequences from dominant intragraft PC clones as well as 24 control mab from peripheral blood PC. Unexpectedly, a majority (21/37) of mAb derived from intragraft PC, but none of the control mAb derived from blood PC, reacted to bilirubin, a by-product of heme degradation. Furthermore, we observed the deposition of bilirubin within lymphocytic aggregates of cardiac grafts with CAV but not in healthy heart tissue. Bilirubin also accumulated in the cytoplasm and the nuclei of smooth muscle cells in the tunica media of coronary arteries with CAV lesions. Lastly, expression of heme-oxygenase-1 and biliverdin reductases in graft-infiltrating macrophages as well as presence of Fe2+ ion in the media of arteries with hyperplasia further supported the implication of heme catabolism in CAV. Collectively, our findings suggest a key role for bilirubin in CAV. The enrichment of PC clones reactive to this metabolite within graft infiltrates indicates that it acts as a prominent target of local antibody responses.