Plasma proteomics and single cell RNAseq identify cellular and soluble mechanisms of graft rejection after HSCT

1. Interferon, TNF and ferroptosis pathways are enriched in graft rejection patients and merit further study as biomarkers/therapeutic targets. 2. Recipient NK cells and T-cells increase during graft rejection, suggesting these recipient cells are the primary drivers of rejection. There are no biomarkers or therapies for graft rejection after hematopoietic stem cell transplant (HSCT) due to a limited mechanistic understanding of this event. We hypothesized that interferon activation from recipient cells drives donor cell elimination during graft rejection. To test this hypothesis, we studied plasma proteomes of HSCT recipients with graft rejection in comparison to febrile HSCT recipients who engrafted. Leading differentially expressed proteins (DEPs) included CXCL11 (q=2e-5) and CXCL10 (q=3.4e-3). The most highly enriched pathway was interferon gamma response (q= 8.59e-10). Interferon gamma, tumor necrosis factor (TNF) and interferon alpha were also the leading upstream regulators of DEPs. Drug network interactomes for interferon gamma and TNF inhibitors confirmed these therapies may inhibit activated pathways. We then performed single cell RNAseq on peripheral blood mononuclear cells (PBMCs) from a graft rejection patient and compared PBMCs at the time of rejection to a prior timepoint after initial engraftment. PBMC transcriptomes were separated by donor (female) and recipient (male) origin, revealing a marked increase in recipient NK cells (3.5% versus 83.7%) and recipient effector memory T-cells (8% versus 91.8%) during rejection. Donor classical monocytes during rejection showed ferroptosis pathway enrichment, an interferon-mediated form of cell death. To our knowledge, this is the first single cell RNAseq study in humans to confirm recipient NK cell and T-cell resurgence during graft rejection after HSCT. We additionally identified promising biomarkers (CXCL11, CXCL10, LAG3), a potential mechanism of donor cell death (ferroptosis) and targetable immunologic pathways (interferon gamma, TNF) meriting further study.