Donor Macrophages Modulate Rejection after Heart Transplantation

Abstract

Background Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T-cells, and have a multitude of adverse effects. The innate immune response plays an essential role in the recruitment and activation of T-cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge regarding donor immune cell types and functions in the setting of cardiac transplantation and no current therapeutics exist for targeting these cell populations. Methods Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and function during acute cellular rejection of transplanted hearts in mice. We performed single cell RNA sequencing on donor and recipient macrophages, dendritic cells, and monocytes at multiple timepoints after transplantation. Based on our single cell RNA sequencing data, we evaluated the functional relevance of donor CCR2+ and CCR2- macrophages using selective cell ablation strategies in donor grafts prior to transplant. Finally, we perform functional validation of our single cell-derived hypothesis that donor macrophages signal through MYD88 to facilitate cellular rejection. Results Donor macrophages persisted in the transplanted heart and co-existed with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2+ and CCR2- macrophage populations and revealed remarkable diversity amongst recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2+ and CCR2- macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. While selective depletion of donor CCR2- macrophages reduced allograft survival, depletion of donor CCR2+ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2+ macrophages were being activated through MYD88/NF-ĸβ signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen presenting cell recruitment, decreased emergence of allograft reactive T-cells, and extended allograft survival. Conclusions Distinct populations of donor and recipient macrophages co-exist within the transplanted heart. Donor CCR2+ macrophages are key mediators of allograft rejection and inhibition of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions.