Type 1 Classical Dendritic Cells Govern Long-Term Cardiac Allograft Acceptance
Acute and chronic rejection continue to represent serious challenges in transplant medicine. Current medical regimens focus on suppressing T-cell responses, lack effectiveness for some patients and impose significant risks of infection and malignancy. Recent studies have suggested that inhibition of co-stimulation pathways between antigen presenting cells (dendritic cells, macrophages, B-cells) and T-cells may serve as an effective strategy to prevent rejection by promoting allograft tolerance as opposed to simply suppressing immune responses. This concept has garnered excitement across the transplant community and demonstrated promising results in human renal transplant studies. A commonly used approach is inhibition of B7-CD28 and CD40-CD40L costimulatory pathways. Blocking these pathways leads to prolonged allograft survival in fully HLA-mismatched mouse heart transplantation models and non-human primates. There are important gaps in knowledge pertaining to mechanisms by which co-stimulation blockade modulates the recipient immune responses and confers donor organ tolerance. The precise immune cell populations and mechanisms responsible for induction and maintenance of donor organ tolerance remain incompletely understood. We have leveraged single cell RNA sequencing to dissect how traditional immunosuppression (cyclosporine) and co-stimulation blockade (CTLA-4 Ig, anti-CD40L neutralizing antibodies) differentially impact the immune landscape of the transplanted heart using a fully HLA-mismatched mouse model (Balb/c donor heart à B6 recipient). Analysis of these datasets demonstrated striking differences between these treatment regimens. Mice that received co-stimulation blockade displayed a marked increase in the abundance of classical dendritic cells (cDC1s and cDC2s). Compared to the cyclosporine treated group, these cells expressed negative regulators of T-cell stimulation including PDL1. Using recipient mice that lack either cDC1s or cDC2s, we found that cDC1s are absolutely required for co-stimulation blockade to confer long-term allograft survival and prevent recipient immune responses against the transplanted donor heart. Overall design: Hearts from a BALB/c donor were transplanted into B6 (Zbtb46gfp/+) recipients treated with conventional immunosuppression (cyclosporine) or co-stimulation blockade (MR1, CTLA4-Ig). After 7 days, allografts were harvested, digested, and single cell suspensions were prepared. 3 allografts from each condition (cyclosporine and co-stimulation blockade) were pooled, and mononuclear phagocytes (MNP) (CD45+Ly6G-CD64+/-Ly6Chi/low) and dendritic cells (CD45+GFP+) were sorted by flow cytometry. 4 separate libraries were generated for single cell RNA sequencing (cyclosporine MNP, cyclosporine GFP+, co-stimulation blockade MNP, co-stimulation blockade GFP+). In a second experiment, hearts from a BALB/c donor were transplanted into B6 recipients (wildtype control or Irf8 +32-/-) treated with co-stimulation blockade. 14 days after transplant, allografts were harvested, digested, and single cell suspensions were prepared. 3 allografts from each condition (wildtype and Irf8 +32-/-) were pooled, and CD45+ DAPI- Ly6G- CD45 IV- immune cells were sorted by flow cytometry. 2 separate libraries were generated for single cell RNA sequencing (WT, IRF8).