Single-Cell Rna Sequencing of the Mouse Isograft and Allograft Lung after Orthotopic Lung Transplantation

P188 Heterogeneity of large and small airway remodeling in human endstage explant cystic fibrosis lungs

Purpose Following lung transplantation, the surgical intervention with ischemic periods and the foreign allograft are the first to trigger a response from the innate immune system, which represents the primary nonspecific defense against “non-self”. In this study, we investigated the diversity of the emerging innate immune cells and the timing of their appearance in a murine orthotopic left lung transplant model. Methods Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6). All mice received daily immunosuppression of 10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone. Serial sacrifice was performed at day 1, 7 and 35 post-transplantation (n=6 at each time point for each group). Left transplanted lungs were harvested, single cell suspension was made and absolute numbers of innate immune cells were quantified by flow cytometry. Results Following transplantation, isografts and allografts showed an increase in neutrophil (p=0.017) and tissue resident NK cell (p=0.002) numbers at day 1, and in eosinophil (p=0.002) numbers at day 7 post-lung transplant. Granulocytes demonstrated similar patterns in isografts and allografts. Allografts demonstrated an increase in antigen presenting cells i.e. patrolling monocytes (Ly6C-) (p=0.002), interstitial macrophages (p<0.0001) and dendritic cells (p=0.005) at day 7 compared to isografts. Classical NK cells were increased in allografts at day 7 versus isografts (p=0.03). All cell numbers returned to baseline isograft values at day 35 post-transplant, except for dendritic cells (p=0.0045). Conclusion Diverse innate immune cell types show different patterns over time post-lung transplantation depending on transplant induced ischemia-reperfusion injury and/or alloimmune response against the foreign graft. These differences may be very important to dissect the various pathophysiological processes triggered by lung transplantation. Following lung transplantation, the surgical intervention with ischemic periods and the foreign allograft are the first to trigger a response from the innate immune system, which represents the primary nonspecific defense against “non-self”. In this study, we investigated the diversity of the emerging innate immune cells and the timing of their appearance in a murine orthotopic left lung transplant model. Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6). All mice received daily immunosuppression of 10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone. Serial sacrifice was performed at day 1, 7 and 35 post-transplantation (n=6 at each time point for each group). Left transplanted lungs were harvested, single cell suspension was made and absolute numbers of innate immune cells were quantified by flow cytometry. Following transplantation, isografts and allografts showed an increase in neutrophil (p=0.017) and tissue resident NK cell (p=0.002) numbers at day 1, and in eosinophil (p=0.002) numbers at day 7 post-lung transplant. Granulocytes demonstrated similar patterns in isografts and allografts. Allografts demonstrated an increase in antigen presenting cells i.e. patrolling monocytes (Ly6C-) (p=0.002), interstitial macrophages (p<0.0001) and dendritic cells (p=0.005) at day 7 compared to isografts. Classical NK cells were increased in allografts at day 7 versus isografts (p=0.03). All cell numbers returned to baseline isograft values at day 35 post-transplant, except for dendritic cells (p=0.0045). Diverse innate immune cell types show different patterns over time post-lung transplantation depending on transplant induced ischemia-reperfusion injury and/or alloimmune response against the foreign graft. These differences may be very important to dissect the various pathophysiological processes triggered by lung transplantation. 

Mapping the Diversity of the Innate Immune System over Time After Left Lung Transplantation in Mice

Immunological Overlap Between Rejection and Infection in Murine Lung Transplantation

Purpose Endothelial cells are the first non-self-barrier encountered by the recipient's immune system after allograft transplantation and thus are involved early during alloimmune response. Our goal was to study processes of endothelial cell injury/activation in a mouse model of lung transplantation. Methods Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6), both received daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone) and were serially sacrificed at day 1, 7 and 35 post-transplant (n=6/timepoint/group). Left transplanted lungs were made into single cell suspension and absolute cell numbers were quantified by flow cytometry for CD31+ endothelial cells, CD45+ leukocytes and MHCI/II, VCAM-1 and ICAM-1 for cell activation. One additional allograft and isograft were scanned with high resolution (HR) ex vivo microCT (µCT) to visualize airways and blood vessels at day 70 post transplantation. Results Endothelial cell numbers decreased from day 1 until day 7 (p=0.03) in allografts and recovered slightly at day 35. While in isografts, endothelial cells seemed to increase non-significantly over time. In contrast, leukocytes significantly increased at day 7 in allografts versus day 1 (p=0.036) and isografts at day 7 (p=0.003). Leukocytes remained stable in isografts over time (p=0.3). Mean fluorescence intensity (MFI) was increased for MHC I/II and VCAM-1 (p=0.003; p<0.0001; p=0.04) on endothelial cells, the MFI of MHC I/II and ICAM-1 on leukocytes was increased in allografts versus isografts (p=0.0002; p=0.006; p=0.002). HR µCT showed normal airway morphology while lumen of arteries and veins was narrowed in allograft compared to isograft. Arterioles and venules were occluded in allograft. Conclusion Destruction of endothelial cells may represent the very first onset of alloimmune response post lung transplantation and warrant further investigations towards diagnostic and therapeutic approaches. Endothelial cells are the first non-self-barrier encountered by the recipient's immune system after allograft transplantation and thus are involved early during alloimmune response. Our goal was to study processes of endothelial cell injury/activation in a mouse model of lung transplantation. Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6), both received daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone) and were serially sacrificed at day 1, 7 and 35 post-transplant (n=6/timepoint/group). Left transplanted lungs were made into single cell suspension and absolute cell numbers were quantified by flow cytometry for CD31+ endothelial cells, CD45+ leukocytes and MHCI/II, VCAM-1 and ICAM-1 for cell activation. One additional allograft and isograft were scanned with high resolution (HR) ex vivo microCT (µCT) to visualize airways and blood vessels at day 70 post transplantation. Endothelial cell numbers decreased from day 1 until day 7 (p=0.03) in allografts and recovered slightly at day 35. While in isografts, endothelial cells seemed to increase non-significantly over time. In contrast, leukocytes significantly increased at day 7 in allografts versus day 1 (p=0.036) and isografts at day 7 (p=0.003). Leukocytes remained stable in isografts over time (p=0.3). Mean fluorescence intensity (MFI) was increased for MHC I/II and VCAM-1 (p=0.003; p<0.0001; p=0.04) on endothelial cells, the MFI of MHC I/II and ICAM-1 on leukocytes was increased in allografts versus isografts (p=0.0002; p=0.006; p=0.002). HR µCT showed normal airway morphology while lumen of arteries and veins was narrowed in allograft compared to isograft. Arterioles and venules were occluded in allograft. Destruction of endothelial cells may represent the very first onset of alloimmune response post lung transplantation and warrant further investigations towards diagnostic and therapeutic approaches. 

C. Hooft

Papers

Single-Cell Rna Sequencing of the Mouse Isograft and Allograft Lung after Orthotopic Lung Transplantation

P188 Heterogeneity of large and small airway remodeling in human endstage explant cystic fibrosis lungs

Mapping the Diversity of the Innate Immune System over Time After Left Lung Transplantation in Mice

Immunological Overlap Between Rejection and Infection in Murine Lung Transplantation

Endothelial Cell Injury and Activation in a Murine Model of Left Lung Transplantation