Session Name: Ischemia Reperfusion & Organ Rehabilitation
Session Date & Time: None. Available on demand.
*Purpose: Analyze the transcriptomics of mouse lung cells with and without latent infection of cytomegalovirus (CMV) before and after transplantation and identify key cell types and molecular pathways responsible for CMV reactivation and dissemination.
*Methods: Orthotropic left lung transplants were performed using Balb/c recipients and donors with or without latent CMV infection. Lung tissues from transplants and naïve controls were harvested for viral RNA analysis and single cell isolation at day 2 post-transplant. After isolating CD31+ and CD45+ cells using fluorescence-activated cell sorting, single cell suspensions were processed using 10X Genomics for barcoding and cDNA library construction for four different conditions at an average of 15500 cells and 41000 reads per cell. The sequenced data were processed using standard 10X Genomics Cell Ranger pipeline and preliminary analyses were carried out using the Loupe browser where cell clusters were identified using well-accepted surface markers. Pathway analyses were done for bulk differences and for different cell types as defined by markers. Additional transplants were treated with small molecule Myd88 inhibitor to determine whether innate immunity plays a role in transplant induced CMV reactivation.
*Results: At 2 days post-transplant, CMV IE-1 mRNA was detected in lung grafts but not in latent lungs not transplanted, suggesting transcriptional activation in the early phase of transplant and ischemia and reperfusion injury as an important contributing factor. Preliminary analysis of clusters show CD31+ lung cells expressing Myd88 increase in number following organ transplant both with and without latent CMV infection. CD31+ lung cells expressing c-Fos are also heightened following transplant. CD11b+ lung cells expressing Myd88 increase in latent CMV infected organs following transplant. CD11b+ lung cells expressing c-Fos were increased following transplantation in CMV infected animals. Pathway analysis of differentiating genes for myeloid cell types shows a number of significant immune response pathways including MIF regulation of innate immunity and communication between innate and adaptive immune cells. Treatment with Myd88 inhibitor decreased the viral DNA copy numbers.
*Conclusions: Results indicate an enhanced innate immune response. The heightened expression of c-Fos suggests possible reactivation and dissemination of CMV in transplanted tissue. Pathway analysis of differentiating genes elucidated a number of significant immune response pathways, including both innate response and communication between innate and adaptive immune cells, suggesting reactivation and dissemination of CMV in these cells following transplantation.
To cite this abstract in AMA style:VanOsdol LA, Han S, Kandpul M, Wang JJ, Qiu L, Zhang ZJ. Single-cell Rna Sequencing of Latent Murine Cytomegalovirus Infected Lung Transplants [abstract]. Am J Transplant. 2021; 21 (suppl 3). https://atcmeetingabstracts.com/abstract/single-cell-rna-sequencing-of-latent-murine-cytomegalovirus-infected-lung-transplants/. Accessed June 15, 2021.
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