*Purpose: Donor and recipient age at the time of lung transplantation (LTx) have been implicated in poor patient outcomes. The underlying mechanisms driving these effects have yet to be fully elucidated. Using a rodent model of LTx and high dimensional mass cytometry techniques, we characterized the rejection responses and immunophenotype of young and old LTx recipients.

*Methods: Balb/c donor lungs were transplanted into C57BL/6 recipients using the following age combinations: 1. Young (10wk)- Young (10wk), 2. Old (44wk)- Old (44wk), 3. Young (10wk old)- Old (44wk), and 4. Old (44wk)- Young (10wk). Grafts were harvested 7 days post-LTx and analyzed for acute rejection (AR). Based on our AR findings we performed a sub-analysis focused specifically on the old recipients. We isolated immune cells from the LTx and performed high dimensional mass cytometry (CyTOF) and mixed lymphocyte reactions (MLR) to evaluate population and functional immune characteristics. Additionally, qPCR was used to determine difference in key T cell cytokines.

*Results: Analysis of AR at 7 days post-LTx demonstrated differences associated with donor and recipient age. Young-young LTx demonstrated the reported grade 4 AR, whereas old-old LTx had an AR score of 1. LTx of old-young also demonstrated grade 3-4. LTx of young lung into an old recipient restored AR grade of 4 in the old recipient, thus suggesting that young donor lung can restore aggressive rejection within the old recipient. Focusing in on the dynamics of old recipients, we demonstrated increased IFN-γ and IL-17 gene expression in young to old transplants versus old to old. qRT-PCR data were corroborated by cytokine ELISA of MLR. To dissect the cellular constitutes associated with these age-related changes we utilized CyTOF cellular analysis and a bioinformatics pipeline for the in-depth characterization of the immune cell subsets. Our unbiased biostatistical approach identified significant increases in alveolar macrophages, CD8+ T cells, dendritic cells, and eosinophils in old-old LTx as compared to young-old recipients, cells that have been associated with tolerance in this model system.

*Conclusions: Here we demonstrate that age effects post-LTx rejection responses, and that rejection is blunted in old recipients of old donor lungs. Further analysis of the immune populations is needed and is ongoing to determine the phenotype and functional significance of the populations we have identified.

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