Lung T Cell Activation in an Outbred Pig Transplant and Ischemia-Reperfusion Injury Model

Purpose Primary graft dysfunction (PGD) contributes to morbidity and mortality after lung transplantation. Ischemia-reperfusion injury (IRI) is a major risk factor for PGD. While the adaptive immune system contributes to IRI, its role is not well understood. The pig lung transplant (LTx) IRI model is useful for pre-clinical testing: however, assessment of immune changes is limited by poor pig-specific reagents. Our objective was to establish strategies for studying changes in adaptive immune cell recruitment and activation in a pig IRI model. Methods Pig lung tissue samples (standardized for size) were collected from separate 4h and 72h pig IRI survival models. Samples included: donor lung post-flush but prior to cold ischemic preservation (pre-CI), donor lung post-CI prior to transplant, post-transplant left allograft with IRI (IRI), and contralateral right native lung (no IRI). Novel porcine flow cytometry panels were developed and used to characterize T lung leukocytes. A one-way ANOVA was used; p Results Both T cell and non-T cell lung tissue leukocytes trended down during CI (post-CI vs. pre-CI) (Fig A, B). After reperfusion, non-T cell leukocytes generally increased (Fig B) without a marked change in CD3 T cells (4h IRI vs. post-CI) (Fig A). The CD4:CD8 ratio increased compared to post-CI in both the allograft and native lung but returned to a CD8 predominant ratio at 72h post-IRI (Fig C). The effector:non-effector T cell ratio increased 4h post-IRI and remained high at 72h post-IRI (Fig D). Concurrently, IRI was linked with a significant decrease in naive CD4 T cells, which were almost undetectable at 72h post-IRI (Fig E). Conclusion In a pig LTx IRI model, substantial T cell activation was seen, supporting the involvement of the adaptive immunity in lung IRI, despite minimal T cell recruitment. The flow cytometric strategies developed will be used to further define adaptive immune responses in pig IRI, effects of IRI-directed therapies, and their role in the development of PGD.