317.7: LEA29Y expression alleviates organ rejection in a porcine heterotopic abdominal heart transplantation model

Background: The T-cell co-stimulation blocker LEA29Y is an established immuno-suppressive drug in transplantation settings. It prevents the activation of T-cells and thus alleviates cellular rejection processes. Here, we are evaluating the effect of local LEA29Y production from a transplanted organ in an allogeneic heterotopic abdominal heart transplantation (HAHT) model in the pig regarding levels of rejection relevant cytokines and immune cell markers in transplanted tissue. Methods: Wildtype (WT) (n=4) or LEA29Y (n=15) expressing hearts from a previously published transgenic pig line (Bähr et al., PLos One, 2016) were transplanted into WT recipient pigs in a non-loaded HAHT model. MHC compatibility was determined by combined haplotype PCR. Grafts were evaluated over a maximum period of 5 weeks post-transplantation without additional immunosuppression. Upon completion of the follow up period, the degree of rejection was initially determined macroscopically and transplants were systematically sampled for assessment at molecular and histological levels. Graft samples of left and right atrium as well as left and right ventricle were snap frozen for RNA extractionand preserved in 4% paraformaldehyde and Tissue Tek. Non-transplanted WT and LEA29Y transgenic heart tissues served as baseline controls. Results: The transplantation setting appears highly robust, with minimal loss of experimental animals during transplantation and the post-operative phase. Stainings for immune cell markers and fibrosis have been adapted for heart tissue sections and established for systematic and quantitative digital assessment. A panel of highly stable cytokine and immune cell marker assays has been developed on cDNA verified for high quality. Preliminary examination shows a general overexpression of cytokines and immune cell surface markers in both transplanted groups in comparison with non- transplanted baseline levels. However, LEA29Y grafts display lower cytokine levels and leukocyte cell markers compared to WT hearts, indicating lower cell infiltration as well as reduced inflammatory reactions intransplanted LEA29Y transgenic hearts. Conclusions: We have successfully established a HAHT model in the pig that allows us to evaluate the effect of local immunosuppression in a transplantation setting. Initial results indicate a protective effect of LEA29Y expression in the cardiac graft based on levels of cytokines and immune cell markers within the transplanted hearts. Further evaluation will allow us to quantitate the protective effect of LEA29Y expression in a transplantation setting. Deutsche Forschungsgemeinschaft TRR127 References: 1. Baehr A.et al. Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs. PLoS One. 2016 May 13;11(5):e0155676. doi: 10.1371/journal.pone.0155676. eCollection 2016.