Evaluation of rejection in LEA29Y expressing hearts in a porcine heterotopic abdominal heart transplantation model

Samjhana Shrestha (München)1, M. Cambra (München)1, B. Reichart (München)2, E. Wolf (Oberschleissheim)3, C. Kupatt (München)1, N. Klymiuk (München)1, S. Michel (München)2, A. Bähr (München)1

1Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I München, Deutschland; 2LMU Klinikum der Universität München Herzchirurgische Klinik und Poliklinik München, Deutschland; 3Lehrstuhl für molekulare Tierzucht und Biotechnologie der LMU München Oberschleissheim, Deutschland


LEA29Y is a potent T-cell co-stimulation blocker in transplantation settings. Here we aimed at testing for the capacity of local LEA29Y abundance to protect the graft. LEA29Y expressing hearts from a published pig line (Bähr et al.,PLos One, 2016) and wild type (WT) hearts were transplanted into wild type recipient pigs in an allogenic heterotopic abdominal heart transplantation (HAHT) model to evaluate the protective effect of local LEA29Y expression

Methods and results
While all WT hearts were profoundly rejected, clinical transplantation outcome in the LEA29Y group showed a high degree of variability (complete rejection (n=5); functional survival with some signs of rejection n=3). To further examine the level of immunological reactions on a molecular basis, we have established a qPCR assay for a range of cytokines and immune cell markers. Graft samples of left and right atrium as well as left and right ventricles were snap frozen for RNA extraction. Non-transplanted WT and LEA29Y transgenic heart tissues served as baseline controls.
The qPCR panel for inflammatory cytokines TNF-α, IFNγ, IL-1α, IL-1β, IL-18, IL-6, IL-10 and TGF-β and immune cell markers CD45, CD68, CYBB was extensively optimized with reference to the four housekeeping genes GAPDH, ACTB, TBP and YWHAZ. The quality of cDNA was extensively monitored  for laying within acceptable range of slope (-3.2 to -3.6) and linearity (R^2=0.99 to 1) in a qPCR dilution row using a GAPDH standard assay. In total, seventy-six samples (RA, RV, LA, LV) were processed (WT transplantation (n=4), LEA29Y transplantation (n=9), WT controls (n=3) and LEA29Y controls (n=3)).
Evaluation shows a general overexpression of cytokines and immune cell surface markers in both transplanted groups compared to non-transplanted baseline levels. All WT transplants express high level of cytokines and immune cell markers. LEA29Y transgenic heart transplants show varied expression of rejection markers that correlates with clinical outcomes. The non-functional LEA29Y hearts exhibit high levels of rejection markers while functional LEA29Y heart transplants exhibit a pattern of lower expression levels for all rejection markers. Notably, one LEA29Y heart shows significantly lower cell infiltration as well as reduced inflammatory reactions after transplantation while also displaying the best function at explantation.

We have successfully established a HAHT model in pigs that allows us to evaluate the impact of local immunosuppression in a transplantation setting. Functional and molecular analysis of rejection provide an indication of a protective effect of LEA29Y expression. Next, we will perform more extensive molecular analysis to elucidate the mechanistic basis of LEA29Y protection in some but not all grafts. Additionally, the systemic impact of local LEA29Y expression in the recipient will be analysed. 
Diese Seite teilen