LEA29Y expression executes a heterogenous protective effect on cardiac transplants in a porcine allograft model

Introduction: The possibility to control graft survival by local T-cell co-stimulation blockade was evaluated in an allogeneic heterotopic abdominal heart transplantation (HAHT) model in the pig. Hearts from wildtype (WT) (n=4) pigs or pigs expressing the belatacept analogon LEA29Y as transgene (n=15) were transplanted into WT pigs. Recipients were gradually tapered off supportive immunosuppression over a maximum period of 5 weeks post-transplantation.
Methods: Blood samples were taken at baseline and at regular intervals throughout the follow-up period. With termination of experiments, samples of the left and right heart were collected and fixed for histology or cryo-conserved for molecular analysis. Non-transplanted WT (n=3) and LEA29Y (n=3) tissues served as baseline controls. Expression of cytokines and immune cell markers was measured by ELISA in blood serum and by qPCR in heart tissue to provide a nuanced determination of rejection levels and examine immune system activation prior to transplantation as a contributing factor in rejection of the graft. SLA mismatch was identified by numerical grading of single nucleotide polymorphisms (SNPs) haplotypes in exon 2 and exon 3 of selected pig MHC (SLA).
Results: While all WT grafts were completely rejected, LEA29Y hearts showed a mixed outcome from 1R to 3R rejection grades in histological assessment, which correlated well with transcript levels of TGFB, TNFA, IFNG, IL1A, IL1B, IL6, IL10 and IL18 cytokines and CD45, CD68 and CYBB immune cell markers in the transplanted hearts. Single nuclei RNAseq profiling of grafts demonstrated a substantial loss of cardiomyocyte in transplants and a reduced expression of main RYR2, MYH7, TNNT2 markers in the remaining cells. Interestingly, 3R-graded LEA grafts appeared more deteriorated than 3R graded WT grafts in the transcriptome analysis. Neither the cumulative nor separate haplotype mismatches in class I (SLA1, SLA2) and class II (SLA-DRB1, SLA-DQA, SLA-DQB) coincided with the graft outcome. In contrast, elevated baseline serum cytokine levels in either recipient or donor animals transpired as a strong negative predictor of LEA29Y protection on the graft.
Conclusions: Pre-existing immune activation may serve as a strong predictive marker for post-transplant rejection responses. Retrospective single nuclei RNAseq analysis will give detailed insight into the underlying mechanisms of immune response pre-disposition and help to determine synergistic angles to support local T-cell co-stimulation blockade.