Background: Healing and scar formation in myocardial remodeling after ischemia/reperfusion (I/R) are complex processes strictly regulated by the immune system and its effector cells. The fusion protein LEA29Y is a commonly used immunosuppressive drug that blocks co-stimulation of T cells and thus restricts immune cell activation. Pigs systematically expressing LEA29Y have been established as a model for general immunosuppression that was used to study I/R remodeling under immuno-compromised conditions.

Methods: LEA29Y transgenic and wildtype (WT) pigs were subjected to a catheter-based procedure of I/R in which the main coronary artery was temporarily occluded by a balloon catheter. Animals were assigned to follow-up periods of 3, 9 and 14 days. Results were compared to transgenic and control animals without intervention to determine baseline conditions. At the end of every experiment infarct size was calculated and the left ventricle (LV) was systematically randomly sampled. Functional measurements by left ventricular angiography as well as electrocardiogram and echocardiography were supplemented by molecular histological assessments. Scar formation and infiltration of immune cells were investigated by picrosirius red and immunohistochemical staining. Peripheral blood mononuclear cells were used for systematic immune cell profiling.

Results: LEA29Y and WT animals displayed similar infarct sizes (26.51±3.26% of LV vs. 25.86±5.373% of LV, p=0.09153). LV angiography demonstrated a significantly higher loss of ejection fraction in transgenic pigs after 3 days (-15.48±1.069 vs. -7.554±1.873, p=0.011), which was not detectable in longer follow-up. Picrosirius red staining showed no difference in scarring between genotypes (day 9: 4.8% of LV vs. 3.8% of LV, p=0.3035). Immunohistochemically, investigations revealed a peak in volume density of neutrophils after 72 h for both groups (0.29% of LV vs. 0.36% of LV, p=0.6515). The same dynamic was seen in T cells, albeit in lower volume densities in LEA29Y animals (0.15% of LV vs. 0.36% of LV, p=0.0026). Macrophages showed a later and not as prominent accumulation in transgenic pigs (day 3: 1.5% of LV vs. 4.12% of LV, p=0.0215; day 9: 3.22% of LV vs. 4.23% of LV, p=0.4358). In contrast, immune cell profiling in the blood yielded higher percentages of T cells (CD4+ in live cells: 63.58±1.217%, vs. 35.29±1.77%, p<0.0001) and CD14+ CD163+ monocytes (48.82±3.409 vs. 32.25±2.735, p=0.0026) for immunosuppressed animals. In LEA29Y pigs, activation markers (ICOS, CD8α) and cytokine production (IFN-γ, IL-17A) were reduced, while TNF-α secretion and the expression of CADM1 were increased.

Conclusions: The lower recruitment to the ischemic area with concomitant higher proportion of circulating cells suggests LEA29Y mediated T cell anergy. This includes a reduced ability to proliferate, differentiate and migrate into injured tissue, which also affects macrophages and leads to an initially higher loss of function after I/R, that seems to be compensated through alternative mechanisms of the immune system.