Background:
The fusion protein LEA29Y, also known as Belatacept, is an immunomodulatory component blocking
co-stimulation in T-cells and thereby working as an immunosuppressant. Pigs systematically expressing
LEA29Y have been established as a model for general immunosuppression. They show a defect in T-
cell maturation and proliferation as well as decreased numbers of B-cells and NK cells. This model can
be used to investigate scar formation and healing after myocardial ischemia/reperfusion (I/R) in
immuno-compromised conditions.

Methods:
To examine possible effects of the impaired immune system in LEA29Y transgenic pigs on scarring and
healing, these pigs as well as wildtype pigs were subjected to a catheter-based procedure of I/R in
which the left anterior descending artery was occluded by a balloon catheter for 45 or 60 minutes
before reperfusion. To detect differences in activation and reaction of the immune system in the
course of time animals with follow up periods of 3, 9 and 14 days were compared to control animals
without any previously intervention. Functional heart measurements were performed before and after
ischemia and at the end of every experiment. Blood sampling underwent the same procedure but was
additionally repeated every 24 hours during the first three days for isolation of PBMCs. The tissue of
the ventricle was systematically sampled to define infarct area, area at risk and control areas.
Experiments are examined regarding functional and histological measurements in the heart as well as
immune cell profiling in the circulation.

Results:
With a HW/BW ratio of about 4,5 g/kg and a infarct size of 35% of LV after three days respectively 20%
of LV after nine days follow up period for all animals no differences between LEA29Y transgenic and
WT pigs can be detected. However, the immuno-compromised animals show a significant higher loss
of EF (LEA29Y: mean -15%, WT: mean -7,5%) after three days. ECG data of implanted event recorder
further indicate a higher range in variability of heart rate in these pigs. In histological analyses Sirius
Red stainings prove an increase of fibrosis up to fourfold only on day 9 in all animals. HE stainings
show an obvious encirclement of the affected areas by immune cells. Immunohistochemistry
displayed the same trend with a peak on day 3 (increased 3 to 4 times) for neutrophils. T-cells also
demonstrated a similar dynamic, albeit at lower numbers in the LEA29Y transgenic pigs.. Whereas
macrophage numbers in the ischemic area are significantly lower and peaking later in the ischemic
area of these animals (LEA29Y: Baseline 0,9%, 3d 1,5%, 9d 3,2% of LV; WT: Baseline 1,1%, 3d 4,1%, 9d
4,2% of LV). In contrast, immune cell profiling in the blood yields higher numbers of T-cells (CD4+ of
live cells: LEA29Y 60%, WT 35%) and monocytes (CD14+ CD163+ cells) in LEA29Y animals. In LEA29Y
T-cells, activation (MFI ICOS in CD8α+ cells) and IFN-γ-, IL-17A- and TNF-α- production is reduced
compared to WT cells.

Summary and Outlook:
The higher numbers of circulating immune cells and the lower recruitment to the ischemic area of
infarcted LEA29Y hearts in heart suggest a distinct anergy or the inability to migrate into affected
tissue, combined with a reduced wound healing. Further studies with immunocompromised pigs
should take this effect into account.