T-cells as a therapeutic target in an acute myocardial infarction pig model

Problem: Following myocardial infarction (MI), both wound healing and long-term survival are frequently impaired. Studies in mice have shown that shifting the balance between Foxp3⁺ CD25⁺ regulatory T cells (Treg) and conventional T cells (Tconv) towards the Treg can enhance healing and improve survival outcomes. However, currently it is unclear whether this can also be achieved in a large animal model under human-like conditions.

Aim: The aim of this project is to evaluate the efficacy of manipulating the Treg/Tconv ratio in a pig model of MI as a key intermediate step before translating this approach from mice to humans. To increase the Treg/Tconv ratio we either preferentially activated Treg over Tconv by injection of a superagonistic anti-CD28 monoclonal antibody (mAb), abbreviated as CD28-SA, or by preferentially inhibiting Tconv versus Treg with an anti-CD28 mAb that blocks ligand binding to CD28 (ligand-blocking anti-CD28 mAb – LBA-CD28). Both approaches had been efficacious in mouse models of MI.

Methods: To induce an MI,  the left anterior descending artery (LAD) was assessed using a C-bow with contrast agent via a femoral arterial approach and occluded with a balloon for 90 minutes mimicking a common ischemia-reperfusion scenario in humans. Echocardiography of the heart (lateral position) was performed on days 0, 3 and 7 measuring stroke volume (SV) through PW Doppler and the diameter of the left ventricular outflow tract below the aortic valve. Therapeutic or control antibodies were applied on day 3 post MI through i.v. infusion by surgeons blinded to the chosen substance. On day 7, pigs were euthanized and samples were taken from spleen, thymus, mediastinal lymph nodes and the heart for flow cytometric analyses of myeloid cells and lymphocytes.

Results: 8 out of 33 animals could not be resuscitated after developing ventricular fibrillation during the MI procedure. The remaining 25 animals were assigned to 5 treatment groups: NaCl infusion (n=2), control mAb 1 mg/kg (n=4), control mAb 10 µg/kg (n=6), CD28-SA (10 µg/kg, n=6), and LBA-CD28 (1 mg/kg, n=7) and remained in the experiment until day 7. Echocardiographic analyses showed no significant differences between the treatment and control groups. Immunologically, CD28-SA infusion increased the proportion of CD25⁺ Foxp3⁺ Treg among heart-infiltrating CD4⁺ T cells (13.8% vs. 6.6% in controls; p<0.05, One-Way-ANOVA und Tukey's post-hoc Test), but not among CD4⁺ T cells of mediastinal lymph nodes, spleen or CD4-single positive thymocytes. LBA-CD28 treatment had no effect on Treg frequencies among CD4⁺ T cells in any of the tissues studied (8.7% in the heart). The increase in the proportion of Tregs among CD4⁺ T cells induced by CD28-SA infusion was accompanied by a reduction in the proportion of TNF-secreting cells among heart-infiltrating myeloid cells.

Conclusions: CD28-SA treatment induced a marked increase in the Treg population, while blocking CD28 did not appear to have a significant effect. To further validate these findings, we are currently establishing histological stainings for Foxp3 and CD4 to enable direct detection of Tregs in the infarcted heart. Additionally, a long-term study has been initiated in which animals will be examined and euthanized 8 weeks after MI to assess the impact of manipulating the Treg/Tconv ratio on wound healing and cardiac function in pigs after MI. 

This study was funded by the DFG within the CRC 1525 ‘Cardio-immune interfaces’ (Project A2).