CD8+ T-cells affect scar formation and maturation post myocardial infarction
S Dasgupta, N Bradley, M Troncoso, P Angel, K Y Deleon-PennellAbstract
Introduction
Myocardial infarction (MI) and subsequent mortality from heart failure is strongly dependent on scar formation and remodeling. A healthy, stiff scar is crucial for maintaining left ventricular (LV) shape and function. Previously, we have shown that CD8+ T-cells have an adverse effect on scar remodeling post-MI. Our data suggests that these adverse effects are mediated by granzymes, or cytotoxic proteases. We have furthermore identified a subset of CD8+ T-cells, CD8^161+ or Innate-Like CD8+ T-cells (ILTs), that are elevated with age. Nonetheless, whether ILTs are involved in scar remodeling post-MI is not known.
Purpose
Study the proteolytic effect of both conventional CD8+ T-cells (CD8^161-) and ILTs. We hypothesize that ILTs impair scar formation and maturation post-MI through protease mediated cleavage.
Methods
MI was induced by permanent ligation of the coronary artery in C57BL6/J (WT; 3-7 mo) and CD8^atm1mak mice (CD8-/-; 3-7 mo). CD8-/- mice were injected with either vehicle or naïve splenic CD8+ T-cells (2x10^6 cells/injection) via tail vein, 4 hours after ligation. Tissue was collected at Day 7 post-MI for spatial proteomics analysis. To determine protease activity, CD8+ T-cells were isolated from the spleen (n=3/sex/group) and plated on a 2% gelatin-coated 96-well plate (10,000 cells/per well). Inhibitors for either granzyme (GZMi; 50µM) or matrix metalloprotease (MMPi; 50µM) were added to test protease specificity. Media alone was used as a negative control and collagenase (6µg/mL) as a positive control. After 24 hours, cleavage of gelatin was determined by staining wells with Coomassie Blue and measuring absorbance at 595 nm.
Results
Imaging mass spectroscopy identified a Col1a2 peptide associated with the collagen gap region that was elevated in CD8-/- mice compared to WT mice. This peptide decreased after re-supplementation with splenic CD8+ T-cells, suggesting that CD8+ T-cells may be facilitating collagen makeup of the infarct scar. Assessment for gelatin cleavage demonstrated that both ILTs and conventional CD8+ T-cells can cleave gelatin (p<0.05) when compared to the negative control. Addition of GZMi and MMPi showed clearance was significantly decreased in both cell types (p<0.05), indicating that both conventional CD8+ T-cells and ILTs secrete GZMs and MMPs and facilitate in gelatin degradation.
Conclusions
Our preliminary data demonstrates that both conventional CD8+ T-cells and ILTs regulate scar remodeling in part through the release of proteases.