Abstract Body: Background: Sustained immune responses are a hallmark of heart failure (HF). Preclinical studies show CD4⁺ T-cell activation and trafficking to the heart during HF. Importantly, CD4⁺ T-cells undergo a pro-inflammatory phenotypic shift during HF facilitating interstitial fibrosis and left-ventricular (LV) remodeling. Despite these preclinical findings, the phenotype and the role of CD4⁺ T-cells in mediating fibrosis and cardiac dysfunction in HF patients is unknown.
Methods: Human heart samples from cardiac transplant and LVAD patients (n=20) as well as cadaveric control samples (n=5) were stained for CD4, CD44 (antigen-experienced T-cell marker), PDCD1 (exhaustion marker), and type I collagen (COL1A1) as a fibrosis marker and analyzed by confocal microscopy.
Results: CD4⁺ T-cells were significantly increased in the failing human hearts compared to the controls (40.0 ± 23.89 vs 0.8 ± 1.304 in controls; p=0.0017). Majority of CD4⁺ T-cells (81.9 ± 16.7%) were localized in the collagen-rich fibrotic areas versus non-fibrotic areas (18.1 ± 16.7%) of failing hearts (p<0.0001). To determine phenotypic differences between CD4⁺ T-cells, we measured PDCD1 (marker for exhaustion) and CD44 (marker for activation) expression on CD4⁺ T-cells present in the failing hearts. Our data show that while all T-cells were positive for PDCD1 and most for CD44, expression of these markers was significantly higher in CD4⁺ T-cells present in fibrotic (1.5- and 1.7-fold, respectively) versus non-fibrotic areas. Importantly, CD4⁺ T-cells and antigen-experienced CD4⁺CD44⁺ T-cells exhibited a positive correlation with COL1A1 levels in failing human hearts (r²=0.7406 and 0.7068, respectively; p<0.0001), highlighting a significant association of activated CD4⁺ T-cell levels with cardiac fibrosis. Single-cell RNA sequencing data further confirmed these findings and showed significantly high expression of pro-fibrotic gene networks in CD4⁺ T-cells present in failing human hearts (p=4.7E-25) as compared to T-cells present in control hearts.
Conclusions: Overall, our data show that CD4⁺ T-cells are enriched in fibrotic areas of failing hearts and exhibit significant phenotypic plasticity dependent upon the local fibrotic milieu.