Cytotoxic CD8+ T Cells Are Involved in the Thrombo-Inflammatory Response during First-Diagnosed Atrial Fibrillation

Figure 1. Activation of CD8+ T cells in patients with first-diagnosed AF. (A) Phenotyping of circulating CD8+ T cells (flow cytometry). Activated cytotoxic T cells (HLA-DR+), naive/early differentiated CD8+ T cells (CD28+CD57−) and matured CD8+ T cells (CD28−CD57+). (B) Increased plasma levels of cytotoxic effector molecules associated with CD8+ T cell function (ELISA). Results are expressed as single values (n = 20/80), mean with SD.

Figure 2. Activation of CD8+ T cells corresponds to biomarkers of cardiac fibrosis and atrial dysfunction in patients with first-diagnosed AF. (A) Increased plasma levels of biomarkers that suggest cardiac fibrosis (sST2, galectin-3) and atrial dysfunction (NT-proANP) (ELISA). Results are expressed as single values (n = 20/80), mean with SD. (B) Correlation matrix comparing CD8+ T cell effector molecules (granulysin, granzymes, sFasL) with sT2, galectin-3 and NT-proANP plasma levels in patients with first-diagnosed AF (n = 80). Pearson correlation coefficients (+1 positive, −1 negative correlation) are shown.

Figure 3. Thrombogenicity corresponds to CD8+ T cell activation. Plasma levels of tissue factor (TF) relative to plasma levels of cytotoxic effector molecules in patients with first-diagnosed AF (ELISA). Results are expressed as single values (n = 80), Pearson correlation coefficients and linear regression lines with 95% CI.

Figure 4. Cardiac PAR1 expression correlates with markers of thrombo-inflammation in patients with AF. Patients underwent transvascular right-ventricular endomyocardial biopsy (EMB). PAR1 gene expression in EMBs: (A) relative to TF gene expression; (B) relative to the distribution of perforin-positive cytotoxic T cells and the extent of cardiac fibrosis. Results are expressed as single values (n = 32), Pearson correlation coefficients and linear regression lines with 95% CI.

Figure 5. CD8+ T cell-mediated effector function in early AF is linked to PAR1 activation. (A,B) Patients with first-diagnosed AF vs. control group. (A) Phenotyping via flow cytometry of T cells in patients with first-diagnosed AF revealed a higher percentage of circulating cells that possess PAR1 (CD3+PAR1+), an increased expression of thrombin-activated PAR1 (as expressed by an increased mean fluorescence intensity (MFI) of thrombin-cleaved PAR1) and a higher percentage of cytotoxic T cells that express PAR1, which was activated by thrombin (CD8+ cleaved PAR1+). (B) Enhanced pro-inflammatory effector function of thrombin-activated CD8+PAR1+ T cells as expressed by staining for IFN-γ and TNF-α. (C) Separate cohort of patients (third cohort) on stable anti-thrombotic therapy. Lower percentage of circulating pro-inflammatory thrombin-activated CD8+PAR1+ T cells under a PAR-directed therapy (FXa/FIIa-inhibitor) when compared to non-PAR-directed therapy (ASA/VKA). Results are expressed as single values (A,B n = 20/80; C n = 20/60), mean with SD.

Figure 6. Frequency of thrombin-activated CD8+PAR1+ T cells is associated with adverse outcomes in patients with first diagnosis of AF. Patients were stratified into two groups according to the occurrence of MACEs (no vs. yes) during follow-up after their first diagnosis of AF. (A) Composite endpoint MACE: first occurrence of cardiovascular death, unplanned re-hospitalisation for AF, un-planned hospitalisation for heart failure (HF), transient ischaemic attack (TIA), ischaemic stroke, acute coronary syndrome (ACS), deep vein thrombosis, peripheral thromboembolism. (B,C) Adverse outcome events that are associated with the disease progression of AF (B) (un-planned re-hospitalisation for AF, un-planned hospitalisation for HF) and thrombo-embolic and atherothrombotic complications (C) (TIA, ischaemic stroke, ACS) are related to CD8+PAR1+ T cells at the time of first diagnosis of AF. Results are expressed as single values (n = 80), mean with SD.