Table 1.
Clinical patient characteristics of the main study group.
Table 2.
Clinical patient characteristics of the time course substudy group.
Figure 1.
Peripheral leukocytosis after primary percutaneus coronary intervention (PPCI).
Absolute number of total leukocytes, neutrophil granulocytes and monocytes (A), as well as lymphocytes and the main T-cell subsets (B) in peripheral blood. Concentration of leukocyte populations are displayed for patients 24 h following PPCI in STEMI (n = 31), 3 months post PPCI (n = 24), and in age-matched healthy Controls (n = 18). 1-way ANOVA with Tukeys' post-hoc test was performed. *p<0.05, ***p<0.001, ns not significant.
Figure 2.
Basic gating strategy for CD4+ T cells.
Viable cells, as distiguished on the basis of Aqua dye staining were gated and CD4+ T cells were selected based on their light scatter properties and CD3 and CD4 co-expression. CD4+ T-cell subpopulations were separated into main subsets based on the CD45RA/CCR7 expression properties, as previously described by Sallusto et al.: NAIVE (CD45RA+CCR7+), CENTRAL MEMORY (CD45RA−CCR7+), EFFECTOR MEMORY (CD45RA−CCR7−) and CD45RA reexpressing „revertant” memory CD4+ T cells (T effector memory CD45RA+ cells, TEMRA). Further sequential gating analysis were perfomed based on the coexpression of co-stimulatory markers CD27/CD28 as well as proliferation/senescence markers CD57, KLRG1, and PD1.
Figure 3.
Distribution of main CD4 T-cell subsets.
Frequencies of CD4+ naive (CD45RA+CCR7+), central memory (CD45RA−CCR7+), effector memory (CD45RA−CCR7−) and terminally differentiated effector memory TEMRA cells (CD45RA+CCR7−) are shown. Groups are identical to Figure 1. 1-way ANOVA with Tukeys' post-hoc test was performed. *p<0.05, ***p<0.001, ns not significant.
Figure 4.
Hierarchical clustering analysis of 13-parameter flow cytometry data.
Dendrogram with heatmap-HCA of 104 events (gated on viable CD3+ T cells) acquired from PBMCs of healthy control and STEMI patient (24 h after PPCI). Heatmap shows relative levels of all 13 parameters (columns) in all 104 events (rows) in color coding (blue, low expression; red, high expression). Dendrogram shows the hierarchy of T cells based on their similarity in all parameters measured. Colored branches of the dendrogram are selected clusters (A). The T-cell subsets from healthy controls (as identified by HCA) are displayed on conventional scatter dot plots (B). HCA revealed that the proliferation marker CD57 is strongly expressed on CD4+ CM T cells in STEMI patients (zoom on cluster of interest in C).
Figure 5.
Increased expression of CD57 in central memory cells indicates acute proliferation in the CD4 T-cell compartment.
Conventional gating analysis confirmed increased expression of proliferation associated markers CD57 on CM CD4+ T cells of STEMI pts (MFI – mean fluorescence intensity). This was reflected by significantly increased frequencies of specific KLRG1−CD57+ CD4 CM+ T cells (A–C). The validity of CD57, PD-1 and KLRG1 as proliferation-associated markers was determined in healthy controls according to Sallusto's model of T-cell differentiation (D). Mean telomere length (mTL) in CD4+ T cells is reduced in patients with STEMI when compared to age-matched controls (E). *p<0.05, **p<0.01, ***p<0.001, ns not significant.
Figure 6.
CD4+CCR7+ T cells are specifically depleted from peripheral blood during myocardial reperfusion.
Time course pilot study showed no significant changes in numbers of monocytes (A) and granulocytes (B) following PPCI. In contrast, T cells decreased by 35% during the first 30 min following reperfusion showing significant relative increase within next 24 hours (C). The absolute cell count analysis revealed a significant decrease in CD4+ T cells during the first 60 min following PPCI (D). This was mainly due to the specific depletion of circulating CD4+CCR7+ cells within the first 30 min following reperfusion (E). *p<0.05, **p<0.01, ns not significant.