Figure 3: In vitro analysis of the effects of recombinant ProTα on CD8⁺ T-cells.

A, Representative gating strategy scatter plots to evaluate markers of cytotoxicity in CD8⁺ T-cells. After cell doublets exclusion, total lymphocytes region was gated based on cell size (forward scatter/FSC) and cell complexity (side scatter/SSC). Dead cells were excluded using a cell viability dye. CD8⁺ high cell population was gated to further analyze the markers of cytotoxicity: FasL, granzyme-B and perforin. Control cultures (non-stimulated, CTRL) are represented by white dots, ProTα stimulated cultures are represented by green dots, estradiol stimulated cultures (E₂) are represented by pink dots, cultures stimulated by both ProTα and E₂ (ProTα+E₂) are represented by red dots. B, Frequency of CD8⁺ cells expressing FasL (CD8⁺FasL⁺), granzyme-B (CD8⁺GzB⁺), perforin (CD8⁺Perf⁺) and (CD8⁺GzB⁺Perf⁺). C, Mean intensity of fluorescence (MFI) of estrogen receptor alpha (ERα) gated in CD8⁺GzB⁺Perf⁺ cells. Representative histogram showing the MFI of ERα in the different culture conditions. D, Representative direct immunofluorescence staining for CD8, GzB and Perf in RHVD valvular lesion. Scale bars = 50 μm. E, Frequency of VLA-1 (CD8⁺GzB⁺Perf⁺VLA-1⁺) and VLA-2 (CD8⁺GzB⁺Perf⁺VLA-2⁺) gated in CD8⁺GzB⁺Perf⁺. Bar graphs show the mean of values in each group (n=10) and standard deviation. Comparisons among non-stimulated and stimulated cell cultures were made using two-way ANOVA followed by Tukey’s test. Statistical significance is indicated in each graph.