Figure 6

⍺-hemolysin differentially affects CD8 + T cells, CD4 + T cells, and Tregs in vitro. (A–E) Splenocytes were cultured in the presence of anti-CD3/anti-CD28 activation for 24 h. These in vitro expanded splenic T cells were subjected to a 24 h treatment of either HLA, the conditioned media from the HLA-treated EO771 cells (HLA-CM), or the conditioned media from the vehicle-treated EO771 cells (CM). For HLA treatment, T cells were incubated with 10 μg/ml of HLA (Low, in blue, n = 4), 50 μg/ml of HLA (High, in red, n = 3), and the control vehicle (Con, in black, n = 4). For HLA-CM and CM treatment, T cells were incubated with low and high doses of HLA-CM or CM (in blue and red, respectively, n = 4, referring to the Methods for details). Since HLA-CM and CM treatment groups share the same control group where T cells were treated with EO771 culture media (Con, in black, n = 4), this control group is duplicated in the figures to serve as a control for both HLA-CM and CM groups. Flow cytometric analysis was performed to enumerate the percentage of CD8⁺ cells (A), CD4⁺ cells (B), and regulatory T cells (Tregs) among viable cells (C), as well as CD8⁺/CD4⁺ (D) and CD8⁺ /Tregs ratios (E). Data are presented as median with quartiles (truncated violin plots). Two-way analysis of variance (ANOVA) with multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.