Fig. 2: rHGF (400 ng/mL) inhibits msCD8⁺ T cell proliferation and promotes apoptosis that mediated by c-Met receptor.

A Analysis of the purity of n-msCD8⁺ T cells isolated using the MACS method with CD8α antibody as assessed by FCM (n = 3). B pn-msCD8⁺ T cells (1 × 10⁵) were cultured and treated with different doses of rHGF for 24 h (n = 3). C CCK-8 assays were performed for the analysis of pn-msCD8⁺ T cell growth between the rHGF-treated and nontreated groups at 24, 48, and 72 h after rHGF administration (n = 3/time point). D(a–g) Immunofluorescence staining for Ki-67⁺ cells among MACS-purified pn-msCD8⁺ T cells within DAPI⁺ cells in the present and absence of rHGF treatment for 72 h (a, c, arrows denote proliferation T cell clones (red), n = 6; TUNEL (green) assay of cell apoptosis after rHGF treatment for 48 (d) and 72 h (b, green, e), scale bar = 200 µM, n = 6; 72-h rHGF (400 ng/mL)-treated pn-msCD8⁺ T cells (g, n = 3) and Jurkat T cells (J) (f, n = 3) were also subjected to gel electrophoresis to detect DNA fragments. E(a, b) Inhibitory effects of an anti-c-Met-Ab on pn-msCD8⁺ T cell proliferation as assessed by CCK-8 assay (a, n = 3) and immunostaining (b, red, boxes, n = 3) 24 h before 400 ng/mL rHGF administration, scale bar = 200 µM. F Immunofluorescence staining for c-Met (red) in pn-msCD8⁺ T cells (green, n = 6, an arrow denotes c-Met-expressing n-msCD8⁺ T cells, brown color after merging) and quantification of c-Met expression by FCM (brown bars, n = 3). Scale bar = 200 µm. At least three independent experiments were performed, and the data are presented as the means ± SDs. The asterisks indicate a statistically significant difference; *p < 0.05 and **p < 0.001 vs. PHA-Ctrls; ^#p < 0.05 and ^##p < 0.001 vs. rHGF; ns represents no significance (Student’s t test).