Fig 2: Inhibition of Fas antagonism by cMet through cHGF/HGF in recipients with acute liver rejection triggers aCD8+ T cell FAS-mediated apoptosis in allogeneic recipients. (Ai, ii) WSIs of liver tissues taken with a multicolor automatic digital slide scanner and analysis (n = 6/group) of FAS (green) and cMet (pink) expression on aCD8+ T cells (red) in liver grafts from allo-OLT model rats on POD 7 (i, bottom box, merged signals are brick brown, shown in Supplementary Figure S6A with an arrow) and comparison of the brick brown-positive cells among total cells with naive livers (i, top box) and the rats (ii, top box) that received cHGF (ii, bottom box). (B) Co-IP was performed with either a Fas or cMet antibody followed by IB for cMet or Fas to assess the association between cMet and Fas on MACS-isolated Lewis rat naive splenic CD8+ T cells; n1 and n2 represent individual cells. (Ci, ii) 125I labeling FAS-L showing FAS on CD8+ T cells formed aggregation with 400 ng/mL (higher levels) rHGF treatment for 72 h that had a significant higher affinity for 125I-labeled Fas-L (i, upper panel, n = 3), and anti-cMet-Ab blocked this interaction (ii, lower panel, n = 3). The data represent the average of three independent experiments with similar results. The red asterisks indicate a statistically significant difference. (Di–iii) Analysis of cMet (i), FADD (ii), and caspase-8 (iii) levels on splenic aCD8+ T cells after treatment with cHGF and anti-cMet-Ab by the LSPR method; n = 6/marker. (Ei, ii) WSIs of human liver tissues with either ARJ (n = 6, i, bottom box) or HA (i, top box) taken with a multicolor automatic digital slide scanner and analysis of FAS (red) and cMet (pink) expression on CD8+ T cells (green) and HA liver tissues (n = 6, i, a top box) and analysis by Co-IP of the cMet-FAS interaction mode on human healthy peripheral MACS-purified CD8+ T cells (ii). (Fi, ii) Analysis of the IHC staining (brown) data for CD8 and HGF in the same field of view of liver tissues from patients with ARJ (i, n = 6) presented in Supplementary Figure S6D (boxes, n1–n3) and HA liver tissues (n = 6) presented in Supplementary Figure S6C (boxes, n1–n3), indicate individuals and the correlation analysis between HGF and CD8 expression (ii) from patients with ARJ ( Supplementary Figure S6D ); the boxes represent a quarter area per stained field. The data are presented as the means ± SDs of at least three independent experiments. Original magnification, ×400; scale bar = 200 µm. *#p < 0.05 vs. the PHA-treated control or cHGF or HA group; **p < 0.001 vs. naive or allo-OLT or HA liver tissues (Student’s t-test, two-way ANOVA).

Fig 3: HGF induced aCD8+ T cell apoptosis in a mechanism mediated by its only receptor cMet. (Ai–iv) Western blot analysis for HGF in liver allografts from recipient rats of the allo-OLT (i) that received FK506 (ii) and cHGF (iii iv) on POD 10–12; n = 6. (Bi–v) IHC staining (brown) for HGF and CD8 in the same area of tissue specimens from the rats subjected to allo-OLT on POD 10–12 (i, boxes) (n = 6/group) and a comparison to the Syn groups on both POD 7 (ii) and 10–12 (iii); the correlations between CD8 and HGF in allo-OLT recipient rats on POD 10–12 (iv), n = 25 points. Western blot analysis was used for the expression of HGF and CD8 in naïve livers (black) and livers from the rats subjected to allo-OLT (red in boxes) on POD 7 and 10–12 (Bv), densitometric quantification showing the decreasing trend in expression. The target protein expression was normalized to the expression of β-actin; n1 and n2 represent two individual samples; n = 3/time point. Original magnification ×200; scale bar = 100 µm. *#p < 0.05 vs. the Syn group. (Ci, ii) Western blot detection for HGF expression in liver allografts from the recipient rats treated with cHGF plus anti-cMet-Ab (i, the same treatment with cHGF but without the IV injection approach) on POD 7 (n = 6) and survival curve with the MST analysis (ii). n = 16. #p < 0.05 vs. the cHGF group (Student’s t-test). (Di–v) IF staining for aCD8+ T cells (red) in the allo-OLT and cHGF groups at the protein (i, boxes, ii, n = 6) and mRNA (RT-qPCR, n = 3) (iii) levels on POD 7 and the cHGF plus anti-cMet-Ab group (iv, a box, v). (Ei, ii) Analysis of the IF staining of Ki-67 (green) within CD8+ T cells (red) in liver allografts from the allo-OLT and cHGF groups on POD 7 and 10–12 shown in Supplementary Figures S4D —i, ii (i, **p < 0.001 vs. allo-OLT); analysis of TUNEL staining (green) within CD8+ T cells (red) in liver allografts from the allo-OLT and cHGF groups on POD 7 and 10–12 shown in Supplementary Figures S4E —i, ii (ii, **p < 0.001 vs. allo-OLT). Analysis of cHGF plus anti-cMet-Ab (solid blue bars) for CD8+ T cell proliferation (Ki-67) and apoptosis (TUNEL) in Supplementary Figures S4D —iii, iv, Eiii, iv (i, ii, #P < 0.05 and #p < 0.05, ##p < 0.001 vs. the cHGF group), Student’s t-test, n = 6. Original magnification, ×400; scale bar = 200 µm. (Fi–v) Analysis of the serum levels of AST, TBiL, and ALB (i); the cytokines IL-2, TNF-α, and IFN-γ (ii) in the naive (light gray bars), Syn (gray bars), allo-OLT (black bars), cHGF (red bars), and cHGF plus anti-cMet-Ab (green bars) subgroups by ELISAs; histological assessment by RAI score [RAISs (0–3) and tRAISs (0–9)] around PVs (a, boxes), BDs (b, boxes) and VEs (c, arrows) from Supplementary Figures S4F —i, ii on POD 7 and POD 10–12 according to Banff’s criteria (iii, #p < 0.05, ##p < 0.001 vs. the cHGF group, Student’s t-test), n = 10/time point, IF staining (n = 6) of cMet (green) in MACS-purified Lewis rat naive splenic CD8 T cells (red) (F—iv, the arrow denotes merged brown staining) and confirmation of cMet expression by Western blot analysis (F—v, n = 3, n1–n3 represent each set of individual cells); the boxes represent one-sixth of whole stained field. Original magnification, ×400; scale bar = 200 µm. The data are presented as the means ± SDs of at least three independent experiments. The asterisks indicate a statistically significant difference.