Acute phase liver graft injury mobilizes regulating B cells after LDLT for HCC patients through TLR4/CXCL10/CXCR3 signaling

Abstract

OBJECTIVE: We aim to investigate the impact of acute-phase liver graft injury on mobilization of circulating regulating B cells (Bregs) in HCC patients after liver transplantation and to explore the molecular mechanism therein. METHODS: From May 2000 to November 2009, 115 HCC recipients were included. The intragraft gene expression profile and Bregs infiltration of the grafts greater (Group 1) and less than 60% (Group 2) of standard liver weight (SLW) were detected by RT-PCR and immunostaining. Circulating Bregs (CD19+CD24hiCD38hi) were compared by FACS analysis after transplantation. Clinical-pathological data including the incidence of tumor recurrence and metastasis were compared. The direct roles of TLR4, CXCL10 and CXCR3 on circulating Bregs mobilization were investigated in TLR4-/-, CXCL10-/- and CXCR3-/- mice models, respectively. The association of intragraft Bregs infiltration and tumor invasiveness were also examined ina rat liver transplantation for liver cancer model. The role of Bregs on liver tumor growth and invasiveness were further studied in a series of in vitro and in vivo functional experiments. RESULTS: The patients were grouped into Group 1 (>= 60% SLW, n=37) and Group 2 (<60% SLW, n=78). Much more patients in Group 2 developed tumor recurrence and lung metastasis [19/78(24.4%) vs 3/37(8%), p=0.04]. Level of circulating Bregs was significantly higher in Group 2 (Week1: 7.02 vs 1.31/10^5PBMC, p=0.03; month3: 5.7 vs 1.3/10^5PBMC, p=0.03) after transplantation. There was more intragraft Bregs infiltration in group 2 indicated by CD20/IL10 staining. Intragraft gene expression of TLR4, CXCL10 and CXCR3 were significantly higher in Group 2 at early phase after transplantation. In rat liver transplantation model, more Bregs infiltration at early phase after transplantation correlated with late phase invasive tumor growth. Levels of circulating Bregs were significantly lower in the mice model with major hepatectomy and hepatic I/R injury using TLR4-/-, CXCL10-/- and CXCR3-/- mice, respectively. Bregs also promoted liver cancer cell proliferation and migration in vitro, and liver tumor growth in vivo. CONCLUSION: A significantly higher population of circulating Bregs, which are mobilized by small-for-size graft injury, may lead to a higher incidence of tumor recurrence and metastasis after LDLT. TLR4/CXCL10/CXCR3 signaling may play important roles on Bregs mobilization.