Donor liver natural killer cells ameliorate liver allograft rejection via inducing apoptosis of alloreactive CD8+ T cells generating CD4+CD25+ regulatory T cells

Abstract

The roles of donor liver natural killer (NK) cells in allogeneic liver transplantation remains controversial. Herein, we investigated the effects of liver NK cells on liver allograft tolerance by using liver NK cells and allogeneic spleen T cell co-culture in vitro experiments and the rat orthotopic liver transplantation model. CD8⁺ T cells exhibited higher apoptotic rate and up-regulated expression of CD95 molecules when co-cultured with allogeneic liver NK cells in vitro, indicating that liver NK cells might induce the apoptosis of CD8⁺ T cells via CD95 in vitro. The rat liver transplant model showed that recipients with donor liver NK cell immunity, had better survival, less damage, and higher apoptotic rate of intrahepatic infiltrating lymphocytes in liver grafts, as well as higher level of peripheral CD4⁺CD25⁺ T cells and lower level of peripheral CD3⁺CD8⁺ T cells. These findings indicate that donor liver NK cells can ameliorate allograft rejection via inducing the CD95-mediated apoptosis of alloreactive CD8⁺ T cells and the generating of CD4⁺CD25⁺ regulatory T cells.

Keywords: Allograft rejection; CD4+CD25+T lymphocytes; Immunoregulation; Liver NK cells; Liver transplantation.

Fig. 1

Determination of apoptosis and CD95 expression in CD4⁺ and CD8⁺ T cells co-culture with NK cells. (A and B) Representative flow cytometry plots using Annexin V-FITC/PI staining for apoptosis in in CD4⁺ (A) and CD8⁺ (B) T cells. (C and D) Flow analyses of CD95 expression in CD4⁺ (C) and CD8⁺ (D) T cells. *: P < 0.5; two-tailed Student’s t-test.

Fig. 2

Measurement of cytokine levels in the culture supernatant. (A and B) ELISA analysis of IL-2, IL-4 and IL-10 levels in the culture media of CD4⁺ (A) and CD8⁺ (B) T cells co-culture with NK cells. *: P < 0.5; two-tailed Student’s t-test.

Fig. 3

Liver function tests and histological analysis in the recipients’ serum. (A)Kaplan-Meier curves for survival of recipient rats in each group. (B-E) The serum levels of ALP (B), bilirubin (C), ALT (D) and AST (E) were measured by ELISA. *: P < 0.05. (F) Representative hematoxylin-eosin images of the liver grafts on day 7 post-transplantation liver (original magnification, ×100). a-c: Representative liver sections of groups A, B and C. (G) The Banff Score of acute liver allograft rejection for liver grafts on days 1, 3 and 7 post-transplantation liver. 9 rats were used in each group. *: P < 0.5; two-tailed Student’s t-test.

Fig. 4

The phenotypic analysis of peripheral T cell subsets of recipients. (A and B) Flow cytometry analyses of percentage value of CD4⁺CD25⁺ (A) and CD3⁺CD8⁺ (B) T cells in peripheral blood. *: P < 0.5; two-tailed Student’s t-test. 9 rats were used in each group.

Fig. 5

Determination of apoptosis and CD95 expression in the liver grafts. (A) TUNLE staining of liver grafts sections. The cells staining brown were judged as being TUNEL (+). (B) Immunohistochemistry images of CD95 protein expression in liver grafts sections. (original magnification, ×200). 9 rats were used in each group.

Fig. 6

Examination of cytokines in recipients’ liver grafts extracts. ELISA analysis of IL-2, IFN-γ, IL-4 and IL-10 levels in the liver graft extracts in group A, B, and C. *: P < 0.5; two-tailed Student’s t-test.