With the incidence reportedly ranging from 30% to 70%, acute cellular rejection (ACR) is one of the most common complications after orthotopic liver transplantation (OLT) 1 2 . The appropriate immunosuppressive therapy for ACR, which is important for reducing morbidity and improving the life quality of recipients, is based on precise diagnoses and grading. At present, the Banff schema is accepted as the diagnostic judge standard for ACR, which is morphologically characterized by lymphocyte infiltration of portal tracts, bile duct damage and endothelitis in portal and hepatic central veins 3 4 5 . However, due to the overlapping histological features between ACR and other complications following liver transplantation, differential diagnoses and severity evaluations for ACR are often difficult. This prompted us to look for some potential methods and molecular markers helpful for diagnosing ACR and evaluating its severity.

It is generally accepted that T cell-mediated immune reactions play a pivotal role in the pathogenesis of ACR, and CD8+ cytotoxic T cells induce target cell death during acute allograft rejection in liver allograft tissues 6 7 8 . Cytotoxic molecules such as perforin, granzyme B and T-cell intracellular antigen-1 (TIA-1) are present in the cytoplasmic granules of cytotoxic T cells and function at the effector end of the acute rejection process 9 . Nevertheless, a study also showed that cytotoxic molecules can also mediate liver graft rejection in the absence of CD8+ T cells 10 . Thus, cytotoxic protein detection might be a sensitive and objective method for predicting acute rejection injury. It has been reported that granzyme B and perforin played predictive roles in acute rejection diagnosis after renal, heart and intestinal transplantation 11 12 13 14 . Moreover, in acute rejection after kidney transplantation, the quantity and intensity of TIA-1 expression are both increased, and this variation can reflect rejection severity to some extent 15 . However, the diagnostic value of these cytotoxic molecules in acute cellular rejection after liver transplantation has not yet been clearly elucidated.

To further evaluate the role of cytotoxic molecules in ACR diagnosis, immunohistological staining of perforin, granzyme B and TIA-1 was performed on allograft liver biopsies. As it was noted that different liver diseases mainly target at different tissues and cells of liver, the positive cells in the portal tract area and lobules were counted separately to investigate the local distribution characteristics of the cytotoxic molecules. Meanwhile, correlations between the numbers of positive cells and the Banff rejection activity index (RAI) were analyzed.

Acute cellular rejection (ACR) is still common after liver transplantation (OLT) despite well developed immunosuppressive agents, with incidence ranging from 30% to 70% in different series. Multivariate analysis showed that recipient age, underlying liver disease, and Child’s class before LT were independently associated with the development of ACR 16 . Although the pathogenesis of ACR remains need to be further elucidated, it is generally accepted that ACR occurrence is mainly due to recognition of donor alloantigen by recipient T lymphocytes. Following recognition and activation, T lymphocytes trigger a series of immunoresponses and effect mechanisms. In most cases, ACR responds well to immunosuppressive treatment. However, this should not lead to an underestimation of its importance because immunosuppressive treatment is associated with increased risk for infections, recurrence of virus hepatitis, and metabolic complications such as diabetes mellitus, hyperlipidemia, and hypertension, etc. On the other hand, the repeated ACR episodes without immunosuppressive treatment or with inadequate immunosuppressive therapy might induce the occurrence of chronic rejection which would result in graft loss. So appropriate immunosuppressive therapy for ACR, is critical for reducing morbidity and improving the life quality of recipients, and makes it necessary to diagnose ACR timely and definitely.

Berman et al. summarized the histopathologic features of ACR in three aspects which constitute the basis of Banff schema: 1) mixed infiltration of inflammatory cells, including mainly mononuclear cells and also various amounts of neutrophils and eosinophils, in the portal area; 2) endothelialitis in portal and hepatic central veins characterized by subendothelial infiltration of inflammatory cells; and 3) bile duct damage with cholangitis and degenerative necrosis of biliary epithelial cells. At present, allograft biopsy remains the ‘gold standard’ for diagnosing ACR, and the Banff schema is accepted by pathologists as the diagnosing and grading criterion for ACR. However, there are overlapping histological features and clinical manifestation between ACR and other complications following liver transplantation 17 , such as ischemic/reperfusion injury, biliary complication, recurrent virus hepatitis, etc. These overlaps make ACR diagnosis and grading often difficult, and urge us to explore some potential methods and molecular markers helpful for diagnosing ACR and evaluating its severity.

Perforin and granzyme B are proteins in the cytoplasmic granules of T lymphocytes and natural killer cells. Upon release by exocytosis, perforin disrupts lipid membranes and granzyme B accesses the cytosol of target cells, subsequently triggering cell death through apoptosis 18 19 . Animal experiments and clinical studies found that perforin and granzyme B were overexpressed after liver transplantation, which suggested a role in pathogenesis of acute rejection 20 21 . Studies have reported that perforin and granzyme B can be sensitive and specific markers for diagnosing ACR 22 23 . Inconsistent with these reports, in our cohort, perforin and granzyme B were expressed widely in liver allograft biopsies with or without ACR, and no difference in expression rate was observed. This inconsistence may arise from differences in the examination methods and judging criteria. Because of the non-utility of the positive rate for ACR diagnosis, in current study, we focused on quantifying the positive cells and determining their distribution in liver tissue. Our results showed that the numbers of perforin- and granzyme B-positive cells in the portal tract area were significantly greater with ACR than with other complications. However, in the lobule area, there were more positive cells with ACR than with BC or OP, but a similar amount of cells to that with I/R injury. These results are consistent with reports stating that ACR involves not only damage to the portal tract but also lobule injury, and that the cytotoxic T cells are also activated during I/R injury 24 25 . Our study indicates that the quantification of perforin- and granzyme B-positive cells in different areas in liver biopsies could be more informative for ACR diagnosis than mere determination of the positive rate.

Unlike perforin and granzyme B, which are expressed in cytotoxic T cells and NK cells, TIA-1, also named granule membrane protein-17 (GMP-17), is expressed not only by cytotoxic T cells and NK cells but also by monocytes and neutrophils 26 27 . TIA-1 is the granule component responsible for inducing DNA fragmentation and apoptosis in cytolytic lymphocyte targets. In acute rejection after kidney transplantation, the quantity and intensity of TIA-1 expression increase, and the degree of this variation can reflect rejection severity to some extent 15 28 29 . However, the diagnostic value of TIA-1 in acute rejection after liver transplantation has not been determined. Our data show that TIA-1 is also expressed widely in liver allograft biopsies with or without ACR. Interestingly, our results showed that the number of TIA-1-positive cells significantly increased in the portal tract area but not in lobules during ACR episodes. By contrast, during other complications such as biliary complications, opportunistic infections, and preservation/reperfusion injuries, TIA-1-positive cells significantly increased in lobules but not in the portal tract area. These opposite alterations may occur because cytotoxic T cells are prevalent in the portal tract in ACR cases, while monocytes and neutrophils, which also release TIA-1, are more popular in lobular in biliary complications, opportunistic infections, and preservation/reperfusion injuries. This distributional difference of TIA-1-positive cells implies that a marked increase in the portal tract combined with insignificant changes in lobules may indicate a diagnosis of ACR. To our knowledge, no previous studies found this pattern of TIA-1 expression in liver grafts. Therefore, the local distribution of TIA-1-positive cells in liver biopsies may provide a morphological means of distinguishing ACR from other complications after liver transplantation.

One study showed that the number of CD8-positive cells correlated with rejection severity in liver allograft tissues.⁸ However, the relationship between rejection severity and the expression of perforin, granzyme B and TIA-1 has not been determined. Our present data show that the numbers of perforin-, granzyme B- and TIA-1-positive cells in the portal tract area correlated with acute rejection severity after liver transplantation. This result is compatible with the notion that the portal tracts are the main targets of ACR and that these cytotoxic molecules are at the effector end of the acute rejection process. Based on our results, we conclude that the identification of perforin, granzyme B and TIA-1 in the portal tract area of liver biopsies would be helpful for determining the severity of ACR.

Immunohistochemical staining has become a routine method in clinical pathological diagnosis. Immunohistochemical assays for perforin, granzyme B and TIA-1 are applicable in most pathology laboratories of large hospitals. ACR pathological diagnoses are based on H&E findings, according to the Banff schema. Our result raise the possibility that immunohistochemical analysis of cytotoxic molecules has the potential to become a supplementary as well as an objective assessment method for ACR diagnosis to be used as an adjunct to the Banff schema in the future.

In conclusion, our results indicate that, though the overall positive rates have nonsense in ACR diagnosis, the quantification and local distribution analysis of cytotoxic molecule positive cells in liver tissue is helpful for differential diagnosis and severity evaluation of ACR following liver transplantation.

ACR: Acute cellular rejection; RAI: Rejection activity index; TIA-1: T-cell intracellular antigen-1; BC: Biliary complications; IRI: Ischemic/reperfusion injuries; OI: Opportunistic infections; UD: Undefined complications.

The authors declare that they have no competing interests.

LC, FT and LT contributed to the research designing, writing of the paper, performing of the research and data analysis. SW, GC and GD participated in the performance of the research. XY participated in research design and the writing of the paper. All authors read and approved the final manuscript.