T cells play an important role in the rejection of allogeneic liver transplantation19, and participate in the recognition of transplantation antigens, immune regulation, cell dissolving effect and other immune responses20,21. In order to study the phenotype and quantity of T cells in different rejection levels after liver transplantation, the changes of CD4+ T cells, CD8+ T cells, Tregs and Th17 in peripheral blood were detected by flow cytometry or immunohistochemistry. After liver transplantation, the levels of blood CD4+ T cells peaked in the acute rejection group and was significantly higher than that of the non-acute rejection group22. Tregs and Th17 are known to be involved in the alloreactive responses in organ transplantation. Wang et al23 investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. The frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index after liver transplantation.
When immune rejection occurs, a large number of T cells infiltrate the graft, such as CD4 and CD8 subsets, which increases with the aggravation of rejection24. The most important histological changes of acute rejection are mixed inflammatory cell infiltration of the confluence area25. Krukemeyer et al26 found that more B lymphocytes and plasma cells migrated to the liver in patients with rejection than in non-rejection patients through liver tissue studies. But these experimental techniques have some limitations. The exact composition of leukocyte infiltration during liver allograft rejection is difficult to comprehend and visualize on the same biopsy slide. With the development of science and technology, multiplex immunofluorescence assays has been favored by researchers.
Multiplexed imaging platforms to simultaneously detect multiple epitopes in the same tissue section have emerged in recent years as powerful tools to study the immune context of organs and tissues27. Multiplex immunofluorescence assays can improve the understanding of the tissue microenvironment, new therapeutic targets, prognostic and predictive biomarkers, and translational studies28. Until now, this new technology has mainly been used in oncology to better characterize immune cell infiltration in tumors in situ29–31. Previous studies have used immunohistochemical or immunofluorescence techniques to detect the changes in CD4+ T cells, CD8+ T cells, Th17, Tregs and other indicators in grafts. However, using serial tissue sections makes the cellular interplay and the precise location of the cells difficult to analyze. We present multiplex immunofluorescence assays that generated readouts for a comprehensive panel of biomarkers from liver biopsies from liver transplantation patients. Our findings revealed increased infiltration of CD4+ T and CD8+ T cells in the acute rejection and non-acute rejection groups as compared with the normal group. The proportion of CD4+ and CD8+ T cells in the acute rejection group was the highest. There were also some interesting note was that it was difficult to observe Tregs in the normal and non-acute rejection groups, but the proportion of Tregs and Th17 was the highest in the acute rejection group. The above results showed that the proportion of CD4+ T cells, CD8+ T cells, Tregs and Th17 increased during acute rejection after liver transplantation, and these cells were involved in the development of acute rejection or immune tolerance after liver transplantation. Calvani et al32detected simultaneously NK cells, macrophages, and T cells to determine their intra- or extravascular localization using multiplex immunofluorescence assays, demonstrating the feasibility and utility of multiplex immunofluorescence imaging to study and better understand the kidney allograft rejection process. The above results show that different types of lymphocytes infiltrate different immune states after organ transplantation. The mechanism through which different immune cells participate in rejection or immune tolerance is different.
When acute rejection occurs, interlobular bile duct and vascular endothelial cells are the primary targets of immune attack33. Many immune cells infiltrate the mixed portal area and hepatic lobules, and cause venous endothelial inflammation and bile duct inflammation34. Similarly, our immunofluorescence staining results showed that infiltrating T cells could be seen in the portal area, including a large number of CD8+ T cells, CD4+ T cells, Tregs and Th17 in the acute rejection group. The number of infiltrating T lymphocytes in the non-acute rejection group was less than that in the acute rejection group. We found a novel phenomenon: more lymphocytes infiltrated hepatic lobules under higher levels of the rejection, but the number of CD4 + sinusoidal endothelial cells decreased significantly. Previous studies have showed that tissue-resident immune cells are important for organ homeostasis and defense, and the epithelium may contribute to these functions directly or by crosstalk with immune cells35. The mechanism of interaction between T cells and sinusoidal endothelial cells in acute rejection of liver transplantation needs further study.
The above results showed that the distribution of infiltrating T cells was different in the hepatic lobules and portal area. Due to the complexity of hepatic lobule structure, it can be divided into three regions according to different metabolic functions. The gene expression patterns of the spatial axis from the central vein to portal area are different, at the same time, its function is gradual10. Because liver lobules contain a spatially polarized immune system "immune zonation", the types and numbers of immune cells infiltrating hepatic lobules are different in different disease states12. Kupffer cells can localize infiltrating neutrophils to the periportal regions of the lobule, limiting damage to cells that reside around the central vein36. It remains unclear whether there are differences in the types and functional status of immune cells in different regions of hepatic lobules under different rejection conditions after liver transplantation. We used multiple immunofluorescence staining to study the infiltration and distribution of T cells in different areas of hepatic lobules, and the spatial distribution of various types of T cells in different areas of hepatic lobules under acute rejection and non-acute rejection after liver transplantation. The results showed that the infiltrated immune cells had some spatial specificity and some exciting phenomena were found after liver transplantation. In the same area of the hepatic lobule, the percentages of CD4+ T cells, CD8+ T cells and Tregs in the rejection group was higher than that in the other two groups, which indicated that the percentage of these T cells increased in each area of the hepatic lobule when rejection occurred. Within three group, percentage of CD4+ T cells, CD8+ T cells and Tregs from periportal zone to perivenous zone were initially increased and then decreased. Interestingly, the percentage of CD8+ T cells from the periportal to perivenous zones increased gradually, the percentage of CD8+ T cells in the perivenous zone was higher than that in the other two zones in the rejection group. Renal allograft biopsies showing c-aABMR show a predominance of infiltrating CD8+ T cells, and increased numbers of interstitial FOXP3+ T cells are associated with inferior allograft survival37. Similarly, the distribution of CD8+ T cells in different regions of hepatic lobules may be closely related to rejection level after liver transplantation.
There were some limitations in our study, such as the small number of clinical samples, the difficulty in obtaining liver biopsy specimens, and the incomplete structure of hepatic lobules in liver biopsy tissue. These deficiencies may have led to instability of the results. Therefore, this study needs to be further improved to find other novel results. In conclusion, we believe that the percentage of CD8+ T cells in different regions of hepatic lobules is closely related to rejection level after liver transplantation. We observed an important heterogeneity in the global composition of the inflammatory burden during liver allograft rejection. Acute rejection of liver transplantation may occur when the percentage of CD8+ T cells in perivenous zone increase, and Tregs increases reactively. The number and proportion of CD8+ T cells increased gradually from the periportal to perivenous zones under different rejection levels. Although the percentage of regional CD4 + T did not reflect the rejection level, the number of CD4+ and CD8+ T cells in different regions was closely related to the rejection level.