Recent studies have shown that host immunoinhibitory factors, such as PD-1 and PD-L1, are exploited to evade the immune response in chronic viral infections [26, 27]. However, the function of the chicken PD-1/PD-L1 pathway has not been well characterized. In order to evaluate immunosuppressive function of PD-1 / PD-L1 pathway, we determined the expression level of IFN-γ mRNA in Lee1 cells which was co-cultured with DF-1 expressing PD-L1. The expression of IFN-γ mRNA was decreased in Lee1 cells co-cultured with DF-1 expressing PD-L1, suggesting that in case of chickens, PD-L1 also interacts with PD-1, and then induces the immunosuppression in PD-1-expressing cells, although further functional analysis concerning the involvement of chickens PD-1/PD-L1 pathway in immunosuppression and tumor formation is required.
The increase in PD-1 expression was reported in the acute phase of HCV and lymphocytic choriomeningitis virus (LCMV) infection [26, 27]. In chickens experimentally infected with MDV-1, the expression of PD-1 mRNA was increased in the early cytolytic phase, but dramatically decreased following the onset of the latent infection (Figure 3A). We previously reported that MDV-1 induces apoptosis in CD4+ T cells during latent phase, and the number of CD4+ T cells is decreased . These observations suggest that PD-1 may be expressed on the CD4+ T cells in the early cytolytic phase, and the expression of PD-1 may be decreased by apoptosis of CD4+ T cells before the onset of MDV latency. In contrast, the increase in PD-L1 mRNA expression was observed in the latent phase (Figure 3B), suggesting that PD-L1 contribute to the establishment and/or maintenance of MDV-1 latency. Further studies, including the identification of PD-1- and PD-L1-expressing cell populations and the expression analysis of PD-1 and PD-L1 on the protein level, are needed to clarify the role of the PD-1/PD-L1 pathway in each phase of the infection.
In the case of HIV infection, some cytokines and viral factors are considered to be responsible for the increase in PD-1 and PD-L1 expressions , and it has been well characterized that PD-L1 expression could be regulated by IFN-γ . In this study, the expression of IFN-γ mRNA was increased in the early cytolytic phase, and this expression kinetics was similar to that of PD-1 mRNA (Figure 3C), suggesting that IFN-γ may be involved in the regulation of PD-1 expression. However, the increase in the PD-1 mRNA expression was not observed in the secondary cytolytic phase despite the increase in the IFN-γ expression. Thus, PD-1 expression may be induced as a consequence of a negative feedback by IFN-γ stimulation during the acute phase. On the contrary, the expression of PD-L1 mRNA was transiently elevated at 7 d.p.i., and then, the expression of IFN-γ mRNA was increased (Figure 3B, C). At 21 d.p.i., however, PD-L1 mRNA expression were increased again although IFN-γ mRNA expression was decreased (Figure 3B, C). The reason for these observations is unknown, but IFN-γ might gradually induce the expression of PD-L1, which, in turn, cause immunosuppression and reduce the IFN-γ expression.
PD-L1 is expressed on a variety of human and murine tumors , whereas tumor-infiltrating lymphocytes highly express PD-1 . In the case of nodular lymphocyte-predominant Hodgkin lymphoma and angioimmunoblastic T-cell lymphoma, tumor cells express PD-1 [32, 33]. Moreover, tumor cells caused by HTLV-1 express both PD-1 and PD-L1, and infiltrating T cells express PD-1, suggesting that PD-L1 expressed on these neoplastic CD4+ T cells induce immunosuppression of infiltrating T cells and contribute to the immune evasion . In the case of MDV-1 infection, both PD-1 and PD-L1 mRNA expression were increased in tumor cells (Figure 4A, B, Figure 7A, B, Figure 8C, Additional file 1: Figure S1). Thus, both PD-1 and PD-L1 may be expressed on MD tumor cells, and may contribute to the immune evasion. Interestingly, the expression of IFN-γ mRNA was increased in tumor lesions (Figure 4)C, and the expression of IFN-γ mRNA in tumor lesions was higher than that in neighboring cells (data not shown). As a positive correlation was shown between IFN-γ and PD-L1 mRNA expressions (Figure 5B), IFN-γ may upregulate the expression of PD-L1 mRNA in tumor cells in an autocrine manner, and subsequently, PD-L1 may enhance the immunosuppression.
Several groups reported that viral proteins such as Nef protein of HIV and core protein of HCV cause the increase in PD-1 expression [23, 24]. In this study, we focused on an MDV-1 oncoprotein, Meq, because Meq regulates the expressions of various genes as a transcription factor and is the most important viral factor related to MD pathogenesis [8, 35]. The expressions of meq and PD-1 mRNA were transiently increased at 14 d.p.i (Figure 3A, D), and in addition, the expression of meq mRNA was positively correlated with PD-1 mRNA expression in tumor lesions (Figure 5A), indicating that Meq may regulate the expression of PD-1. However, a positive correlation between the expression of meq and PD-L1 was not observed in the spleens and tumors from MDV-1-infected chickens. Since various viral factors are involved in MDV-1 oncogenesis and pathogenesis , other factors may correlate with the expressions of host immunoinhibitory molecules.
In summary, we demonstrated that host immunoinhibitory factors, PD-1 and PD-L1 were expressed in the spleens of MDV-1-infected chickens, and their expressions were showed different kinetics in each phase of the infection (Additional file 2: Figure S2). Furthermore, these factors were expressed in MD-derived tumors (Additional file 2: Figure S2). These results suggest that the PD-1/PD-L1 pathwayis involved in the immunosuppression and tumor formation by MDV-1.