| Costimulatory signals play an important role in regulating the function of T lymphocytes at different stages of immune response. Programmed death 1 is a member of the B7 family, and programmed death ligand 1(PD-L1) is one of the ligand of Programmed death 1. PD-1 is mainly expressed on activated T, B, monocyte surface. The negative inhibitory signals mediated by PD-1/PD-L1 participate in a variety of diseases, through inhibiting the function of T and B lymphocyte. In addition to membrane-bound form, it has been demonstrated that many costimulatory molecules also have soluble isoform, such as CD28, CTLA-4(cytotoxic T lymphocyte-associated antigen-4), CD80, CD86, B7-H3 and B7-H4, the biological and clinical significance of these soluble factors remains to be investigated. We hope to elucidate the role and machanisim of PD-1/s PD-L1 pathway in regulating the function of T lymphocyte so as to provide a new strategy for immune therapy of human disease.In this study, we observed the effect of adopting different concentrations of s PD-L1 on regulating the function of T lymphocyte and the change of PD-1 cytoplasmic domain signal adaptor molecule tyrosine protein kinase(SHP)-1, SHP-2 and phosphorylated SHP-1, phosphorylated SHP-2, and its biological significance was discussed so as to reveal the effect of inhibiting the function of T lymphocyte by PD-1/s PD-L1 costimulatory signal pathway and to further explore mechanism of s PD-L1 negative regulation of T cell function.Part I The effect of soluble PD-L1 via PD-1/PD-L1 pathway in regulating the functions of T lymphocyteObjective To study the effect of s PD-L1 on the proliferation, cell cycle and apoptosis of T lymphocyte.Methods The level of PD-1 expressed on membrane surface of T lymphocyte from peripheral blood of healthy donors was detected by flow cytometry. The effects of different concentrations of s PD-L1 on regulating the proliferation of T lymphocyte was studied by CCK-8 method and the most suitable concentration was determined. The effect of s PD-L1 on regulating the cell cycle and apoptosis of T lymphocytes was studied by flow cytometry.Results Low expression of PD-1 was found on resting T lymphocyte from human peripheral blood with flow cytometry and up-regulated PD-1 was observed on the surface of T lymphocyte stimulated with PHA. s PD-L1 could markedly inhibit the proliferation of T lymphocyte by PD-1/s PD-L1 pathway(P=0.017) when the concentration of s PD-L1 Ig was set to 250 ng/ml. The proliferation of T lymphocyte could be partly recovered by blocking the negative costimulatory signal using PD-L1 antibody(P=0.035); s PD-L1 was found to promote the percentage of T lymphocyte in G1 phase significantly(P=0.000) in cell cycle study, whereas by blocking the inhibitory signal using PD-L1 antibody, the percentage of T cells in G1 phase was significantly reduced(P=0.014); In addition, s PD-L1 was found to significantly increase the apoptosis rate of T lymphocyte(P=0.001), whereas the effect could also be reversed by adding blocking antibody against PD-L1(P=0.002).Conclusion Soluble PD-L1 can inhibit the proliferation of T lymphocytes, promoting cell cycle arrest in the G1 phase and apoptosis. s PD-L1 may play a crucial role in the negative regulation of T cell function in cellular immunity.Part II The significance of adapter molecule SHP-1, SHP-2 in PD-1/s PD-L1 pathway in inhibiting the function of T lymphocytesObjective To detect the serum level of s PD-L1 in different age groups of healthy donors and analyze the amount of signal adaptor molecule Src homology 2 domain-containing tyrosine phosphatase(SHP)-1 and SHP-2 recruited so as to elucidate the mechanism of s PD-L1 in inducing the negative signal via the pathway of PD-1/s PDL1.Methods One hundred and eight healthy volunteers from Health Checkup Center of The Second Affiliated Hospital of Soochow University from June 2013 to May 2014 were enrolled. The serum level of s PD-L1 in 20 to 30, 31 to 50, 51 to 70 age groups were detected by ELISA kit previously developed by our study group. T lymphocyte from human peripheral blood of the healthy donors was separated by immunomagnetic beads. The cells to be treated were divided into five groups: Group A: resting T lymphocytes, Group B: activated T lymphocytes, Group C: activated T lymphocytes + s PD-L1 Ig, Group D: activated T lymphocytes + s PD-L1 Ig + membrance-bound immunoglobulin(m Ig G), Group E: activated T lymphocytes + s PD-L1 Ig + anti-PD-L1 antibody(2H11). The phosphorylation level of programmed death 1(PD-1) signaling motif tyrosine was measured by Western-blot. Furthermore, the amount of signal adaptor molecule Src homology 2 domain-containing tyrosine phosphatase(SHP)-1 and SHP-2 was studied by immunoprecipitation.Results The serum level of s PD-L1 molecule was(0.766±0.154) ng/ml in peripheral blood of 20-30 years old age group(0.866±0.201) ng/ml in 31-50 years old age group and(1.047±0.453) ng/ml in 51-70 years old age group. Significant statistical difference was found between every two age groups(P=0.000;P=0.001;P=0.000 respectively). The expression of SHP-1, SHP-2 in all groups expressed in gray value had no significant differences(P>0.05); while the expression level of p-SHP-1 and p-SHP-2 in group C was significantly higher than those in group B(P=0.000; P=0.000); Moreover, the expression level of p-SHP-1 in group E was significantly lower than that in group D(P=0.028)Conclusion The expression level of s PD-L1 in healthy human peripheral blood is related with age. The expression level of p-SHP-1, p-SHP-2 expression increased in s PD-L1 group compared with not join in s PD-L1 and this phenomenon maybe associated with the PD-1/s PD-L1 inhibitory signal transduction in the pathway. By specifically blocking the inhibitory signal with PD-L1 antibody, the function of p-SHP-1 can be partly restored. Hence, p-SHP-1 may play an important role in conveying inhibitory signal in PD-1/s PD-L1 pathway.To summarize, we can come to the following results in this study:(1) T lymphocytes proliferation can be inhibited by adding s PD-L1, arrested in the G1 phase in cell cycle study, resulting in more T lymphocytes to undergo apoptosis. Anti-PD-L1 blocking antibody can partly restore the function of T lymphocytes. These phenomena signify that increased concentration of s PD-LI may convey negative costimulatory signal to T lymphocytes by binding PD-L1.(2) The expression level of s PD-L1 in peripheral blood of healthy human beings is related with age. s PD-L1 promotes the expression level of p-SHP-1, p-SHP-2. The anti-PD-L1 blocking antibody can partly restore the expression of p-SHP-1. The phosphorylation of SHP-1 contributes to the inhibitory signaling of PD-1/s PD-L1 pathway, furthermore inhibit the function of T lymphocytes... |
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