CMV Infection And SHLA-G5 Expression In Renal Transplant Recipients And Study On CMV Related Immune Escape Mechanism

Infection is the main complication after renal transplantation and it is also one of the common causes of renal allograft dysfunction.Cytomegalovirus(CMV)is one of the important pathogens of infection.The matching degree of human major histocompatibility complex(MHC)is a key to determine the success of renal transplantation,at the same time,MHC itself is also an important molecule in anti-viral cellular immunity.The purpose of our study was to observe the expression change about human leukocyte antigen-G(HLA-G)of non-classical MHC classⅠmolecule during CMV infection after renal transplantation,and to analyze the effects of CMV infection on renal allograft function and immune status in recipients.We also verified the effect of CMV UL111A gene-encoded interleukin-10(cmvIL-10)on the expression of HLA-G in target cells,and explored possible immune escape mechanism of CMV initially.Our study included 59 kidney transplant recipients who had undergone renal transplantation in The 8th Medical Center of Chinese PLA General Hospital.They were grouped according to CMV infection status:CMV-DNA load>1×10~3 copies/ml,CMV-DNA(+)group(n=29);CMV-DNA load<1×10~3 copies/ml,CMV-DNA(-)group(n=30).30 healthy volunteers were healthy control group(H group).The levels of serum creatinine(Scr)and blood urea nitrogen(BUN)in CMV-DNA(+)group were 245.47±168.33μmol/ml and 18.49±13.70μmol/ml,respectively,which were significantly higher than those in CMV-DNA(-)group(105.21±43.28μmol/ml,P<0.001;10.02±6.13 mmol/ml,P=0.001)and H group(55.10±13.45μmol/ml,P<0.001;4.31±1.41 mmol/ml,P<0.001),indicating that CMV infection is one of the causes of renal allograft dysfunction.The count of lymphocyte subsets in each group was detected by flow cytometry.The results were as follows:the number of total T cells,CD3~+CD4~+T cells,CD3~+CD8~+T cells,B cells,NK cells and the ratio of CD4~+/CD8~+in CMV-DNA(+)group was 405.64±370.09 cells/μl,184.99±184.57 cells/μl,195.74±203.18 cells/μl,101.25±115.22 cells/μl,53.10±51.86 cells/μl and 1.13±0.71 respectively,which were all significantly lower than those in CMV-DNA(-)group(1330.62±594.57cells/μl,P<0.001;737.57±402.45 cells/μl,P<0.001;551.05±240.74cells/μl,P<0.001;192.58±142.54 cells/μl,P=0.004;131.11±96.21cells/μl,P=0.002;1.42±0.70,P=0.017)and H group(1463.64±352.10cells/μl,P<0.001;827.94±232.53 cells/μl,P<0.001;605.02±191.45cells/μl,P<0.001;248.39±160.76 cells/μl,P<0.001;295.20±225.08cells/μl,P<0.001;1.47±0.47,P=0.013),illustrating that the immune function of recipients in CMV-DNA(+)group was decreased significantly.The expressions of soluble human leukocyte antigen-G5(sHLA-G5)and interleukin-10(IL-10)detected by ELISA in CMV-DNA(+)group were:124.29±71.82 U/ml and 67.22±69.09 U/ml,respectively,which were significantly increased than those in CMV-DNA(-)group(35.22±25.23U/ml,P<0.001;15.17±37.61 pg/ml,P<0.001)and H group(39.74±21.18U/ml,P<0.001;28.74±11.89 pg/ml,P<0.001),indicating that increased expressions of sHLA-G5 and IL-10 may be closely related to the risk of CMV infection.In vitro experiments,we cultured CMV-susceptible human renal proximal tubule epithelial cell line HK-2 cells.When the cells grew to 80%confluence,the EGFP-labeled plasmid containing CMV UL111A gene and the control plasmid containing only EGFP were transfected into HK-2 cells.After 48 hours of action,cellular RNA and protein were extracted with Trizol method and RIPA lysate,respectively.The extracted cellular RNA was converted into cDNA by reverse-transcripted PCR,and real-time quantitative PCR detected that HLA-G mRNA in UL111A-transfected cells was significantly increased compared with cells transfected with control plasmid(P=0.009);Western blotting showed that HLA-G protein was also increased in cells transfected with UL111A gene compared with cells transfected with control plasmid(P=0.023),indicating that cmvIL-10encoded by CMV UL111A gene can upregulate HLA-G expression at the levels of transcription and translation,which may be one of the ways for CMV to achieve immune escape.In summary,this study found that sHLA-G5 increased significantly when renal transplantion recipients suffered from CMV infection.This may be one of the mechanisms in latent or active infection of CMV,providing important evidence and ideas for the diagnosis and prevention of CMV infection.In vitro experiments,the expression of HLA-G in HK-2 cells increased after CMV UL111A gene transfection,suggesting that cmvIL-10 may achieve viral immune escape by promoting HLA-G expression,which provides a new direction for further studying the possible mechanism of CMV immune escape.