| The human leukocyte antigen-G (HLA-G) was initially found on the maternal-fetal interface. It protects the fetus from the surveillance and exclusion of the maternal immune system. It is an immune tolerance molecule and involved in immune response through various ways and contributed to the immunosuppressive effect. Therefore, HLA-G has got much attention in the field of organ transplantation. It's reported that HLA-G play a role in organ transplantation rejection and transplants'function. HLA-G has the membrane type and the soluble type. Both the structure and function of the soluble HLA-G5 (soluble human leukocyte antigen-G, sHLA-G) are definite. But it's not reported wheather sHLA-G5 could be regarded as the biological marker for the prognosis of renal transplantation. Moreover, the relationship of the concentration of sHLA-G5 and its effect is not clarified. Scientists also don't know if anti-IL-2 receptor monoclonal antibody could affect the expression of sHLA-G5 at the early stage of renal transplantation. We would focus on these aspects.First of all, 215 renal transplantation recipients were divided into two groups, including F group (kidney functions stable, n = 173) and AR group (acute rejection, n = 42). Healthy donors were H group (control group, n = 69). The results indicated that mHLA-G1 expressed low on the CD4+, CD8+, CD19+ and CD16+56+ lymphocyte subsets surface in the peripheral blood of preoperative and postoperative renal transplantation recipients, and there was no statistical differences. Before operation sHLA-G5 in F, AR, H group was 33.43±21.46 ng/ml, 31.90±20.12 ng/ml and 24.94±13.19 ng/ml, respectively. The difference between group F and H was statistically significant (p <0.05). However, there's no significant difference between group F and AR(p> 0.05), as well as between group H and AR (p> 0.05). But sHLA-G5 was higher in group F than that in group AR during the following 12-week dynamic observation after renal transplantation. Histochemistry analysis of renal tissue from 21 renal transplantation recipients with acute rejection indicated that HLA-G5 expressed negatively in 17 patients. According to the ROC curve, the optimal cutoff value of sHLA-G5 predicting acute rejection was 139.0ng/ml, which showed high sensitivity and specificity. Western blot was applied to detect the expression of sHLA-G5. The result indicated that sHLA-G5 increased gradually in the F group, significantly higher than that in the AR group. RT-PCR analysis also showed that sHLA-G5 mRNA in peripheral blood increased gradually after operation in group F, while lower in group AR .According to whether or not with active CMV infection, 215 renal transplantation recipients was divided into CMV (+) group and CMV (-) group. The expression of mHLA-G1 in peripheral blood was low in both CMV (+) group and CMV (-)group. When CMV-pp65 was positive, there was no significant change of mHLA-G1. In CMV (+) group, when CMV-pp65 was positive, namely active CMV infection, CD14+ mHLA-G1+ cells in peripheral blood increased significantly, reaching 45.53±17.32%, when CMV-pp65 was negative, it decreased to 10.22±5.78%. The expression of sHLA-G5 increased significantly in CMV (+) group. The optimal cutoff value of sHLA-G5 predicting active CMV infection was 202.9ng/ml, with high diagnostic accuracy. HLA-G was positive in the kidney tissue of 10 patients out of 12 patients with active CMV infection. Western blot analysis indicated that sHLA-G5 expressed significantly higher in CMV (+) group than that in CMV (-) group. RT-PCR analysis revealed the similar result.Next, prokaryotic expression plasmid PET28a-HLA-G was constructed to express sHLA-G5 in E.coli BL21 (DE3) bacteria. After enough protein was obtained, it was used in mixed lymphocyte culture system with different concentration to explore its effect on T cell proliferation, and the relationship between concentration of sHLA-G5 and its effect, so as to provide theoretical basis for clinical research. Mixed lymphocyte culture was conducted with 9 pairs of living kidney donors and recipients. CD3+ T cells of the recipients in the mixed lymphocyte culture were labled with CFSE, then cells were collected for proliferation analysis 7 days later. The results indicated that the prokaryotic expressed sHLA-G5 protein could inhibit the allogeneic T cell proliferation. In the mixed lymphocyte culture system, when sHLA-G5 was less than 0.25μg/ml, the inhibition rate increased with the upregulation of concentration, and it was not correlated with the concentration level when the concentration reached 1.0μg/ml, although there was still difference compared with the control group. Further flow cytometry analysis indicated that CD4lowT cells and CD8lowT cells increased with the increase of concentration of sHLA-G5. Proliferation of CD4+ T cells and CD8+ T cells was inhibited, but CD4lowT cells and CD8lowT cells increased significantly.Finally, we studied the effect of anti-IL-2 receptor monoclonal antibody on the expression of sHLA-G5, and tried to find out whether the production and increase of sHLA-G5 indicated a new acting mechanism of anti-IL-2 receptor monoclonal antibody. The results showed that the expression of sHLA-G5 increased after using the first dose of anti-IL-2 receptor monoclonal antibody. On 1d, 4d, 1w, 2w post operation, sHLA-G5 of recipients injected the antibody expressed significantly higher than that of those without using the antibody. In addition, preoperative use of the first dose of anti-IL-2R monoclonal antibodies affected the conversion of cytokines in peripheral blood of renal transplantation. The decrease of IFN-γ, TNF-αand the increase of IL-10 provided the micro-environment for the upregulation of sHLA-G5. After the use of the second dose, IL-10 and IL-4 increased, resulting in Th2 dominant shift in the peripheral circulation of the renal transplantation recipients.These results mentioned above demonstrated that in the peripheral blood of renal transplantation recipients, the main subtype of HLA-G was sHLA-G5. The expression level of sHLA-G5 post operation in the F group was higher than that in the H group and AR group. ROC curve analysis indicated that 139.0ng/ml was the cutoff value of sHLA-G5 to predict acute rejection. and 202.9ng/ml was the cutoff value of sHLA-G5 to predict active CMV infection. Renal transplantation recipients in the CMV(+) appeared to have functional kidney when using less or no immunosuppressive agent. The possible mechanism is that sHLA-G5 expression significantly increased in peripheral blood in the CMV (+) group, positive expression of HLA-G in epithelial cells of the graft tubular suggested that sHLA-G5 expression could protect renal function. HLA-G5 protein was expressed in prokaryotic expression system and purified with nickel affinity chromatography. Then effective renaturation was conducted and sHLA-G5 proteins were obtained with purity of 90%. The effect of sHLA-G5 in the mixed lymphocyte culture system was correlated with its concentration. In a certain range, it's dose-dependent, but not direct correlated. When the concentration was higher than 1.0ng/ml, its ability to inhibit the proliferation of T cells did not increase, but decrease. Using anti-IL-2R monoclonal antibodies in the early stage after renal transplantation could induce the upregulation of sHLA-G5. Anti-IL-2R monoclonal antibody could affect the drift of cytokines in peripheral blood of renal transplantation recipients, which is beneficial for protecting the transplants and for reducing the risk of acute rejection. Preoperative use of the first dose of anti-IL-2R monoclonal antibodies affected the conversion of cytokines in peripheral blood of renal transplantation. The decrease of IFN-γ, TNF-αand the increase of IL-10 provided the micro-environment for the upregulation of sHLA-G5. After the use of the second dose, IL-10 and IL-4 increased, resulting Th2 dominant shift in the peripheral circulation of the renal transplantation recipients. This regulation promotes protective immunity, thereby reducing the risk of rejection. |
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