The Clinical Study Of ATP Bioluminescence As A Measure Of CD4～+ T Cell Proliferation In Early Renal Transplantation

Successful management of the transplant recipient currently required lifelong immunosuppression of the patient to avoid graft rejection. While calcineurin inhibitors had dramatically improved graft survival, the patient was at increased risk of drug toxicity, opportunistic infections and cancer. In addition, basal immune reactivity to therapy, surgical trauma, anesthesia, and known pharmacokinetic differences on drug metabolism had all been demonstrated to influence immunity, yet their impact was not measured. Until now, no assay was designed to assess the global immune response in patients receiving immunosuppressive drugs which was reflective of their changing clinical course. Traditionally, lymphoprolifertion(LPA) had been used an in vitro model for cell-mediated immunity but difficult to standardize. Sottong PR designed an assay using ATP bioluminescence as a measure of CD4~+ cell proliferation, In healthy adults undergoing vaccination with tetanus, the assay gave comparing results with LPA. To assess the clinical value of this assay after renal transplantation, we followed up 52 cases of cadaveric kidney transplant completed in our center and 50 healthy adults. The cell-mediated immunity status and its relative factors in recipients and healthy adults were analyzed.Objective To establish an assay using ATP bioluminescence as a measure of CD4~+ cell proliferation, to assess the clinical value of the immune cell function assay measuring global cell-mediated immunity after early renal transplantation and during pulmonary infection, to discuss the clinical feasibility on directing the regulation of immunosuppression dosages. Methodology Clinical information of 52 cadaveric renal allograft recipients(6 cases with pulmonary infection after operation among 52 recipients)and 50 apparently healthy adults was collected, two whole blood samples with sodium heparin anticoagulated vacutainer tubes were drawn from healthy adults and transplant patients(0d, 3d, 7d, 14d ,30d and during infection). One sample was used for immune cell function assay, the assay combining cell stimulation withPHA-L, CD4 cell selection with anti-human CD4 monoclonal antibody coated magnetic particales, and quantification of metabolic markers(e.g. ATP Adenosine Triphosphate) with bioluminescence measured CD4+ T cell early proliferative responses. The other sample was used for flow cytometry, the percentages and absolute CD4 counts (ALCs) and ratio of CD4+ cell to CD8+cell were analyzed. With the help of SPSS 10.0 software, the linear calibration curve and regression equation of ATP standard samples were founded between ATP concentrations and relative light units (RLU) of bioluminescence, the results of whole blood samples were analyzed by two-tailed t-tests to assess the statistical significance of differences(P<0.05), the association was assessed using Pearson analysis. Results ATP concentrations vs. RLU values were plotted on a log-log scale, and linear regression analysis generated a calibration equation(Y=0.75X+2.37, r=0.9948, P<0.05) and its curve. The average ATP concentration of healthy adults and patients before renal transplantation were 516±257ng/ml and 404±190ng/ml respectively(P<0.05), the average ATP concentration of antibody-inducing therapy group among patients was 393±203ng/ml and significantly lower than that of healthy adults group. ATP concentrations vs. time since transplant: ATP concentration was at the lowest level by the first week(147±96ng/ml), then increased slowly, but it was significantly lower than that of pretransplantation by the fourth week (PO.01), especially in the antibody-inducing therapy group. ATP concentrations and ratio of CD4 to CD8 discreased during infection and increased after cure, while ALCs did not significantly change. ATP concentrations and ALCs by flow cytometry: the average ALC of healthy adults and patients before renal transplantation was 735±370 cells/ul and 499±240 cells/ul respectively (P<0.05), the average ALC of patients with antibody-inducing therapy was significantly lower than that of patients without antibody-inducing therapy after organ transplantation(e.g. 60 cells/ul and 312 cells/ul respectively by the first week). In addition, no direct correlation was observed between ALCs and ATP concentrations^O.l), no direct correlation was observed between ratio and ATP concentrationsO^O.l). Comparison of immunosuppression regimes: although there was no difference before organ transplantation, the average ATP concentration ofpatients with antibody-inducing therapy was significantly lower than that of patients without antibody-inducing therapy after organ transplantation. No statistically significant difference between cyclosporine-treated patients and tacrolimus-treated patients when patients with antibody-inducing therapy was excepted, nonetheless, the former demonstrated on average ATP concentration greater responses to PHA stimulation than the later. In addition, no direct correlation was observed between ATP concentrations and therapeutic drug trough levels in whole blood(r2<0.1). Conclusions The amount of ATP presented in stimulated blood specimens was a measure of early lymphocyte responses. Our tests made clear that ATP concentration was an effective marker reflecting cell-mediated immunity during early renal transplantation and infection. CD4+ cell numbers and their functional activity as measured by ATP were independent variables. Recipients using antibody-inducing therapy and tacrolimus demonstrated low immune responses to PHA stimulation, which accorded with their clinically therapeutic effects. No direct correlation was observed between the amount of ATP and therapeutic drug trough level, emphasizing the importance of measuring the aggregate effect of the drug(s) on immune system parameters. In short, we got the message of early immune reconstruction of kidney transplant recipients by the changes of ATP level of CD4+ cells. The immune cell function assay reflecting the cell-mediated immunity of kidney transplantation might take the place of traditional LPA, it would be beneficial to direct the regulation of calcineurin inhibitor dosages.