The Correlation Between ATP Concentration In CD4~+ T Cells And Immune State After Human Kidney Transplantation

Acute cellular rejection (ACR) after organ transplantation is the most important problem for the recipients and also is the obstacle to the long survival. Immunosuppressants after transplantation for life time are the main methods to overcome this problem. So it is important to monitor the immunosuppressive state of the patient after organ transplantation.Currently,the methods of monitoring immunosuppressive state after organ transplantation includes appraisal of general state, renal function, pathological examination of graft,the concentration of immunosuppressants and immunosuppressive state of recipients.General state of recipients and renal function are not special and the same change can be observed when infection and other complication occurred.Pathological examination of graft is the gold criteria to determine whether ACR presents or not, but it is a traumatic examination and some related complications may occur. The concentration of immunosuppressants is foundation to appraise immunotolerance and adjust clinical medication. However, ACR occurred inevitably in many patients because of individual difference of pharmacokinetics. Lymphocyte count and the proportion of lymphocyte subset are easy and feasible methods to appraise immunosuppressive state. The detect of cytokine, such as IL-2, IL-4, IL-6, IL-8, Fas L, TNF, IFN, ICAM-1 are useful to diagnose ACR after organ transplantation, a quantitative index of cytokine is not available to determine whether ACR present or not.CD4 positive T lymphocyte plays an important role in ACR after organ transplantation and is target of immunosuppressive therapy. The function of immune cell depends on the ATP supply directly or indirectly, so detection of the concentration of ATP can reflect the function of immunocytes. ATP-CVA method is an highly sensitive method to detect ATP quantitatively. Fundamental principle is with the presence of ATP, luciferase catalyzes luciferine and fluorescence is detected by fluorometer. Linear relation exists between the value of fluorescence and ATP concentration, the ATP concentration of sample can be calculated through standard curve.In this study, ATP-CVA was used to measure the intracellular ATP concentration of CD4 positive T cell stimulated by PHA to reflect the immune function. Renal recipients were assyed the immune state using ATP-CVA pre and post transplant. The object of this study is to further explore a rational supporting titration of immunosuppressive therapy, predict the occurrence of rejection; so as to reduce the risk of renal recipients suffering from drug toxicity effect, opportunistic infection and cancer and improve their living quality and survival.In our study, ATP-CVA was used to measure the post-renal transplant immune function. Diluted whole blood samples were placed in 96 well microtiter plates and incubated with the phytohemagglutinin-L (PHA-L) for 15-18 hours.After the incubation, CD4+T cells were immunoselected using monoclonal antibody (McAb)-coated magnetic particles and lysed to release intracellular ATP. Released ATP was detected using firefly luciferin/luciferase system and quantified using an ATP calibration curve.Before using this assay to test the samples, the ATP calibration curve must be determined firstly. The ATP standard substance were diluted with nutrient into fiver concentrations:1,10,100,1000 and 10000ng/ml.200ul of each diluted standard specimen was dispensed into three wells after mixed with the same volume Celltiter-Gloms luorescent reagent. Relative light unit (RLU) were measured by luminometer within an hour after mixture. ATP calibration concentrations vs. RLU values were plotted on a log-log scale, and linear regression analysis generated a calibration curve. There was a good liner relation when ATP concentrations varied from1ng/ml to 10000ng/ml,Y=0.75X+2.37(the correlation coefficient r2 =0.99452).These data confirmed that the assay could be used to measure the immune function because the released intracellular ATP could be quantified using the ATP calibration curve.ATP concentrations in CD4+T cells of the recipients undergoing renal transplantation were(398.17±108.21)ng/ml before operation, significantly lower than normal volunteers(437.25±56.16)ng/ml, (P＜0.05), reached the lowest in the first week after operation, especially in the recipients subject to antibody-inducing therapy, then increased slowly on the post-operation week 2, but still significantly lower than pre-operation by the fourth week (329.64±67.57) ng/ml (P＜0.05), especially in the recipients subject to antibody-inducing therapy。There were 7 patients who occurred acute rejection,the level of ATP increased(308.81±69.43)ng/ml, and was lower than pre-operation(456.21±102.06) ng/ml, (P>0.05), then decreased slowly with effective treatment after two weeks(258.36±34.76)ng/ml, still higher than that of the first week after operation (184.09±42.63)ng/ml (P＜0.05)In 18 patients with cytomegaloviral pneumonia, the level of ATP decreased(89.13±8.85)ng/ml, and was slightly lower than that of the first week after operation (95.84±12.38)ng/ml, there was statistical significance (P＜0.0001), significantly lower than pre-operation(381.42±101.32)ng/ml (P＜0.0001),decreased especially when pathogenetic condition was aggravated, then increased slowly with effective treatment, still lower than pre-operation in the second week after operation (96.74±8.60)ng/ml (P＜0.05)In conclusion, monitoring the immune function post renal transplantation by ATP-bioluminescence assay in our study proved that it can be used to measure the CMI function post-renal transplantation.Perhaps the important observation from this trail is that the level of tacrolimus by immunoassay of whole blood is not correlated with the degree of biological immunosuppresion as measured by the PHA-induced ATP levels, this emphasize the importance of having a functional readout the global immunosurpression. Maybe using this assay to measure the immune function of renal recipients and monitoring the drug levels in blood can achieve a better rational supporting individualized titration of immunosuppressive therapy.