TRIM-00879; No of Pages 6 Transplant Immunology xxx (2013) xxx–xxx
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Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients Xu-Zhen Wang a,1, Zhan-Kui Jin c,1, Xiao-Hui Tian a,b,1, Wu-Jun Xue a,b,⁎, Pu-Xun Tian a,b, Xiao-Ming Ding a,b, Jin Zheng a,b, Yang Li a, Xin Jing a, Zi-Zhen Luo a a b c
Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, China Institute of Organ Transplantation, Xi'an Jiaotong University, Xi'an, Shaanxi, China Department of Orthopaedics, Shaanxi Provincial People's Hospital (The Third Affiliated Hospital of Medical College of Xi'an Jiaotong University), Xi'an, Shaanxi, China
a r t i c l e
i n f o
Article history: Received 12 September 2013 Received in revised form 28 October 2013 Accepted 28 October 2013 Available online xxxx Keywords: Acute rejection Adenosine triphosphate (ATP) Kidney transplantation
a b s t r a c t Background: Peripheral blood CD4+ T cell adenosine triphosphate (ATP) release has been reported to be an adjunct tool to evaluate global cellular immune response in solid-organ transplant recipients. However, the correlation between the ATP level and rejection was controversial. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of acute rejection (AR) episodes, determine the predicting value of intracellular ATP level for AR in kidney transplant (KT) recipients. Patients and methods: In the period of October 2011 to October 2012, 140 KT recipients were recruited and followed for six months after transplantation. Patients were categorized into stable group and AR group according to their clinical course. Whole blood samples were collected pretransplantation, and at 7, 14, 21, and 28 days, and at 2, 3, 4, 5 and 6 months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7 days after intravenous anti-rejection therapy started, and on the day when AR reversed. The intracellular ATP in CD4+ T cells was detected by ImmuKnow Immune Cell Function Assay according to the manufacturer's instruction. The absolute number of CD4+ T cells and the trough levels of tacrolimus and cyclosporine were also measured. Results: The ATP level detected on the day AR occurred (627.07 ± 149.85 ng/ml) was obviously higher than that of the stable group (320.48 ± 149.11 ng/ml, P b 0.05). ATP value decreased to 265.35 ± 84.33 ng/m at the end of anti-rejection therapy, which was obviously lower than that measured on the day before the anti-rejection therapy started (665.87 ± 162.85 ng/ml, P b 0.05). ROC analysis revealed that increased intracellular adenosine triphosphate level showed better sensitivity and specificity than those obtained using single time point detection (89.5% vs 85.0%;95.0% vs 88.9%). The best cutoff value was 172.55 ng/ml. A positive correlation between the intracellular ATP level and absolute CD4 + T cell number (r = 0.656, P b 0.001) was found in the patients with CD4+ T cell counts b 200/μl. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Kidney transplantation has been successfully used as an effective therapeutic option for patients with end-stage kidney diseases. With the introduction of a variety of immunosuppressive agents, the graft and patient survival rate after kidney transplantation has been significantly improved during the past decade. However, acute rejection (AR), which usually occurs on days 7–14 in the early posttransplantation period [1,2], remains a major obstacle for the long term survival in kidney transplant (KT) recipients. Currently,
⁎ Corresponding author at: Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, 277 Yanta West Road, Xi'an, Shaanxi 710061, China. Tel.: +86 29 85323721; fax: +86 29 85323718. E-mail address: [email protected] (W.-J. Xue). 1 Contributed equally to this work.
allograft rejection is taken into consideration only when continuous and irreversible deterioration of renal function occurs, and the gold standard-biopsy procedure usually takes up several days. As a result, anti-rejection therapy starts relatively late as kidney damage has already been established. Hence, convenient and noninvasive approaches for predicting and diagnosing AR would be of considerable value to improve outcomes in KT recipients. The Cylex ImmuKnow Cell Function Assay may be a promising tool for post-transplant immune monitoring. In 2002, the U.S. Food and Drug Administration (FDA) approved this assay for evaluating global cellular immune response in solid-organ transplant recipients [3]. It assesses the activity of CD4+ T cells by measuring the concentration of intracellular adenosine triphosphate (ATP) after stimulation with phytohemagglutinin (PHA) in vitro. It is expected that this assay could be beneficial in monitoring the net immune state in transplant recipients and lead to better patient management and evidence-based individualization.
0966-3274/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.trim.2013.10.008
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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X.-Z. Wang et al. / Transplant Immunology xxx (2013) xxx–xxx
Based on a multicenter study in American, Kowalski et al. classified immune response of transplant recipients into three zones: strong (≥ 525 ng/ml ATP), moderate (226–524 ng/ml ATP), and low (≤ 225 ng/ml ATP) [4]. They reported that ATP values higher than 525 ng/ml were associated with an increased risk of AR. Thereafter, most of the following studies used ATP level at 525 ng/ml as a threshold to investigate the predictive role of intracellular ATP values at single time point for AR. Several studies have shown that the high intracellular ATP levels can identify patients at risk for rejection [5–7], whereas others argue against its predictive capacity [8]. Therefore, the correlation between ATP level and rejection is still undetermined. 2. Objective We hypothesized that patients with high intracellular ATP level in stimulated CD4+ T cells measured by the Cylex ImmuKnow Cell Function Assay are at increased risk of AR episodes. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of AR episodes.
Inc., Columbia, MD, USA), washed and lysed to release intracellular ATP. Subsequently, a luciferin/luciferase reagent was added and released ATP was measured by a luminometer (Turner Biosystems, Sunnyvale, CA, USA). The concentration of ATP was expressed as ng/ml. 3.4. Flow cytometry and absolute CD4+ T cell counting Before intracellular ATP detection, part of the blood samples were stained for lymphocyte surface markers and then sorted by flow cytometry (FACSCalibur, BD, USA) to obtain the ratio of CD4+ T lymphocytes to the total peripheral blood leukocytes, which was then used to calculate the absolute number of CD4+ T cells by multiplying the total leukocyte number. Blood routine tests were performed to obtain the leukocyte number in the blood samples. FITC-labeled mouse anti-human CD3 +, PE-labeled mouse anti-human CD4+ and PElabeled mouse anti-human CD8+ monoclonal antibodies were from BD (USA). 3.5. Immunosuppressive agent concentration monitoring The trough levels of tacrolimus and cyclosporine were measured in the whole blood samples using a microparticle enzyme immunoassay (MEIA) according to the manufacturer's instructions (Abbott Diagnostics, North Chicago, IL, USA).
3. Materials and method 3.1. Patients This is a single center prospective study that was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Medical College of Xi'an Jiaotong University. The informed consent was obtained from all study participants. A total of 140 KT recipients were recruited from October 2011 to October 2012, and all were first-time transplant recipients. All kidney grafts were from either living donors or cardiac deceased donors. Patients were excluded when suffered infection or delayed graft function during the six month follow-up after transplantation. All patients received induction therapy with rabbit anti-human T lymphocyte immunoglobulin (ATG) and steroids. Subsequent immunosuppressive maintenance regimens were usually standard triple therapy, which consisted of tacrolimus (FK506) or cyclosporine A (CsA), combined with mycophenolate (MMF) and prednisone. AR was defined clinically by an acute rise in serum creatinine and urine output decrease, which was confirmed by a kidney allograft biopsy. Rejection episodes were treated with methylprednisolone at a dose of 500 mg by intravenous injection once daily for three consecutive days. ATG was used in the patients with refractory acute rejection episodes.
3.6. Statistical analysis All data were recorded as the mean ± standard deviation. Continuous variables were compared using Student's t test. Categorical variables were compared using the chi-square or the Fisher's exact test. Correlations between the intracellular ATP and other variables were determined using the Pearson's correlation coefficients, with correlation expressed as r values. Receiver operator characteristic (ROC) analysis was performed to assess the potential of intracellular ATP for distinguishing patients with and without rejection. All statistical calculations and tests were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA), GraphPad Prism software (GraphPad Prism Software Inc., San Diego, CA, USA), and MedCalc (MedCalc Software, Mariakerke, Belgium). A P value less than 0.05 was considered statistically significant. 4. Results 4.1. Intracellular ATP level in Chinese healthy adults and kidney transplant recipients At the endpoint of this follow-up, 19 patients experienced AR episodes and 121 patients were categorized into stable group. Demographic details for these patients are
3.2. Sample collection The anticoagulated whole peripheral blood samples were collected from all recipients pre-transplantation, and at 7, 14, 21, and 28 days, and at 2, 3, 4, 5 and 6 months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7 days after intravenous anti-rejection therapy started, and on the day when AR reversed. 3.3. Cylex immune cell function assay analysis +
The intracellular ATP in CD4 T cells was detected by Cylex ImmuKnow Cell Function Assay (Cylex, Inc., Columbia, MD, USA) according to the manufacturer's instruction. Briefly, 100 μl whole blood (1:4 dilution) was added to wells of a 96-well microtiter plate, and incubated overnight (15–18 h) with or without PHA at 37 °C in a 5% CO2 incubator. On the following day, CD4+ T cells were selected by antihuman CD4 monoclonal antibody-coated magnetic particles (Dynal, Oslo, Norway) and a strong magnet (Cylex Magnet tray 1050; Cylex
Table 1 Baseline characteristics of the kidney transplant recipients. Variable
SG (n = 121)
ARG (n = 19)
P value
Recipient age(year; mean ± SD) Gender (male; female) Donor age (year; mean ± SD) Type of transplant Deceased donor Living related Pre-transplant PRA, n (%) PRA b 10% 10% ≤ PRA b 50% HLA A, B, DR MM (number) Cold ischemia time (hour) Immunosuppression protocol CsA + MMF + Pred FK + MMF + Pred
38.70 ± 7.20 89:32 52.1 ± 8.75
31.90 ± 5.23 12:7 45.1 ± 6.27
0.46 0.41 0.76 0.31
72 49
12 7
120 1 2.45 ± 0.75 0.98 ± 0.43
17 2 2.53 ± 0.46 1.03 ± 0.57
32 89
4 15
0.09
0.37 0.95 0.78
Abbreviations: SG, stable group; ARG, acute rejection group; PRA, panel reaction antibody; MM, mismatches; HLA, human leukocyte antigen; FK506, tacrolimus; MMF, mycophenolate mofetil; Pred, prednisone; CsA, cyclosporine A.
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
X.-Z. Wang et al. / Transplant Immunology xxx (2013) xxx–xxx shown in Table 1. 50 healthy Chinese people were categorized into healthy control group. Their average age was 34.50 ± 6.32 years old in the range from 19 to 53 years old. The intracellular ATP level of healthy controls was 421.293 ± 131.61 ng/ml. The preoperative intracellular ATP level of patients in the stable group was 368.91 ± 145.98 ng/ml, significantly lower than that of the healthy controls (P b 0.05). The preoperative intracellular ATP level of patients in the AR group was 538.76 ± 279.22 ng/ml, which was significantly higher than that of the patients in stable group (P b 0.05), but similar to that of healthy controls (P N 0.05). 4.2. Dynamic changes of intracellular ATP level in KT recipients in the six month follow-up We observed that the intracellular ATP levels changed dynamically after kidney transplantation, as shown in Fig. 1. The intracellular ATP level decreased immediately after transplantation in all KT recipients and then altered differently in the stable and AR groups. In the stable group, the intracellular ATP level increased at 14 days postoperation, and thereafter maintained at an average level of 289.49 ± 100.96 ng/ml. On the other hand, the intracellular ATP level of AR group increased remarkably at 28 days, decreased until the third month post-operation and fluctuated smoothly in the subsequent 3 months. The intracellular ATP levels at 14, 21, and 28 days, and 2, 3, 4, 5, and 6 months post-transplantation did not differ statistically between the two groups. However, patients in the AR group had higher intracellular ATP levels at 7 days posttransplantation (431.36 ± 137.31 ng/ml, P b 0.05), compared to the patients in the stable group (184.23 ± 84.40 ng/ml). 4.3. Association between the intracellular ATP level and AR in kidney transplantation At the end of the 6 months post-transplantation follow-up, 19 patients experienced biopsy-proven AR, among whom, 4 suffered from acute humoral rejection and 15 suffered from acute cellular rejection. The median time when AR occurred was 32 days, with a range from 3 to 89 days post-transplantation. Because ATP level changed dynamically post-transplantation, we used ATP values of the stable group measured no more than three days before or after AR occurrence as the corresponding controls for correlation analysis between ATP level and AR. The intracellular ATP level detected on the day AR occurred (627.07 ± 149.85 ng/ml) was obviously higher than that of the stable group (320.48 ± 149.11 ng/ml, P b 0.05) and the healthy control group (421.293 ± 131.61 ng/ml, P b 0.05) (Fig. 2A). After anti-rejection therapy, AR was reversed in all patients. We also measured the intracellular ATP levels of patients who suffered AR episode on the day before as well as 3 and 7 days after the start of anti-rejection therapy, and at the end when AR was reversed. ATP value was 665.87 ± 162.85 ng/ml before starting antirejection therapy, which first decreased to 228.11 ± 127.44 ng/ml at 3 days (P b 0.05) and increased to 320.51 ± 99.48 ng/ml (P b 0.05) at 7 days after therapy. At the end of anti-rejection therapy, the intracellular ATP level decreased to 265.35 ± 84.33 ng/ml, which was obviously lower than that measured on the day before anti-rejection therapy started (P b 0.05, Fig. 2B). 4.4. Predictive role of intracellular ATP level for AR An ROC curve analysis was performed to evaluate the availability of ATP level at each time point in the first month post-transplantation for predicting AR occurrence. The results of the analysis showed that the intracellular ATP levels at several time points,
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including preoperative and 14, 21, and 28 days post-operation were failed to predict the subsequent AR episodes (data not shown). The high intracellular ATP level at 7 days post-transplantation showed the best sensitivity and specificity to identify AR patients in the early stage post-transplantation, as shown in Fig. 3A. The best cutoff ATP value was 274.57 ng/ml, with the area under ROC curve being 0.928, sensitivity of 85.0% and specificity of 88.9%. Previous literatures have suggested the intracellular ATP level of 525 ng/ml to be the threshold for AR occurrence in KT recipients. However, we found that the intracellular ATP levels in some patients were less than 525 ng/ml when AR occurred. Interestingly, we observed that their intracellular ATP levels indeed increased compared with that measured before AR episodes. Moreover, a gradually increasing trend was observed even earlier. Therefore, we generated a ROC curve using the data obtained by deducting the intracellular ATP value measured right before the incidence of AR from the intracellular ATP value detected when AR occurred, and found that the sensitivity and specificity were 89.5% and 95.0% respectively to predict subsequent AR episodes. The best cutoff value was 172.55 ng/ml, with the area under ROC curve being 0.929 (Fig. 3B). 4.5. Parallel detection of intracellular ATP, tacrolimus trough levels and absolute CD4+ T cell counts The correlation of tacrolimus trough level in the whole blood with intracellular ATP level was analyzed, as the majority of patients in this study received tacrolimus therapy. However, no correlation (r = −0.078, P = 0.469) was observed (Fig. 4). We also found that there was no significant correlation between the intracellular ATP level and absolute CD4+ T cell counts in the patients with CD4+ T cell counts N200/μl (r = 0.365, P = 0.577). Furthermore, we analyzed the intracellular ATP level of 32 samples obtained from the patients with CD4+ T cell counts b200/μl, and found a positive correlation between the intracellular ATP level and absolute CD4+ T cell number (r = 0.656, P b 0.001, Fig. 5).
5. Discussion Our study was based on 1497 blood samples from 50 healthy individuals and 140 KT recipients during the 6-month follow-up after transplantation. To our knowledge, this is the most comprehensive study to analyze the utility of Cylex ImmuKnow Cell Function Assay in predicting AR episodes in KT recipients. We demonstrated for the first time that the increased intracellular ATP level rather than the intracellular ATP level at a single time point could be a useful index to predict acute rejection in KT recipients. As shown in the study, the average intracellular ATP level in healthy adults was 421.293 ± 131.61 ng/ml, similar to that in healthy Americans (433 ± 148 ng/ml) [4]. The average pre-transplantation intracellular ATP level in KT recipients was 368.91 ± 145.98 ng/ml, significantly lower than that of healthy controls. This is not surprising considering the impaired immune cell function in uremia patients [9]. However, there were wide inter- and intra-individual
Fig. 1. Dynamic changes of intracellular ATP levels in SG and ARG during the six-month follow-up of post-kidney transplantation. The data are expressed as mean ± SD.
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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Fig. 2. (A) Intracellular ATP level in the AR group was significantly higher than that of the stable group and healthy controls (P b 0.05). (Horizontal line in the box = median, box = interquartile range, whiskers = full value range). (B) Nineteen patients in the AR group showed continuously increased intracellular ATP levels till day 0 (before anti-rejection therapy) and significantly decreased ATP levels during the therapy. The data are expressed as mean ± SD. * denotes P less than 0.05 compared with the intracellular ATP on day 0.
variations in intracellular ATP levels. Even in healthy controls, there were several individuals with ATP levels which remained less than 100 ng/ml after repeated testing, indicating the complexity of the immune system and the diversity of individuals. Therefore, we suggest that the preoperative intracellular ATP value should be considered as the baseline for objective evaluation of the immune status in KT recipients. We found that the high intracellular ATP levels were significantly correlated with AR episodes in KT recipients. The average intracellular ATP level on the day AR occurred was obviously higher than that of the stable group. In addition, the intracellular ATP levels decreased significantly after the reversal of AR, implying that intracellular ATP monitoring could be applicable to assess the effectiveness of anti-rejection therapy. Contrary to our study, some previous studies failed to find an association between the intracellular ATP levels and AR episodes. De Paolis P et al. observed that only one recipient experienced an AR episode in the total 17 KT recipients with elevated intracellular ATP values [10]. Torio A et al. reported that only 12% of AR patients were included in the strong immune response zone [11]. Serban G et al. found that more than 40% of the samples showed moderate to high values during the first 3 months post-transplantation, and no correlation between ATP value and rejection was demonstrated [12]. However, all these studies set ATP level of 525 ng/ml at a single time point as an index to predict AR. In fact, there were very wide inter- and intra-individual variations in ATP values. The intracellular ATP also exhibited dynamic changes over time post-transplantation. We doubt whether the intracellular ATP level at a single time point is suitable to reflect the immune status,
which was also pointed out by Huskey J et al. [13] and Israeli M et al. [14,15]. We demonstrated that the trend of intracellular ATP increasing rather than a value at a single time point was more helpful to identify patients at higher risk of rejection. Several previous studies have shown that the high intracellular ATP level (≥ 525 ng/ml) at a single time point lacked both sensitivity and specificity for prediction of AR episodes [10,11,16]. In our large population study and the long-term serial testing scheme, we observed that patients with low intracellular ATP level (b 525 ng/ml) still had strong cellular immune responses and experienced AR episodes. In addition, longitudinal analysis revealed a significant increase in the intracellular ATP levels in AR patients, compared to that measured right before AR episodes. ROC analysis indicated that the sensitivity and specificity to predict AR episodes were 89.5% and 95.0% respectively, when the increase of intracellular ATP level was higher than 172.55 ng/ml. Otherwise, the intracellular ATP levels detected at 14, 21, and 28 days post-operation all failed to predict the subsequent AR episodes. The intracellular ATP level at 7 days posttransplantation showed the best sensitivity and specificity to identify AR patients at the early stage, but with lower sensitivity of 85.0% and specificity of 88.9%. These findings suggested that the trend of intracellular ATP increasing rather than ATP level at a single time point could predict the subsequent rejection timely and accurately. We also found that the peripheral blood trough tacrolimus concentration was not correlated with intracellular ATP level, consistent with previous studies [17–19]. It indicates that blood concentration of immunosuppressive agents could not reflect factually
Fig. 3. (A) ROC curves of intracellular ATP levels at day 7 of post-kidney transplantation in predicting acute rejection. (B) ROC analysis showing the increase of intracellular ATP level for predicting subsequent acute rejection in kidney transplant recipients. AUC, area under the ROC curve.
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
X.-Z. Wang et al. / Transplant Immunology xxx (2013) xxx–xxx
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In conclusion, we demonstrated that the high intracellular ATP level had a significant correlation with AR episodes in KT recipients and the trend of intracellular ATP increasing rather than the intracellular ATP level at a single time point could be used to predict the subsequent rejection episodes timely and accurately. However, our data were collected from recipients during the early post-transplant period. Further studies with longer follow-up time, larger sample size, and multicenter participation should be performed to evaluate the clinical utility of the Cylex ImmuKnow Cell Function Assay in monitoring immune status in transplant recipients. Competing interests The authors declare that they have no competing interests. Acknowledgments
Fig. 4. Correlation between the tacrolimus concentration and intracellular ATP level in kidney transplant recipients(r = −0.078, P = 0.469).
the immunosuppressive degree in KT recipients, which was also supported by other studies using cytokine-based measures of T-cell function [20,21]. Based on our data, we suggest that intracellular ATP level could be used as an additional tool to evaluate the degree of immunosuppression for individual management of immunosuppressive agents. Although a prospective randomized clinical trial in liver transplant recipients has utilized modulated immunosuppressive protocols according to the intracellular ATP level [22], further studies are needed to estimate the utility of ImmuKnow assay in tapering immunosuppressive agents in KT recipients. We observed that intracellular ATP level can be extremely low in patients with remarkably low absolute CD4+ T cell count, which was in agreement with Xue F et al.'s study performed in liver graft recipients [23]. It was reported that the low intracellular ATP level was associated with a higher risk of infection, suggesting the presence of overimmunosuppression [24–26]. In our study, however, no clinical features of infection existed in 140 KT recipients. This suggests that it is inappropriate to classify the patients into over-immunosuppression group immediately when an extremely low intracellular ATP value is detected. The absolute CD4 + T cell count should be taken into account simultaneously.
Fig. 5. Correlation between the absolute CD4+ cell number and intracellular ATP level in kidney transplant recipients with CD4+ cell counts of b200/μl (r = 0.656, P b 0.001).
This work was supported by the grants from The National Basic Research Program of China (973 program) (No. 2009CB522407) and the National Nature Science Foundation of China (Nos. 30972947, 81100179). We also thank Zhen Wan, MD, for his constructive suggestions and editorial assistance. References [1] Nashan B, Moore R, Amlot P, Schmidt AG, Abeywickrama K, Soulillou JP. Randomised trial of basiliximab versus placebo for control of acute cellular rejection in renal allograft recipients. CHIB 201 International Study Group. Lancet 1997;350(9086):1193–8. [2] Choi BS, Shin MJ, Shin SJ, Kim YS, Choi YJ, Kim YS, et al. Clinical significance of an early protocol biopsy in living-donor renal transplantation: ten-year experience at a single center. Am J Transplant 2005;5(6):1354–60. [3] Kowalski RJ, Post DR, Mannon RB, Sebastian A, Wright HI, Sigle G, et al. Assessing relative risks of infection and rejection: a meta-analysis using an immune function assay. Transplantation 2006;82(5):663–8. [4] Kowalski R, Post D, Schneider MC, Britz J, Thomas J, Deierhoi M, et al. Immune cell function testing: an adjunct to therapeutic drug monitoring in transplant patient management. Clin Transplant 2003;17(2):77–88. [5] Cadillo-Chávez R, de Echegaray S, Santiago-Delpín EA, Rodríguez-Trinidad AT, Camacho-Carrazo B, Alfaro T, et al. Assessing the risk of infection and rejection in Hispanic renal transplant recipients by means of an adenosine triphosphate release assay. Transplant Proc 2006;38(3):918–20. [6] Cabrera R, Ararat M, Soldevila-Pico C, Dixon L, Pan JJ, Firpi R, et al. Using an immune functional assay to differentiate acute cellular rejection from recurrent hepatitis C in liver transplant patient. Liver Transpl 2009;15(2):216–22. [7] Dong JY, Yin H, Li RD, Ding GS, Fu ZR, Wu YM, et al. The relationship between adenosine triphosphate within CD4(+) T lymphocytes and acute rejection after liver transplantation. Clin Transplant 2011;25(3):E292–6. [8] Ling XT, Xiong J, Liang WH, Paul MS, Wu LW, Ju WQ, et al. Can immune cell function assay identify patients at risk of infection or rejection? a meta-analysis. Transplantation 2012;93(7):737–43. [9] Betjes MG. Immune cell dysfunction and inflammation in end-stage renal disease. Nat Rev Nephrol 2013;9(5):255–65. [10] De Paolis P, Favaro A, Piola A, Martini F, Cristiana G, Agrati C, et al. “ImmuKnow” to measurement of cell-mediated immunity in renal transplant recipients undergoing short-term evaluation. Transplant Proc 2011;43(4):1013–6. [11] Torio A, Fernandez EJ, Montes-Ares O, Guerra RM, Perez MA, Checa MD. Lack of association of immune cell function test with rejection in kidney transplantation. Transplant Proc 2011;43(6):2168–70. [12] Serban G, Whittaker V, Fan J, Liu Z, Manga K, Khan M, et al. Significance of immune cell function monitoring in renal transplantation after Thymoglobulin induction therapy. Hum Immunol 2009;70(11):882–90. [13] Huskey J, Gralla J, Wiseman AC. Single time point immune function assay (ImmuKnow) testing does not aid in the prediction of future opportunistic infections or acute rejection. Clin J Am Soc Nephrol 2011;6(2):423–9. [14] Israeli M, Yussim A, Mor E, Sredni B, Klein T. Preceeding the rejection: in search for a comprehensive post-transplant immune monitoring platform. Transpl Immunol 2007;18(1):7–12. [15] Israeli M, Ben-Gal T, Yaari V, Valdman A, Matz I, Medalion B, et al. Individualized immune monitoring of cardiac transplant recipients by noninvasive longitudinal cellular immunity tests. Transplantation 2010;89(8):968–76. [16] Hwang S, Kim KH, Song GW, Yu YD, Park GC, Kim KW, et al. Peritransplant monitoring of immune cell function in adult living donor liver transplantation. Transplant Proc 2010;42(7):2567–71. [17] Israeli M, Klein T, Sredni B, Avitzur Y, Mor E, Bar-Nathen N, et al. ImmuKnow: a new parameter in immune monitoring of pediatric liver transplantation recipients. Liver Transpl 2008;14(6):893–8. [18] Mizuno S, Hamada T, Nakatani K, Kishiwada M, Usui M, Sakurai H, et al. Monitoring peripheral blood CD4 + adenosine triphosphate activity after living donor liver transplantation: impact of combination assays of immune
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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X.-Z. Wang et al. / Transplant Immunology xxx (2013) xxx–xxx
[19]
[20]
[21]
[22]
function and CYP3A5 genotype. J Hepatobiliary Pancreat Sci 2011;18(2):226–32 [discussion 32–4]. Sampson VB, Dunn SP, Rymeski B, Malatack J, Rong NH, Flynn L, et al. Failure of immunosuppressive drug levels to predict T-cell reactivity in pediatric transplant patients. J Pediatr Surg 2008;43(6):1134–41. Schulick RD, Weir MB, Miller MW, Cohen DJ, Bermas BL, Shearer GM. Longitudinal study of in vitro CD4+ T helper cell function in recently transplanted renal allograft patients undergoing tapering of their immunosuppressive drugs. Transplantation 1993;56(3):590–6. Ahmed M, Venkataraman R, Logar AJ, Rao AS, Bartley GP, Robert K, et al. Quantitation of immunosuppression by tacrolimus using flow cytometric analysis of interleukin-2 and interferon-gamma inhibition in CD8(−) and CD8(+) peripheral blood T cells. Ther Drug Monit 2001;23(4):354–62. Ravaioli M, Morelli C, Del Gaudio M, Zanello M, Berardi S, Ercolani G, et al. Immunosuppression monitoring by Cylex ImmuKnow test after liver trans-
[23]
[24]
[25]
[26]
plantation: preliminary results of randomized prospective trial [abstract]. Transpl Int 2011;24(Suppl. 2):227. Xue F, Zhang J, Han L, Li Q, Xu N, Zhou T, et al. Immune cell functional assay in monitoring of adult liver transplantation recipients with infection. Transplantation 2010;89(5):620–6. Husain S, Raza K, Pilewski JM, Zaldonis D, Crespo M, Toyoda Y, et al. Experience with immune monitoring in lung transplant recipients: correlation of low immune function with infection. Transplantation 2009;87(12):1852–7. Sanchez-Velasco P, Rodrigo E, Valero R, Ruiz JC, Fernandez-Fresnedo G, Lopez-Hoyos M, et al. Intracellular ATP concentrations of CD4 cells in kidney transplant patients with and without infection. Clin Transplant 2008;22(1):55–60. Zhou T, Xue F, Han LZ, Xi ZF, Li QG, Xu N, et al. Invasive fungal infection after liver transplantation: risk factors and significance of immune cell function monitoring. J Dig Dis 2011;12(6):467–75.
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients Xu-Zhen Wang a,1, Zhan-Kui Jin c,1, Xiao-Hui Tian a,b,1, Wu-Jun Xue a,b,⁎, Pu-Xun Tian a,b, Xiao-Ming Ding a,b, Jin Zheng a,b, Yang Li a, Xin Jing a, Zi-Zhen Luo a a b c
Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, China Institute of Organ Transplantation, Xi'an Jiaotong University, Xi'an, Shaanxi, China Department of Orthopaedics, Shaanxi Provincial People's Hospital (The Third Affiliated Hospital of Medical College of Xi'an Jiaotong University), Xi'an, Shaanxi, China
a r t i c l e
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Article history: Received 12 September 2013 Received in revised form 28 October 2013 Accepted 28 October 2013 Available online xxxx Keywords: Acute rejection Adenosine triphosphate (ATP) Kidney transplantation
a b s t r a c t Background: Peripheral blood CD4+ T cell adenosine triphosphate (ATP) release has been reported to be an adjunct tool to evaluate global cellular immune response in solid-organ transplant recipients. However, the correlation between the ATP level and rejection was controversial. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of acute rejection (AR) episodes, determine the predicting value of intracellular ATP level for AR in kidney transplant (KT) recipients. Patients and methods: In the period of October 2011 to October 2012, 140 KT recipients were recruited and followed for six months after transplantation. Patients were categorized into stable group and AR group according to their clinical course. Whole blood samples were collected pretransplantation, and at 7, 14, 21, and 28 days, and at 2, 3, 4, 5 and 6 months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7 days after intravenous anti-rejection therapy started, and on the day when AR reversed. The intracellular ATP in CD4+ T cells was detected by ImmuKnow Immune Cell Function Assay according to the manufacturer's instruction. The absolute number of CD4+ T cells and the trough levels of tacrolimus and cyclosporine were also measured. Results: The ATP level detected on the day AR occurred (627.07 ± 149.85 ng/ml) was obviously higher than that of the stable group (320.48 ± 149.11 ng/ml, P b 0.05). ATP value decreased to 265.35 ± 84.33 ng/m at the end of anti-rejection therapy, which was obviously lower than that measured on the day before the anti-rejection therapy started (665.87 ± 162.85 ng/ml, P b 0.05). ROC analysis revealed that increased intracellular adenosine triphosphate level showed better sensitivity and specificity than those obtained using single time point detection (89.5% vs 85.0%;95.0% vs 88.9%). The best cutoff value was 172.55 ng/ml. A positive correlation between the intracellular ATP level and absolute CD4 + T cell number (r = 0.656, P b 0.001) was found in the patients with CD4+ T cell counts b 200/μl. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Kidney transplantation has been successfully used as an effective therapeutic option for patients with end-stage kidney diseases. With the introduction of a variety of immunosuppressive agents, the graft and patient survival rate after kidney transplantation has been significantly improved during the past decade. However, acute rejection (AR), which usually occurs on days 7–14 in the early posttransplantation period [1,2], remains a major obstacle for the long term survival in kidney transplant (KT) recipients. Currently,
⁎ Corresponding author at: Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, 277 Yanta West Road, Xi'an, Shaanxi 710061, China. Tel.: +86 29 85323721; fax: +86 29 85323718. E-mail address: [email protected] (W.-J. Xue). 1 Contributed equally to this work.
allograft rejection is taken into consideration only when continuous and irreversible deterioration of renal function occurs, and the gold standard-biopsy procedure usually takes up several days. As a result, anti-rejection therapy starts relatively late as kidney damage has already been established. Hence, convenient and noninvasive approaches for predicting and diagnosing AR would be of considerable value to improve outcomes in KT recipients. The Cylex ImmuKnow Cell Function Assay may be a promising tool for post-transplant immune monitoring. In 2002, the U.S. Food and Drug Administration (FDA) approved this assay for evaluating global cellular immune response in solid-organ transplant recipients [3]. It assesses the activity of CD4+ T cells by measuring the concentration of intracellular adenosine triphosphate (ATP) after stimulation with phytohemagglutinin (PHA) in vitro. It is expected that this assay could be beneficial in monitoring the net immune state in transplant recipients and lead to better patient management and evidence-based individualization.
0966-3274/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.trim.2013.10.008
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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X.-Z. Wang et al. / Transplant Immunology xxx (2013) xxx–xxx
Based on a multicenter study in American, Kowalski et al. classified immune response of transplant recipients into three zones: strong (≥ 525 ng/ml ATP), moderate (226–524 ng/ml ATP), and low (≤ 225 ng/ml ATP) [4]. They reported that ATP values higher than 525 ng/ml were associated with an increased risk of AR. Thereafter, most of the following studies used ATP level at 525 ng/ml as a threshold to investigate the predictive role of intracellular ATP values at single time point for AR. Several studies have shown that the high intracellular ATP levels can identify patients at risk for rejection [5–7], whereas others argue against its predictive capacity [8]. Therefore, the correlation between ATP level and rejection is still undetermined. 2. Objective We hypothesized that patients with high intracellular ATP level in stimulated CD4+ T cells measured by the Cylex ImmuKnow Cell Function Assay are at increased risk of AR episodes. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of AR episodes.
Inc., Columbia, MD, USA), washed and lysed to release intracellular ATP. Subsequently, a luciferin/luciferase reagent was added and released ATP was measured by a luminometer (Turner Biosystems, Sunnyvale, CA, USA). The concentration of ATP was expressed as ng/ml. 3.4. Flow cytometry and absolute CD4+ T cell counting Before intracellular ATP detection, part of the blood samples were stained for lymphocyte surface markers and then sorted by flow cytometry (FACSCalibur, BD, USA) to obtain the ratio of CD4+ T lymphocytes to the total peripheral blood leukocytes, which was then used to calculate the absolute number of CD4+ T cells by multiplying the total leukocyte number. Blood routine tests were performed to obtain the leukocyte number in the blood samples. FITC-labeled mouse anti-human CD3 +, PE-labeled mouse anti-human CD4+ and PElabeled mouse anti-human CD8+ monoclonal antibodies were from BD (USA). 3.5. Immunosuppressive agent concentration monitoring The trough levels of tacrolimus and cyclosporine were measured in the whole blood samples using a microparticle enzyme immunoassay (MEIA) according to the manufacturer's instructions (Abbott Diagnostics, North Chicago, IL, USA).
3. Materials and method 3.1. Patients This is a single center prospective study that was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Medical College of Xi'an Jiaotong University. The informed consent was obtained from all study participants. A total of 140 KT recipients were recruited from October 2011 to October 2012, and all were first-time transplant recipients. All kidney grafts were from either living donors or cardiac deceased donors. Patients were excluded when suffered infection or delayed graft function during the six month follow-up after transplantation. All patients received induction therapy with rabbit anti-human T lymphocyte immunoglobulin (ATG) and steroids. Subsequent immunosuppressive maintenance regimens were usually standard triple therapy, which consisted of tacrolimus (FK506) or cyclosporine A (CsA), combined with mycophenolate (MMF) and prednisone. AR was defined clinically by an acute rise in serum creatinine and urine output decrease, which was confirmed by a kidney allograft biopsy. Rejection episodes were treated with methylprednisolone at a dose of 500 mg by intravenous injection once daily for three consecutive days. ATG was used in the patients with refractory acute rejection episodes.
3.6. Statistical analysis All data were recorded as the mean ± standard deviation. Continuous variables were compared using Student's t test. Categorical variables were compared using the chi-square or the Fisher's exact test. Correlations between the intracellular ATP and other variables were determined using the Pearson's correlation coefficients, with correlation expressed as r values. Receiver operator characteristic (ROC) analysis was performed to assess the potential of intracellular ATP for distinguishing patients with and without rejection. All statistical calculations and tests were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA), GraphPad Prism software (GraphPad Prism Software Inc., San Diego, CA, USA), and MedCalc (MedCalc Software, Mariakerke, Belgium). A P value less than 0.05 was considered statistically significant. 4. Results 4.1. Intracellular ATP level in Chinese healthy adults and kidney transplant recipients At the endpoint of this follow-up, 19 patients experienced AR episodes and 121 patients were categorized into stable group. Demographic details for these patients are
3.2. Sample collection The anticoagulated whole peripheral blood samples were collected from all recipients pre-transplantation, and at 7, 14, 21, and 28 days, and at 2, 3, 4, 5 and 6 months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7 days after intravenous anti-rejection therapy started, and on the day when AR reversed. 3.3. Cylex immune cell function assay analysis +
The intracellular ATP in CD4 T cells was detected by Cylex ImmuKnow Cell Function Assay (Cylex, Inc., Columbia, MD, USA) according to the manufacturer's instruction. Briefly, 100 μl whole blood (1:4 dilution) was added to wells of a 96-well microtiter plate, and incubated overnight (15–18 h) with or without PHA at 37 °C in a 5% CO2 incubator. On the following day, CD4+ T cells were selected by antihuman CD4 monoclonal antibody-coated magnetic particles (Dynal, Oslo, Norway) and a strong magnet (Cylex Magnet tray 1050; Cylex
Table 1 Baseline characteristics of the kidney transplant recipients. Variable
SG (n = 121)
ARG (n = 19)
P value
Recipient age(year; mean ± SD) Gender (male; female) Donor age (year; mean ± SD) Type of transplant Deceased donor Living related Pre-transplant PRA, n (%) PRA b 10% 10% ≤ PRA b 50% HLA A, B, DR MM (number) Cold ischemia time (hour) Immunosuppression protocol CsA + MMF + Pred FK + MMF + Pred
38.70 ± 7.20 89:32 52.1 ± 8.75
31.90 ± 5.23 12:7 45.1 ± 6.27
0.46 0.41 0.76 0.31
72 49
12 7
120 1 2.45 ± 0.75 0.98 ± 0.43
17 2 2.53 ± 0.46 1.03 ± 0.57
32 89
4 15
0.09
0.37 0.95 0.78
Abbreviations: SG, stable group; ARG, acute rejection group; PRA, panel reaction antibody; MM, mismatches; HLA, human leukocyte antigen; FK506, tacrolimus; MMF, mycophenolate mofetil; Pred, prednisone; CsA, cyclosporine A.
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
X.-Z. Wang et al. / Transplant Immunology xxx (2013) xxx–xxx shown in Table 1. 50 healthy Chinese people were categorized into healthy control group. Their average age was 34.50 ± 6.32 years old in the range from 19 to 53 years old. The intracellular ATP level of healthy controls was 421.293 ± 131.61 ng/ml. The preoperative intracellular ATP level of patients in the stable group was 368.91 ± 145.98 ng/ml, significantly lower than that of the healthy controls (P b 0.05). The preoperative intracellular ATP level of patients in the AR group was 538.76 ± 279.22 ng/ml, which was significantly higher than that of the patients in stable group (P b 0.05), but similar to that of healthy controls (P N 0.05). 4.2. Dynamic changes of intracellular ATP level in KT recipients in the six month follow-up We observed that the intracellular ATP levels changed dynamically after kidney transplantation, as shown in Fig. 1. The intracellular ATP level decreased immediately after transplantation in all KT recipients and then altered differently in the stable and AR groups. In the stable group, the intracellular ATP level increased at 14 days postoperation, and thereafter maintained at an average level of 289.49 ± 100.96 ng/ml. On the other hand, the intracellular ATP level of AR group increased remarkably at 28 days, decreased until the third month post-operation and fluctuated smoothly in the subsequent 3 months. The intracellular ATP levels at 14, 21, and 28 days, and 2, 3, 4, 5, and 6 months post-transplantation did not differ statistically between the two groups. However, patients in the AR group had higher intracellular ATP levels at 7 days posttransplantation (431.36 ± 137.31 ng/ml, P b 0.05), compared to the patients in the stable group (184.23 ± 84.40 ng/ml). 4.3. Association between the intracellular ATP level and AR in kidney transplantation At the end of the 6 months post-transplantation follow-up, 19 patients experienced biopsy-proven AR, among whom, 4 suffered from acute humoral rejection and 15 suffered from acute cellular rejection. The median time when AR occurred was 32 days, with a range from 3 to 89 days post-transplantation. Because ATP level changed dynamically post-transplantation, we used ATP values of the stable group measured no more than three days before or after AR occurrence as the corresponding controls for correlation analysis between ATP level and AR. The intracellular ATP level detected on the day AR occurred (627.07 ± 149.85 ng/ml) was obviously higher than that of the stable group (320.48 ± 149.11 ng/ml, P b 0.05) and the healthy control group (421.293 ± 131.61 ng/ml, P b 0.05) (Fig. 2A). After anti-rejection therapy, AR was reversed in all patients. We also measured the intracellular ATP levels of patients who suffered AR episode on the day before as well as 3 and 7 days after the start of anti-rejection therapy, and at the end when AR was reversed. ATP value was 665.87 ± 162.85 ng/ml before starting antirejection therapy, which first decreased to 228.11 ± 127.44 ng/ml at 3 days (P b 0.05) and increased to 320.51 ± 99.48 ng/ml (P b 0.05) at 7 days after therapy. At the end of anti-rejection therapy, the intracellular ATP level decreased to 265.35 ± 84.33 ng/ml, which was obviously lower than that measured on the day before anti-rejection therapy started (P b 0.05, Fig. 2B). 4.4. Predictive role of intracellular ATP level for AR An ROC curve analysis was performed to evaluate the availability of ATP level at each time point in the first month post-transplantation for predicting AR occurrence. The results of the analysis showed that the intracellular ATP levels at several time points,
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including preoperative and 14, 21, and 28 days post-operation were failed to predict the subsequent AR episodes (data not shown). The high intracellular ATP level at 7 days post-transplantation showed the best sensitivity and specificity to identify AR patients in the early stage post-transplantation, as shown in Fig. 3A. The best cutoff ATP value was 274.57 ng/ml, with the area under ROC curve being 0.928, sensitivity of 85.0% and specificity of 88.9%. Previous literatures have suggested the intracellular ATP level of 525 ng/ml to be the threshold for AR occurrence in KT recipients. However, we found that the intracellular ATP levels in some patients were less than 525 ng/ml when AR occurred. Interestingly, we observed that their intracellular ATP levels indeed increased compared with that measured before AR episodes. Moreover, a gradually increasing trend was observed even earlier. Therefore, we generated a ROC curve using the data obtained by deducting the intracellular ATP value measured right before the incidence of AR from the intracellular ATP value detected when AR occurred, and found that the sensitivity and specificity were 89.5% and 95.0% respectively to predict subsequent AR episodes. The best cutoff value was 172.55 ng/ml, with the area under ROC curve being 0.929 (Fig. 3B). 4.5. Parallel detection of intracellular ATP, tacrolimus trough levels and absolute CD4+ T cell counts The correlation of tacrolimus trough level in the whole blood with intracellular ATP level was analyzed, as the majority of patients in this study received tacrolimus therapy. However, no correlation (r = −0.078, P = 0.469) was observed (Fig. 4). We also found that there was no significant correlation between the intracellular ATP level and absolute CD4+ T cell counts in the patients with CD4+ T cell counts N200/μl (r = 0.365, P = 0.577). Furthermore, we analyzed the intracellular ATP level of 32 samples obtained from the patients with CD4+ T cell counts b200/μl, and found a positive correlation between the intracellular ATP level and absolute CD4+ T cell number (r = 0.656, P b 0.001, Fig. 5).
5. Discussion Our study was based on 1497 blood samples from 50 healthy individuals and 140 KT recipients during the 6-month follow-up after transplantation. To our knowledge, this is the most comprehensive study to analyze the utility of Cylex ImmuKnow Cell Function Assay in predicting AR episodes in KT recipients. We demonstrated for the first time that the increased intracellular ATP level rather than the intracellular ATP level at a single time point could be a useful index to predict acute rejection in KT recipients. As shown in the study, the average intracellular ATP level in healthy adults was 421.293 ± 131.61 ng/ml, similar to that in healthy Americans (433 ± 148 ng/ml) [4]. The average pre-transplantation intracellular ATP level in KT recipients was 368.91 ± 145.98 ng/ml, significantly lower than that of healthy controls. This is not surprising considering the impaired immune cell function in uremia patients [9]. However, there were wide inter- and intra-individual
Fig. 1. Dynamic changes of intracellular ATP levels in SG and ARG during the six-month follow-up of post-kidney transplantation. The data are expressed as mean ± SD.
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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Fig. 2. (A) Intracellular ATP level in the AR group was significantly higher than that of the stable group and healthy controls (P b 0.05). (Horizontal line in the box = median, box = interquartile range, whiskers = full value range). (B) Nineteen patients in the AR group showed continuously increased intracellular ATP levels till day 0 (before anti-rejection therapy) and significantly decreased ATP levels during the therapy. The data are expressed as mean ± SD. * denotes P less than 0.05 compared with the intracellular ATP on day 0.
variations in intracellular ATP levels. Even in healthy controls, there were several individuals with ATP levels which remained less than 100 ng/ml after repeated testing, indicating the complexity of the immune system and the diversity of individuals. Therefore, we suggest that the preoperative intracellular ATP value should be considered as the baseline for objective evaluation of the immune status in KT recipients. We found that the high intracellular ATP levels were significantly correlated with AR episodes in KT recipients. The average intracellular ATP level on the day AR occurred was obviously higher than that of the stable group. In addition, the intracellular ATP levels decreased significantly after the reversal of AR, implying that intracellular ATP monitoring could be applicable to assess the effectiveness of anti-rejection therapy. Contrary to our study, some previous studies failed to find an association between the intracellular ATP levels and AR episodes. De Paolis P et al. observed that only one recipient experienced an AR episode in the total 17 KT recipients with elevated intracellular ATP values [10]. Torio A et al. reported that only 12% of AR patients were included in the strong immune response zone [11]. Serban G et al. found that more than 40% of the samples showed moderate to high values during the first 3 months post-transplantation, and no correlation between ATP value and rejection was demonstrated [12]. However, all these studies set ATP level of 525 ng/ml at a single time point as an index to predict AR. In fact, there were very wide inter- and intra-individual variations in ATP values. The intracellular ATP also exhibited dynamic changes over time post-transplantation. We doubt whether the intracellular ATP level at a single time point is suitable to reflect the immune status,
which was also pointed out by Huskey J et al. [13] and Israeli M et al. [14,15]. We demonstrated that the trend of intracellular ATP increasing rather than a value at a single time point was more helpful to identify patients at higher risk of rejection. Several previous studies have shown that the high intracellular ATP level (≥ 525 ng/ml) at a single time point lacked both sensitivity and specificity for prediction of AR episodes [10,11,16]. In our large population study and the long-term serial testing scheme, we observed that patients with low intracellular ATP level (b 525 ng/ml) still had strong cellular immune responses and experienced AR episodes. In addition, longitudinal analysis revealed a significant increase in the intracellular ATP levels in AR patients, compared to that measured right before AR episodes. ROC analysis indicated that the sensitivity and specificity to predict AR episodes were 89.5% and 95.0% respectively, when the increase of intracellular ATP level was higher than 172.55 ng/ml. Otherwise, the intracellular ATP levels detected at 14, 21, and 28 days post-operation all failed to predict the subsequent AR episodes. The intracellular ATP level at 7 days posttransplantation showed the best sensitivity and specificity to identify AR patients at the early stage, but with lower sensitivity of 85.0% and specificity of 88.9%. These findings suggested that the trend of intracellular ATP increasing rather than ATP level at a single time point could predict the subsequent rejection timely and accurately. We also found that the peripheral blood trough tacrolimus concentration was not correlated with intracellular ATP level, consistent with previous studies [17–19]. It indicates that blood concentration of immunosuppressive agents could not reflect factually
Fig. 3. (A) ROC curves of intracellular ATP levels at day 7 of post-kidney transplantation in predicting acute rejection. (B) ROC analysis showing the increase of intracellular ATP level for predicting subsequent acute rejection in kidney transplant recipients. AUC, area under the ROC curve.
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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In conclusion, we demonstrated that the high intracellular ATP level had a significant correlation with AR episodes in KT recipients and the trend of intracellular ATP increasing rather than the intracellular ATP level at a single time point could be used to predict the subsequent rejection episodes timely and accurately. However, our data were collected from recipients during the early post-transplant period. Further studies with longer follow-up time, larger sample size, and multicenter participation should be performed to evaluate the clinical utility of the Cylex ImmuKnow Cell Function Assay in monitoring immune status in transplant recipients. Competing interests The authors declare that they have no competing interests. Acknowledgments
Fig. 4. Correlation between the tacrolimus concentration and intracellular ATP level in kidney transplant recipients(r = −0.078, P = 0.469).
the immunosuppressive degree in KT recipients, which was also supported by other studies using cytokine-based measures of T-cell function [20,21]. Based on our data, we suggest that intracellular ATP level could be used as an additional tool to evaluate the degree of immunosuppression for individual management of immunosuppressive agents. Although a prospective randomized clinical trial in liver transplant recipients has utilized modulated immunosuppressive protocols according to the intracellular ATP level [22], further studies are needed to estimate the utility of ImmuKnow assay in tapering immunosuppressive agents in KT recipients. We observed that intracellular ATP level can be extremely low in patients with remarkably low absolute CD4+ T cell count, which was in agreement with Xue F et al.'s study performed in liver graft recipients [23]. It was reported that the low intracellular ATP level was associated with a higher risk of infection, suggesting the presence of overimmunosuppression [24–26]. In our study, however, no clinical features of infection existed in 140 KT recipients. This suggests that it is inappropriate to classify the patients into over-immunosuppression group immediately when an extremely low intracellular ATP value is detected. The absolute CD4 + T cell count should be taken into account simultaneously.
Fig. 5. Correlation between the absolute CD4+ cell number and intracellular ATP level in kidney transplant recipients with CD4+ cell counts of b200/μl (r = 0.656, P b 0.001).
This work was supported by the grants from The National Basic Research Program of China (973 program) (No. 2009CB522407) and the National Nature Science Foundation of China (Nos. 30972947, 81100179). We also thank Zhen Wan, MD, for his constructive suggestions and editorial assistance. References [1] Nashan B, Moore R, Amlot P, Schmidt AG, Abeywickrama K, Soulillou JP. Randomised trial of basiliximab versus placebo for control of acute cellular rejection in renal allograft recipients. CHIB 201 International Study Group. Lancet 1997;350(9086):1193–8. [2] Choi BS, Shin MJ, Shin SJ, Kim YS, Choi YJ, Kim YS, et al. Clinical significance of an early protocol biopsy in living-donor renal transplantation: ten-year experience at a single center. Am J Transplant 2005;5(6):1354–60. [3] Kowalski RJ, Post DR, Mannon RB, Sebastian A, Wright HI, Sigle G, et al. Assessing relative risks of infection and rejection: a meta-analysis using an immune function assay. Transplantation 2006;82(5):663–8. [4] Kowalski R, Post D, Schneider MC, Britz J, Thomas J, Deierhoi M, et al. Immune cell function testing: an adjunct to therapeutic drug monitoring in transplant patient management. Clin Transplant 2003;17(2):77–88. [5] Cadillo-Chávez R, de Echegaray S, Santiago-Delpín EA, Rodríguez-Trinidad AT, Camacho-Carrazo B, Alfaro T, et al. Assessing the risk of infection and rejection in Hispanic renal transplant recipients by means of an adenosine triphosphate release assay. Transplant Proc 2006;38(3):918–20. [6] Cabrera R, Ararat M, Soldevila-Pico C, Dixon L, Pan JJ, Firpi R, et al. Using an immune functional assay to differentiate acute cellular rejection from recurrent hepatitis C in liver transplant patient. Liver Transpl 2009;15(2):216–22. [7] Dong JY, Yin H, Li RD, Ding GS, Fu ZR, Wu YM, et al. The relationship between adenosine triphosphate within CD4(+) T lymphocytes and acute rejection after liver transplantation. Clin Transplant 2011;25(3):E292–6. [8] Ling XT, Xiong J, Liang WH, Paul MS, Wu LW, Ju WQ, et al. Can immune cell function assay identify patients at risk of infection or rejection? a meta-analysis. Transplantation 2012;93(7):737–43. [9] Betjes MG. Immune cell dysfunction and inflammation in end-stage renal disease. Nat Rev Nephrol 2013;9(5):255–65. [10] De Paolis P, Favaro A, Piola A, Martini F, Cristiana G, Agrati C, et al. “ImmuKnow” to measurement of cell-mediated immunity in renal transplant recipients undergoing short-term evaluation. Transplant Proc 2011;43(4):1013–6. [11] Torio A, Fernandez EJ, Montes-Ares O, Guerra RM, Perez MA, Checa MD. Lack of association of immune cell function test with rejection in kidney transplantation. Transplant Proc 2011;43(6):2168–70. [12] Serban G, Whittaker V, Fan J, Liu Z, Manga K, Khan M, et al. Significance of immune cell function monitoring in renal transplantation after Thymoglobulin induction therapy. Hum Immunol 2009;70(11):882–90. [13] Huskey J, Gralla J, Wiseman AC. Single time point immune function assay (ImmuKnow) testing does not aid in the prediction of future opportunistic infections or acute rejection. Clin J Am Soc Nephrol 2011;6(2):423–9. [14] Israeli M, Yussim A, Mor E, Sredni B, Klein T. Preceeding the rejection: in search for a comprehensive post-transplant immune monitoring platform. Transpl Immunol 2007;18(1):7–12. [15] Israeli M, Ben-Gal T, Yaari V, Valdman A, Matz I, Medalion B, et al. Individualized immune monitoring of cardiac transplant recipients by noninvasive longitudinal cellular immunity tests. Transplantation 2010;89(8):968–76. [16] Hwang S, Kim KH, Song GW, Yu YD, Park GC, Kim KW, et al. Peritransplant monitoring of immune cell function in adult living donor liver transplantation. Transplant Proc 2010;42(7):2567–71. [17] Israeli M, Klein T, Sredni B, Avitzur Y, Mor E, Bar-Nathen N, et al. ImmuKnow: a new parameter in immune monitoring of pediatric liver transplantation recipients. Liver Transpl 2008;14(6):893–8. [18] Mizuno S, Hamada T, Nakatani K, Kishiwada M, Usui M, Sakurai H, et al. Monitoring peripheral blood CD4 + adenosine triphosphate activity after living donor liver transplantation: impact of combination assays of immune
Please cite this article as: Wang X-Z, et al, Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.10.008
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X.-Z. Wang et al. / Transplant Immunology xxx (2013) xxx–xxx
[19]
[20]
[21]
[22]
function and CYP3A5 genotype. J Hepatobiliary Pancreat Sci 2011;18(2):226–32 [discussion 32–4]. Sampson VB, Dunn SP, Rymeski B, Malatack J, Rong NH, Flynn L, et al. Failure of immunosuppressive drug levels to predict T-cell reactivity in pediatric transplant patients. J Pediatr Surg 2008;43(6):1134–41. Schulick RD, Weir MB, Miller MW, Cohen DJ, Bermas BL, Shearer GM. Longitudinal study of in vitro CD4+ T helper cell function in recently transplanted renal allograft patients undergoing tapering of their immunosuppressive drugs. Transplantation 1993;56(3):590–6. Ahmed M, Venkataraman R, Logar AJ, Rao AS, Bartley GP, Robert K, et al. Quantitation of immunosuppression by tacrolimus using flow cytometric analysis of interleukin-2 and interferon-gamma inhibition in CD8(−) and CD8(+) peripheral blood T cells. Ther Drug Monit 2001;23(4):354–62. Ravaioli M, Morelli C, Del Gaudio M, Zanello M, Berardi S, Ercolani G, et al. Immunosuppression monitoring by Cylex ImmuKnow test after liver trans-
[23]
[24]
[25]
[26]
plantation: preliminary results of randomized prospective trial [abstract]. Transpl Int 2011;24(Suppl. 2):227. Xue F, Zhang J, Han L, Li Q, Xu N, Zhou T, et al. Immune cell functional assay in monitoring of adult liver transplantation recipients with infection. Transplantation 2010;89(5):620–6. Husain S, Raza K, Pilewski JM, Zaldonis D, Crespo M, Toyoda Y, et al. Experience with immune monitoring in lung transplant recipients: correlation of low immune function with infection. Transplantation 2009;87(12):1852–7. Sanchez-Velasco P, Rodrigo E, Valero R, Ruiz JC, Fernandez-Fresnedo G, Lopez-Hoyos M, et al. Intracellular ATP concentrations of CD4 cells in kidney transplant patients with and without infection. Clin Transplant 2008;22(1):55–60. Zhou T, Xue F, Han LZ, Xi ZF, Li QG, Xu N, et al. Invasive fungal infection after liver transplantation: risk factors and significance of immune cell function monitoring. J Dig Dis 2011;12(6):467–75.
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