ImmuKnow: A New Parameter in Immune Monitoring of Pediatric Liver Transplantation Recipients

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1 LIVER TRANSPLANTATION 14: , 2008 ORIGINAL ARTICLE ImmuKnow: A New Parameter in Immune Monitoring of Pediatric Liver Transplantation Recipients Moshe Israeli, 1,3 * Tirza Klein, 1,5 * Benjamin Sredni, 3 Yaron Avitzur, 4,5 Eitan Mor, 2 Nathan Bar-Nathen, 2 Ran Steinberg, 4,5 Gabriel Dinari, 4,5 and Rivka Shapiro 4,5 1 Tissue Typing Laboratory and 2 Organ Transplantation Department, Rabin Medical Center, Petach-Tikva, Israel; 3 Cancer, AIDS & Immunology Research Institute, Goodman Faculty of Life-Sciences, Bar-Ilan University, Ramat-Gan, Israel; 4 Institute of Gastroenterology, Hepatology and Nutrition, Schneider Children s Medical Center of Israel, Petach-Tikva, Israel; and 5 Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Lifelong immunosuppression is mandatory for optimal graft and patient survival following liver transplantation. Nevertheless, graft rejection or numerous adverse events associated with overimmunosuppression or underimmunosuppression cannot be completely avoided. The ImmuKnow assay measures cell-mediated immunity and is able to discern between conditions of overimmunosuppression and underimmunosuppression. The aim of this study was to evaluate the ImmuKnow assay in the evaluation of the immune function in pediatric liver transplant recipients and to assess its correlation with the patients clinical and biochemical status. Eighty-nine whole blood samples were collected from 23 liver transplant recipients that were 1 to 18 years old. The net state of immune function was determined by the quantitative measurement of the intracellular adenosine 5-triphosphate level in CD4 lymphocytes after phytohemagglutinin stimulation. Comprehensive clinical data were correlated with the ImmuKnow assay results. In 23 of the 28 samples collected during clinical quiescence, ImmuKnow results were correlated with the clinical status, expressing the patient s moderate immune function. However, a correlation between measured therapeutic drug levels and clinical quiescence was found in only 18 of the 28 samples. In 6 patients who suffered from clinical complications, ImmuKnow measurements showed a wide range of deviations, expressing the unstable immunological status of these patients. In conclusion, the ImmuKnow assay correlates with the clinical status of livertransplanted children. It serves as a reliable and unique parameter of the cellular immune function. We conclude that the ImmuKnow assay, together with existing clinical tools, may allow for the immune monitoring of pediatric liver recipients. Liver Transpl 14: , AASLD. Received August 2, 2007; accepted December 6, In the last 2 decades, major progress has been achieved in the prolongation of graft and patient survival because of the development of effective and potent immunosuppressing medications. Nevertheless, major drawbacks of these medications are their toxic side effects, their nonspecific mechanism of action, and the absence of a reliable method of tailoring an individual treatment scheme according to the immune function at every time point. The currently used medication protocol for pediatric liver transplant recipients is mainly based on a calcineurin inhibitor (such as tacrolimus or cyclosporine) as a single drug, which is sometimes combined with Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP, adenosine 5-triphosphate; FK, tacrolimus; GGT, gamma glutamyl transpeptidase; GVHD, graft-versus-host disease; IS, immunosuppression; Ped Tx, pediatric transplantation; PHA, phytohemagglutinin; PTLD, posttransplant lymphoproliferative disease. This work was conducted as part of the fulfillment of a Ph.D. thesis performed by Moshe Israeli at Bar-Ilan University. This work was partly supported by the Safdié Institute for AIDS and Immunology Research, the Dave and Florence Muskovitz Chair in Cancer Research, the Frieda Stollman Cancer Memorial Fund, the Dorsha Wallman Cancer Research Endowment, and Teva-Medical, Ltd. *These authors contributed equally to this study. Address reprint requests to Moshe Israeli, M.Sc., Tissue Typing Laboratory, Rabin Medical Center (Beilinson Campus), Zabotinski Road, Petach-Tikva, Israel. Telephone: ; FAX: ; israelimoshe@yahoo.com or mosheis@clalit.org.il DOI /lt Published online in Wiley InterScience ( American Association for the Study of Liver Diseases.

2 894 ISRAELI ET AL. steroids or mycophenolate mofetil, depending on the patient s condition. Immunosuppression drugs are commonly administered as a lifelong treatment. Today, the monitoring of immunosuppressive treatment efficacy is based on the measurement of liver enzymes and liver function tests along with blood drug levels. The major challenge of the clinician is to avoid overimmunosuppression, which may cause hazardous side effects such as nephrotoxicity or malignancies, or underimmunosuppression, which may lead to a consequent graft loss. The goal of immunosuppression is to suppress the production or function of T cell lymphocytes. However, each patient has a unique pharmacokinetic behavior and distinctive characteristics of the immune system leading to an individual response to immunosuppression. Not all immunosuppression drug levels are measurable in routine clinical workup, and there is no tool enabling measurement of the additive effects of different factors on the immune function of the patient. Therefore, a reliable and comprehensive immune function test is essential for posttransplant patient immune monitoring, regardless of the immunosuppression regimen. The ImmuKnow assay, which has been approved by the Food and Drug Administration, belongs to the new generation of tests that directly measure the immune function of T cells and has been shown to reliably discern between immune profiles of overimmunosuppression and underimmunosuppression. 1 The ImmuKnow test has been reported to be a user-friendly, noninvasive, in vitro assay, and its effectiveness as an immune monitoring tool for organ transplantation recipients has been demonstrated. 2 Comprehensive reports have described its utilization in the immune monitoring of adult transplant recipients, 3 and studies smaller in scale have reported its efficacy in pediatric renal transplant recipients. 4 This study assesses the ImmuKnow assay s relevance and reliability in the immune monitoring of pediatric liver transplant recipients. PATIENTS AND METHODS Patient Samples Eighty-nine whole blood samples were collected from 23 liver transplant recipients with a mean interval time of 5.3 years after liver transplantation (range: 0-10 years) from either a cadaver or a living donor. The mean patient age during the study was 9.7 years (range: 1-18 years). Informed consent in writing was obtained from each patient s parent or legal guardian. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the review committee of the Schneider Children s Medical Center and the Israeli Ministry of Health. ImmuKnow Immune Function Assay The Food and Drug Administration approved Immu- Know assay directly measures cellular immune function by quantification of intracellular adenosine 5-triphosphate (ATP) levels in stimulated T cells. The assay s ability to discern between immune profiles of overimmunosuppression and underimmunosuppression has been reported in previous publications. Whole blood (100 L of a 1:4 dilution) is tested in quadruplicate with or without phytohemagglutinin (2.5 g/ml) overnight (15-18 hours in a 5% CO 2 incubator at 37 C). Anti-human CD4 monoclonal antibody coated magnetic particles (Dynal, Oslo, Norway) are added to immunoselect CD4 cells from both stimulated and nonstimulated wells. After the selected CD4 cells are washed on a strong magnet (Cylex catalog number 1050), a lysing reagent is added to release intracellular ATP. A luciferin/luciferase mixture is then added to the cell lysate. Within 10 minutes after the addition of the enzyme, the bioluminescent product is measured in a luminometer (Turner BioSystems, Sunnyvale, CA; Fig. 1). The amount of light emitted (emission maximum: 562 nm) is compared with a calibration curve generated with ATP calibrators (0, 1, 10, 100, and 1000 ng/ml). The concentration of ATP (ng/ml) in each sample is then calculated from the calibration curve with an Excelbased program. Replicate samples with a calculated percentage coefficient of variation greater than 20% are included in the calculation if a single value is within 3 standard deviations of the mean value of all wells. Clinical Evaluation Patient clinical status was monitored by pediatric hepatologists at our center (Schneider Children s Medical Center of Israel). Monitoring included a complete blood count, liver function tests, blood drug levels, and serology for cytomegalovirus and Epstein- Barr virus. Liver biopsy was performed according to the patient s clinical and biochemical status. Rejection was determined according to biopsy-proven pathological analysis. Stable patients were defined as patients who were in good clinical status and had normal liver function tests. Statistical Analysis Statistical analysis was performed with StatPac software, version 3.0 (StatPac, Inc., Minnesota). The matched pair t test was used in situations in which 2 measurements were taken for each examined factor. The independent group t test was used to determine if there was a difference between 2 means taken from different samples, assuming unequal variance. The F test was used to test if 2 population variances were equal or significantly different. RESULTS An Immune Response Zone for Pediatric Liver Recipients The median ImmuKnow level measured from 28 samples collected from stable pediatric liver recipients was 300 ng

3 IMMUKNOW: PEDIATRIC LIVER IMMUNE MONITORING 895 Immune Response (ATP ng/ml) (0 to 18 years of age) Strong Moderate Low Figure 1. Basic concept diagram of the innovative Food and Drug Administration approved ImmuKnow assay. Abbreviations: ATP, adenosine 5-triphosphate; PHA, phytohemagglutinin. of ATP/mL (standard deviation: 102). Fifty percent of the measurements were found between 251 and 387 ng of ATP/mL, between the 25th and 50th percentiles. We found no significant difference between the immune function of stable liver pediatric recipients over or under 12 years. (Fig. 2; P 0.3). The age of 12 years was selected to serve as the apparent boundary following the footsteps of the initial publication about Immu- Know testing in a pediatric patient population. 8 Correlation of ImmuKnow Results with Stable Immunological Function In 23 of the 28 samples collected during clinical quiescence, ImmuKnow results were correlated with the clinical status, expressing the patient s moderate immune function. The ImmuKnow correlation was defined as an ATP measurement within the moderate range (between 195 and 395 ng of ATP/mL). The average deviation from the overall measurement median was 57% (standard deviation: 28%). On the other hand, a correlation between measured therapeutic drug levels and clinical quiescence was found in only 18 of the 28 samples (Fig. 3). A drug-level correlation was defined as a tacrolimus trough level between 10 and 15 ng/ml in the first 3 postoperative months and between 5 and 8 ng/ml 3 months posttransplant. The average deviation from the overall measurement median was 53% (standard deviation: 30%, P 0.05, F test). Correlation of ImmuKnow Results with an Unstable Clinical Course 0 ImmuKnow showed a noteworthy correlation with the clinical course of patients suffering from clinical complications. In 6 of the unstable patients, ImmuKnow monitoring was performed 3 times or more (average: 4.3 samples per patient), and this allowed a reliable depiction of the dynamics of their immune status over 3 separate time points. Throughout the longitudinal follow-up, ImmuKnow results expressed extreme shifts of 42% to 6000% in these 6 patients suffering from an unstable clinical course (see Table 1). For example, Fig. 4 describes the clinical course of a 2-year-old male who was treated with pulse steroids for biopsy-proven acute rejection 6 months post-transplantation. Clinical and pathological symptoms were ambiguous, and this prevented the clinicians from determining whether they were the result of posttransplant lymphoproliferative disorder or graft-versus-host disease. Lowering of the immunosuppression dosage caused an event of acute rejection. A retrospective analysis with the ImmuKnow results that were collected throughout the entire period showed the correspondence of the shifting results with the unsteady clinical course. DISCUSSION Age < 12 Age > 12 n=19 n=9 Figure 2. Cellular immune responses of samples collected from stable pediatric liver transplant recipients do not show any significant age dependence within the pediatric patient population (P 0.3). Abbreviation: ATP, adenosine 5-triphosphate. Many traits and characteristics of the immune system in the pediatric age group are distinct from those in adults. Children have higher numbers of T and B cells, a higher CD4 /CD8 T cell ratio, and others. 6 It is possible that these disparities account for increased alloimmune responsiveness and may be to a certain extent responsible for the higher rates of acute rejection that have been observed in children. Improved monitoring and management of the immune function in pediatric recipients could assist in confronting these higher rates of graft rejection. 7,8 A previously published evaluation of the ImmuKnow assay for immune monitoring of pediatric renal recipients showed that children under the age of 12 had significantly lower immune function levels than adults. For adults, the ImmuKnow assay zones of strong, moderate, and low immune function correspond to 525, 225 to 525, and 225 ng of ATP/mL, respectively. In children younger than 12 years, it was found that the

4 896 ISRAELI ET AL. Figure 3. Clinical quiescence of pediatric liver recipients as reflected through ImmuKnow and FK level measurements. The zone of moderate immune function and proper drug level is darkened. ImmuKnow results reliably express a patient s stability. Abbreviations: ATP, adenosine 5-triphosphate; FK, tacrolimus. corresponding zones are 395, 175 to 395, and 175 ng of ATP/mL. The median value for healthy adults is 415 ng of ATP/mL, and in renal transplant recipient adults, it is 258. The median level among healthy children younger than 12 years is 295 ng of ATP/mL, and for stable pediatric renal transplant patients, it is 165 ng of ATP/mL. 5 No findings have been reported regarding ImmuKnow levels among pediatric liver transplant recipients. The immunosuppressive regimen is substantially different in liver transplant recipients compared to renal transplant recipients because of the physiologic and immunologic characteristics of the transplanted organ. The currently used medication protocol for pediatric liver transplantation recipients is mainly a single drug protocol based on tacrolimus or cyclosporine, which is seldom combined with steroids or mycophenolate mofetil, depending on the patient s condition. The differences in immunosuppression protocols between renal and liver transplant recipients support the importance of our study in ImmuKnowbased immune monitoring of liver-transplanted children. In this study, we found a significant difference between the immune function values measured from stable pediatric renal transplant recipients and the values measured from stable pediatric liver transplant recipients, the latter being significantly higher. The median ImmuKnow level measured among 50 stable pediatric renal recipients was 165 ng of ATP/mL (standard deviation: 119), whereas the median among 28 stable liver recipients was 300 ng/ml (standard deviation: 102, P 0.001; Fig. 5). 5 This finding could be attributed to the more aggressive immunosuppression used in renal transplant recipients because of the vulnerability of the kidney to rejection. Indeed, it is possible that pediatric renal transplant recipients may be overimmunosuppressed. This could explain the fact that infectious problems are one of the major complications in the long-term follow-up of renal transplant children. The reported increase in hospitalizations due to infections in the pediatric renal transplant populations would suggest that this is true. 9 In our study, we found that pediatric liver transplant recipient immune responses are age-independent. Within the pediatric patient population (age 12 years), we did not identify any age-related effect on the immune function. An identical finding has been reported for pediatric renal transplant recipients: the immune responses of stable pediatric renal recipients do not show age dependence either. 5 The ImmuKnow assay showed a better correlation than drug-level measurements in patients going through a stable posttransplant clinical course. Druglevel measurements did not reliably reflect the immunological quiescence of these patients but rather illustrated a misleading depiction of these patients as either overimmunosuppressed or underimmunosuppressed. The deviation of the ImmuKnow results from the expected moderate range was less pronounced and not as common as that of the drug-level measurement (18% outliers versus 29% outliers, respectively; see Fig. 3). The standard deviation of the ImmuKnow measurements from the expected range describing quiescence was found to be statistically different and noticeably narrower than that of the drug-level measurement (P 0.05, F test). Hence, the ImmuKnow monitoring provided more reliable evidence of the patient s stable clinical course than the drug-level measurements. This finding was anticipated because drug-level measurement indeed does not measure any trait or parameter of the patient s immune system. It solely provides important data about the chemical concentration of the drug in the peripheral blood circulation. The Immu- Know assay, by looking at the actual responsiveness of the patient s CD4 lymphocytes, enables a dependable evaluation of the patient s immune function. Six patients who suffered from clinical complications

5 IMMUKNOW: PEDIATRIC LIVER IMMUNE MONITORING 897 TABLE 1. Summary of Clinical Data and ImmuKnow Level Shifts in Patients Going Through a Unstable Clinical Course Patient Number Clinical Status Liver Enzymes Drug Level (FK) Subsequent ImmuKnow Results Overall Change 4 Chronic graft failure AST fold increase ALT 482 GGT 96 5 Acute rejection (IS AST fold increase withdrawal, PTLD) ALT 403 GGT IS modification AST fold increase ALT 43 GGT IS modification AST * fold increase following repeated infections ALT 50 GGT IS modification AST fold increase ALT 53 GGT Suprahepatic AST fold decrease abscess (1 month post-transplant) ALT 29 GGT 95 NOTE: The median interval between tests was 20 days. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; FK, tacrolimus; GGT, gamma glutamyl transpeptidase; IS, immunosuppression; PTLD, posttransplant lymphoproliferative disease. *Cyclosporine (Neoral). ImmuKnow results are presented as nanograms of adenosine 5-triphosphate per milliliter. Figure 4. Clinical course of a 2-year-old male liver transplant recipient. A retrospective analysis with the ImmuKnow results demonstrates the correspondence of the shifting ATP measurements with the unsteady clinical course. Abbreviations: ATP, adenosine 5-triphosphate; GVHD, graft-versus-host disease; PTLD, posttransplant lymphoproliferative disease. were monitored longitudinally over 3 or more Immu- Know tests. A retrospective analysis of the unstable clinical course of these 6 patients showed that the ImmuKnow results provide a reliable indication of the cellular immune status throughout the clinical episode. The assay provides an insight into the immunological events that may bring about clinical complications at the present time or in the future. It is possible that using this assay together with other diagnostic tools could warn the clinician of immunological imbalance or

6 898 ISRAELI ET AL. (0 to 18 years of age) (0 to 11 years of age) 600 Immune Response (ATP ng/ml) P< Strong 395 Moderate Stable Liver Ped Tx n=28 Stable Renal PedTX n=50 Low Figure 5. The cellular immune function of stable pediatric liver transplant recipients is significantly higher than that of renal transplant recipients. Abbreviations: ATP, adenosine 5-triphosphate; Ped Tx, pediatric transplantation. instability prior to the manifestation of clinical symptoms. This pilot study demonstrates that in patients with clinical and immunological quiescence and in patients suffering from a unstable clinical course, the Immu- Know assay correlates with the clinical status of liver transplant children. Therefore, this assay could serve as a reliable and unique parameter of the cellular immune function. Further studies are warranted in order to confirm our preliminary findings. REFERENCES 1. 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: 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: Israeli M, Yussim A, Mor E, Sredni B, Klein T. Preceding the rejection: in search for a comprehensive post-transplant immune monitoring platform. Transpl Immunol 2007;18: Gautam A, Morrissey PE, Brem AS, Fischer SA, Gohh RY, Yango AF, et al. Use of an immune function assay to monitor immunosuppression for treatment of post-transplant lymphoproliferative disorder. Pediatr Transplant 2006;10: Hooper E, Hawkins DM, Kowalski RJ, Post DR, Britz JA, Brooks KC, et al. Establishing pediatric immune response zones using the Cylex ImmuKnow assay. Clin Transplant 2005;19: Comans-Bitter WM, de Groot R, van den Beemd R, Neijens HJ, Hop WC, Groeneveld K, et al. Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations. J Pediatr 1997;130: Danovitch GM. Handbook of Kidney Transplantation. 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; Khera N, Janosky J, Zeevi A, Mazariegos G, Marcos A, Sindhi R. Persistent donor-specific alloreactivity may portend delayed liver rejection during drug minimization in children. Front Biosci 2007;12: Dharnidharka VR, Stablein DM, Harmon WE. Post-transplant infections now exceed acute rejection as cause for hospitalization: a report of the NAPRTCS. Am J Transplant 2004;4: