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Bone Marrow Transplantation (2010) 45, 527–533 & 2010 Macmillan Publishers Limited All rights reserved 0268-3369/10 $32.00

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ORIGINAL ARTICLE

Assessment of CD4 T-lymphocyte reactivity by the Cylex ImmuKnow assay in patients following allogeneic hematopoietic SCT B Gesundheit1, E Budowski1, M Israeli2, MY Shapira1, IB Resnick1, R Bringer1, Y Azar1, S Samuel1, L Dray1, A Amar3, D Kristt4 and R Or1 1 Department of Bone Marrow Transplantation, Cancer Immunotherapy & Immunobiology Research Center, Hadassah Hebrew University Medical Center, Jerusalem, Israel; 2CAIR Institute, Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel; 3Laboratory of Histocompatibility and Immunogenetics, Hadassah Hebrew University Medical Center, Jerusalem, Israel and 4 Molecular Pathology Program, Laboratory of Histocompatibility and Immunogenetics, Rabin Medical Center, Petach Tikvah, Israel

After allogeneic hematopoietic SCT (alloHSCT), immunosuppressed patients are susceptible to opportunistic infections, and uncontrolled function of the graft can result in GVHD. Accurate immune monitoring may help early detection and treatment of these severe complications. Between October 2005 and November 2007, a total of 170 blood samples were collected from 40 patients after alloHSCT in the Hadassah Hebrew University Medical Center and from 13 healthy controls. We utilized the Cylex ImmuKnow assay for CD4 ATP levels to compare known clinically immunocompromised vs immunocompetent patients after alloHSCT. We also compared the reconstitution of WBC count to the ImmuKnow results and clinical status. The patients’ clinical course correlated with the stratification of immune response established by the ImmuKnow assay for solid organ transplantation (immunocompetent vs immunocompromised), and this often differed from their WBC count. On the basis of our observations, we conclude that the ImmuKnow assay is a simple and fast immune-monitoring technique for patients undergoing alloHSCT, with potential to predict clinical course and facilitate prompt management of postHSCT complications. The assay should be evaluated prospectively in clinical trials. Bone Marrow Transplantation (2010) 45, 527–533; doi:10.1038/bmt.2009.182; published online 31 August 2009 Keywords: Immune reconstitution after HSCT; infectious complication; immune function and monitoring

Introduction Patients who undergo allogeneic hematopoietic SCT (alloHSCT) remain severely immunocompromised for a

Correspondence: Dr R Or, Hadassah University Hospital, Department of Bone Marrow Transplantation, Cancer Immunotherapy and Immunobiology Research Center, Hadassah Hebrew University Medical Center, P.O.B. 12000, Jerusalem, 91120, Israel. E-mail: [email protected] Received 26 January 2009; revised 15 May 2009; accepted 17 May 2009; published online 31 August 2009

prolonged period of time after the procedure and it can take several weeks or even months for the new hematopoietic graft to achieve normal blood counts,1 particularly after haploidentical HSCT.2 Functionally, as long as the new graft is immunologically immature, patients after HSCT are susceptible to opportunistic infections and increased risk of relapse of their underlying disease on the one hand,3 whereas on the other, uncontrolled function of the hematopoietic graft may cause acute or chronic GVHD, with high morbidity and mortality. Controlled graft-vs-host activity that mediates the suppression and eradication of the underlying malignancy by means of its associated graft-vs-tumor effect is, however, crucial for the long-term success of alloHSCT. Although the intensity of the conditioning regimen defines the success of the HSCT, it is detrimental to the immunological function of the donor cells, which is vital for the elimination of the recipient’s malignant cells. Therefore, accurate immune monitoring is essential for the assessment and appropriate management of immune-compromised patients after alloHSCT. Currently, post-alloHSCT laboratory tests that assess engraftment consist primarily of complete blood count (CBC) and chimerism studies,4 but there are no routine functional tests that evaluate the immune function of the new graft. The Cylex ImmuKnow assay, a laboratory test approved by the FDA in 2002, determines the cellular immune function in immunosuppressed patients by quantitatively measuring the intracellular ATP levels in PHAstimulated CD4 þ T-helper (Th) cells in whole blood. Although this is not a specific immunological response, studies of adult organ transplantation have shown that results of the ImmuKnow assay correlate with the clinical states of both over- or under- immune-suppressed patients. This has proven clinically useful in the management of these patients and for identifying the risks of graft failure or infections before the development of clinical signs and symptoms.5,6 These studies describe the contribution of the ImmuKnow assay in monitoring patients’ immune status during immunosuppressive treatment after kidney,7–9 liver,10 lung,11,12 small bowel13 and pancreas14,15 transplantation. Kowalski et al.16,17 have reviewed the role of the ImmuKnow assay in relation to solid organ transplantation

Cylex immune-reconstitution assay after allogeneic HSCT B Gesundheit et al

528 Table 1

Patients’ characteristics and their consecutive Cylex levels

ATP level / Months from BMT 1 Patient No. Diagnosis Age (years) Gender 1 SAA 18 M 2 AML 46 M AML 55 F 3 AML 47 M 4 5 NHL 40 F AML 10 M 6 TM M / 8 7 AML 50 M 8 F 9 3 TM 10 TM 4 F 11* MM 52 M / / 12 Ovarian Ca 50 F 13* ALL 4 F 14 CML 42 M 15* ALL 13 M 16* ALD 12 M 17 SAA 33 M F 18* AML 19* ALL 16 M F 41 20* AML M / 21 ALD 7 64 22 AML M F 23 AM 13 M 24 TM 4 25 HD 59 M 19 26* AML M 27 MDS 61 M / M 28 AML 54 29 ALL 28 M 30* MDS 57 M 31* Breast Ca 34 F 32 AML M 46 / 33 TM 20 M 34 MM 39 M 35* NHL 29 M / 36* Testicular Ca 30 M 59 / 37 AML M controls 13 healthy controls

2

3

4

5

6

7

8

9

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Outcome

12

at 12-13 months at 13 months, at 15 Relapse at 2 month at 18 month - high Remained low at 14 month

× at 12-13 months

× × × × × /

×

/ × ×

×

× × × × × × × × × × 1

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x x x x ×

Death of Progressive Disease at 9 month Death from complications of HSCT at 2 month Relapse at 13 month and Death from Relapse at 14 month Death from cGvHD after 3.5 years

× × × × × ×

Contiuos infections (months 0-12) Contiuos infections Death from Relapse at 6 month Death of Progressive Disease at 4 month Rejection at 4 month and Death due to infection at 8 month Death from Relapse at 4 month Rejection at 1 month and Death from Sepsis at 4 month Relapse at 5 month and Death from VOD at 6 month

× × × × × × × × × × × × ×

Death from Sepsis at 5 month Rerlapse at 1 monsth and Death from Sepsis at 2 month Death from Rejection at 2 month Death from Sepsis at 7 month Death from Sepsis at 3 month Death from MOF at 4 month Death from aGvHD at 2 month Death from Sepsis at 2 month Death from Sepsis at 2 month Death from Sepsis at 2 month Death from Sepsis at 2 month Death from MOF at 1 month Death from Relapse at 1 month

12

Cylex Levels from 3 Long-Term Follow-Up patients Patient No. 1 2 3

Diagnosis Age (years) Gender ATP level / Months from BMT - 6y post BMT 41 F MDS - 1y-2y post BMT, post 2y AML 35 F 2y-3y post BMT NHL 50 M

Abbreviations: ALD ¼ adrenoleukodystrophy; HD ¼ Hodgkin’s lymphoma; HSCT ¼ hematopoietic SCT; MDS ¼ myelodysplastic syndrome; MM ¼ multiple myeloma; MOF ¼ multi-organ failure; NHL ¼ non-Hodgkin’s lymphoma; SAA ¼ severe aplastic anemia; TM ¼ thalassemia major. * ¼ Haplo-identical donor for HSCT; kk ¼ very low ATP levels (o50 ng/ml); k ¼ low ATP levels (50–225 ng/ml), 2 ¼ normal ATP levels(225–525 ng/ml); m ¼ high ATP levels (4525 ng/ml).

in depth. Although many studies support the potential use of ImmuKnow assay in organ transplantation, its contribution remains controversial and therefore is not uniformly accepted. However, the role of ImmuKnow assay has not yet been studied for patients undergoing alloHSCT although immediate assessment of their immune function could have important relevance for their clinical management. The goal of this study was to evaluate the potential contribution of the ImmuKnow assay to the management of patients following HSCT. Our specific aims were to compare known clinically immunocompromised and immunocompetent patients by ImmuKnow assay, and to compare the reconstitution of their WBC with their clinical status and the results of the ImmuKnow assay. For some of the patients no correlation was found between their clinical immune status and ImmuKnow results, so two of these cases were further evaluated for the relation between their ImmuKnow levels and donor/recipient engraftment status, using a DNA STR assay to estimate chimerism levels. Bone Marrow Transplantation

Materials and methods Patients Between October 2005 and November 2007, a total of 170 blood samples were collected from 40 patients (28 men; 12 women) with various malignant (31) and nonmalignant (9) diseases, who underwent alloHSCT at the Hadassah Hebrew University Medical Center following engraftment of their donor hematopoietic stem cells (Table 1). Twelve of these patients received haploidentical transplants, which are known to cause severe immunosuppression and infectious complications. The median age of the patients was 34 years (range 3–64 years). From 22 patients, 4 or more blood samples were taken, 3 samples were taken from 7 patients and 2 samples from 11 patients, resulting in an average of 3.9 (2–9) samples per patient. Thirty-seven patients were first tested shortly after engraftment followed by regular tests up to 12 months after alloHSCT. For assessment of immune reconstitution several years after alloHSCT, three individuals were tested following an


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average interval of 3 (range 1–5.5) years post-alloHSCT and continued during a average follow-up of 4 (range 2–6.5) years along with other routine tests after alloHSCT including CBC and chimerism studies. Thirteen healthy individuals were tested as a normal control group.

response were stratified as follows: low (0–225 ng/ml), normal (225–525 ng/ml) or high (4525 ng/ml).19

Cylex ImmuKnow assay methodology The FDA-approved ImmuKnow assay measures cellular immune function directly by quantification of intracellular ATP levels in stimulated T cells (Figure 1). The assay’s ability to discern between immune profiles of over- and under-immunosuppression has been reported in previous publications in the clinical context of organ transplantations.18 The methodology is as follows: Whole blood, collected in sodium heparin tubes (100 ml of a 1:4 dilution), is incubated in quadruplicate with or without PHA mitogen (2.5 mg/ml) for 15–18 h in a 5% CO2 incubator at 37 1C. Anti-human CD4 monoclonal Ab-coated magnetic particles (Dynal, Oslo, Norway) are added to immunoselected CD4 cells from both the stimulated and nonstimulated wells. After washing the selected CD4 cells on a strong magnet (Cylex Cat. 1050) a lysing reagent is added to release intracellular ATP. A luciferin/luciferase mixture is then added to the cell lysate. Within 10 min after addition of the enzyme, the bioluminescent product is measured in a luminometer (Turner BioSystems, Sunnyvale, CA, USA). 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 using an Excel-based 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 s.d. of the mean value of all wells.

The results of the ImmuKnow assay tested on our 40 patients after alloHSCT were, on the whole, in keeping with their clinical course and correlated with the established stratification of the immune response known from the literature on solid organ transplantations, as described under the heading, ‘Stratification of immune response in solid organ transplantations according to ImmuKnow assay’ in the Materials and methods section above. Based on their ImmuKnow assay levels, 12 patients who achieved normal immune reconstitution 7 months following HSCT were defined as immunocompetent (Table 1, Figure 2a). They engrafted within the first month following transplantation, achieving normal ranges of WBC, but their ATP levels reflected only gradual immune reconstitution, with moderate ATP concentrations being reached 6 months later. There was no significant correlation between WBC and ATP levels (r ¼ 0.65). Overall survival for the immunocompetent group was 83% (10 out of 12 patients) at a follow-up of 13 months. The results of one 18-year old patient with severe aplastic anemia (SAA) after alloHSCT from his HLA-matched sibling are representative of the immunocompetent patients’ group (Figure 2b). He was not treated with chemotherapy before his alloHSCT. His chimerism status post transplant with 80–96% donor cells was quantified by STR studies, but the ImmuKnow assay reflects normal immune function. At day þ 147 post-alloHSCT the chimerism status showed 80% donor, but his ATP levels reached normal levels of 249 ng/ml (normal ¼ 225–525 ng/ml) and he did not experience any infectious complications during his post-alloHSCT course. In regard to our 25 immunocompromised patients, as defined by their low ImmuKnow assay ATP levels, recurrent infections and rejection or relapse of the underlying disease were observed (Table 1; Figure 3a). The ImmuKnow assay showed that normal immune reconstitution was not achieved even during transient normalization

Stratification of Immune Response in Solid Organ Transplantations according to ImmuKnow assay From studies on solid organ transplantations, the immune response was assessed by the levels of ATP activity measured with ImmuKnow assay. These levels of immune

• Whole blood • Sodium heparin • Maintain at room temperature • Test within 30 hrs

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Incubate 15-18 hours

LYMPHOCYTE STIMULATION w/ PHA ATP

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Figure 1

MAGNETIC SEPARATION OF CD4 CELLS

MEASURE LIGHT INTENSITY

Technical Methodology of the Cylex ImmuKnow Assay. A schematic diagram describing the laboratory protocol of the Cylex ImmuKnow assay. Bone Marrow Transplantation


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Results of Cylex ImmuKnow Assay and WBC in immunocompetent patients after alloHSCT: (a) For the 12 immuno-competent patients, WBC returned to normal within one month after HSCT, but the Cylex levels showed normal ranges that reflected restored immunocompetence only after an additional 6 months. Ten of the 12 immunocompetent patients (83%) survived at a follow-up of 13 months. (b) Representative results of Cylex ImmuKnow Assay from one 18-year old patient with severe aplastic anemia who underwent HSCT and engrafted at day 12 with normal blood counts, while his Cylex ImmuKnow assay showed normal values at day +147. Chimerism studies post HSCT showed donor status between 80–96%. His post-HSCT course was uneventful with no infectious complications.

of the WBC count; this reflects a lack of significant correlation between WBC and ATP (r ¼ 0.60). Their generally decreased ATP levels predicted these patients’ clinical complications more accurately than their CBC. Overall survival for the immunocompromised group was 12% (3 out of 25 patients) at a follow-up of 12 months. The results of one 41-year-old patient with AML after matched alloHSCT are representative of the immunocompromised patients’ group (Figure 3b). Despite the presence of 100% donor hematopoietic stem cells according to repeated STR studies, functionally the patient’s newly transplanted immune system was extremely compromised, as documented by her low ATP levels. This patient subsequently suffered from recurrent infections and died of sepsis at day þ 228 post-alloHSCT. Bone Marrow Transplantation

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Figure 3 Results of Cylex ImmuKnow Assay and WBC in immunocompromised patients after alloHSCT. (a) In our 25 immuno-compromised patients, normal ranges of WBC were seen within one month, but Cylex ImmuKnow assay did not show moderate ATP ranges at any point of time, thereby reflecting prolonged immuno-suppression and constituting a more accurate marker of clinical complications than the WBC. Only 3 of the 25 immuno-suppressed patients (12%) survived 12 months’ follow-up after HSCT. (b) Representative results of Cylex ImmuKnow assay from one 41year old patient with AML. After her 2nd HSCT she engrafted at day 16 according to her neutrophil counts, but had consistently low ATP levels. Chimerism studies post HSCT showed 100% donor status. She suffered from recurrent infections and died on day +228 from sepsis.

Regarding the type of donors, it is worth mentioning that of our 12 patients who underwent alloHSCT from haploidentical donors, 10 (83%) experienced severe immunosuppression according to the ImmuKnow assay, and only 2 (17%) regained immunocompetence (Table 1, haploidentical HSCT are marked with asterisk). Of our 21 patients transplanted from matched related donors, 9 (43%) remained immunosuppressed and 12 (57%) regained immunocompetence. Of our six patients transplanted from matched unrelated donors (MUD), three (50%) became immunocompetent and three remained immunosuppressed. The ATP levels of the three long-term follow-up patients measured by ImmuKnow assay at an average interval of 3 (range 1–5.5) years after alloHSCT were normal or higher than normal (Table 1). Regarding GVHD, no significant increase was found in the ATP levels of patients who


531 Patients with GvHD (n=21) 700

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Figure 4 Results of Cylex ImmuKnow Assay for 21 patients with acute and/or chronic GVHD.

developed only chronic (3), only acute (13) or both acute and chronic (5) GVHD; also, there was no significant difference in ATP levels between the immunocompromised and immunocompetent patients with acute (162.22 ng/ml; s.d. 121.957) and chronic (133.63 ng/ml; s.d. 158.535) GVHD (Figure 4). As for our control group, we performed one ImmuKnow assay on each of our 13 healthy volunteers, and the results obtained were compatible with the normal levels (225–525 ng/ml) of immune function established in the literature for the ImmuKnow assay. Samples from different volunteers were used as controls for each patient assay. In the future, it might be preferable to use the same individual volunteer for the same sequential patient.

Discussion This is the first time that the ImmuKnow assay, a simple and fast monitoring technique for the immune function has been investigated in patients undergoing alloHSCT. Our study was based on 170 blood samples from 40 patients during the first 12 months following alloHSCT, 3 long-term follow-up patients tested at an average interval of 3 (1–5.5) years after alloHSCT and 13 healthy individuals. Prolonged immune suppression after alloHSCT has major clinical implications, and our research provides the first analysis of immune reconstitution dynamics based on the ATP levels in CD4 cells measured by ImmuKnow assay, during the first 12 months following alloHSCT. ImmuKnow assay is currently being used on recipients of solid organ transplants to monitor their immune response and, consequently, to indicate the adjustments required to attain optimum treatment. The use of the ImmuKnow assay for patients after alloHSCT, however, constitutes a new aspect in relation to this test. Although in patients undergoing organ transplantation the immune system is pharmacologically suppressed to achieve tolerance, in HSCT patients the immune system is, in fact, the transplanted organ, and our use of the ImmuKnow assay was to measure the gradually improving function of the new graft. Our findings qualify the test as a fast, sensitive and simple means of monitoring the immune function of patients after alloHSCT both by analysis of

their ATP levels and by correlating these findings with their WBC and chimerism studies. It is interesting to note that the results of the repeated ImmuKnow assays carried out on our 40 patients during the 12 months after alloHSCT (Table 1) correlated with the established stratification of the immune response defined in the literature on solid organ transplantations. Also, more often than not they correlated with the patients’ clinical condition and they were predictive to some extent of the complications our patients developed, their clinical outcome and survival. During the first month after HSCT the vast majority of patients were extremely immune-suppressed; during the first 2 months after alloHSCT no statistically significant difference was observed between the groups of immunocompetent and immunocompromised patients; from the second to the fifth month, extremely low ImmuKnow levels (o50 ng/ml) were indicative of very poor clinical outcome, whereas normal ImmuKnow levels from the seventh month onwards were predictive of good outcome. Our three longterm follow-up patients tested at an average interval of 3 (range 1–5.5) years after alloHSCT, had normal or increased ImmuKnow levels in keeping with their fully recovered immune system. The normal Cylex levels of our 13 healthy controls were important because they confirmed the normal ImmuKnow measurements obtained from our HSCT patients. The ImmuKnow levels of patients who underwent alloHSCT from haploidentical donors (Table 1, marked with asterisk) showed that they experienced significantly deeper immune suppression and poorer clinical outcome than patients transplanted from matched related or unrelated donors; these findings are in keeping with the well-known severe immune suppression and consecutively very poor outcome of haploidentical alloHSCT. Our group was too small and their diagnostic and preHSCT therapeutic background too diverse to analyze significant differences statistically between patients undergoing myeloablative vs nonmyeloablative HSCT conditioning regimens.20 Low levels of ATP shown by the ImmuKnow assay are predictive of recurrent infections, relapse and poor prognosis due to the suboptimal immunological function of the graft even with a reasonable WBC, whereas normal levels shown by ImmuKnow assay predict few clinical complications even with a reduced WBC. WBC per se is not a reliable measure of immunocompetence because the majority of these cells are likely to be neutrophils. Most interestingly, in our two representative patients for the immune competent and immune compromised groups, respectively (Figures 2b and 3b), the results of the ImmuKnow assay document a most interesting and important clinical contribution to the chimerism studies: normal ATP levels even in the absence of full donor status demonstrate a satisfactory recovery of immune status (2b), whereas decreased ATP levels even in the presence of full donor chimerism reflect reduced immune function leading to recurrent infections (3b). Due to the small number of patients studied, and the fact that many of our patients died, relapsed, were lost to follow-up or did not consent to donate blood for our research due to clinical complications, we were unable to Bone Marrow Transplantation


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take blood samples at planned time points following transplantation. Therefore, it is difficult to establish a clear cause–effect relationship. Even so, we trust that these preliminary results may provide new insights and directions for future research in the area of immune function after HSCT. Our findings are compatible with the previously reported finding by R Kowalski et al.5 that low ATP seems to indicate a higher risk of infection, whereas a higher than normal ATP level is compatible with rejection of the graft in the context of organ donation. For HSCT patients, high ATP levels might theoretically signal the risk of GVHD; however, in our study we could not confirm a correlation between acute and/or chronic GVHD and increased ATP levels by ImmuKnow assay. Indeed, our findings suggest that there may be an association between low ImmuKnow levels and GVHD (Figure 4); this is in keeping with the known connection between GVHD and immunoincompetence after alloHSCT.22 More intensive, consecutive ImmuKnow assays at shorter intervals may perhaps improve the assessment and monitoring of GVHD, and perhaps even predict the onset of the disease; this is a possibility worth future investigation. All our patients engrafted after alloHSCT. A future study to assess patients who did not engraft and to compare their ImmuKnow assays with their pretransplant levels might also be of interest. Clinically, the ImmuKnow assay may prove helpful in determining the immune function of the graft collected from the donor for alloHSCT, and in evaluating the biological potential of cord blood for alloHSCT. Currently, no pharmacological agents are available to improve the immune function of severely compromised patients. In our preliminary study of the contribution of ImmuKnow assay to patients undergoing alloHSCT, we focused on their sequential ATP levels in correlation with their clinical course. Data from future, larger studies relating to the patients’ diagnosis, previous treatments and various conditioning regimens, along with a comparison with other immune function tests after alloHSCT23 may help to define and establish the clinical merit of this simple and fast assay as a standard test for optimal monitoring of the immune function of patients following alloHSCT.

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Conflict of interest 14

The authors declare no conflict of interest.

Acknowledgements This study was partly supported by Teva Medical Ltd. We thank Mrs Sharon Pantel-Bakst and Dr Mario Baras for data analysis and statistical evaluation; Mrs Lilly Schaechter, Mrs Yifat Saydoff and Mr Jaakov Bar for their help with the preparation of the article.

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