Cellular immune function monitoring after allogeneic haematopoietic cell transplantation: evaluation of a new assay

Authors


Correspondence: M. Israeli, home address: 5 Odem Street, Modiin 71725, Israel; laboratory address: Tissue Typing Laboratory, Rabin Medical Center (Beilinson campus), Zabotinski Road, Petach-Tikva 49100, Israel

E-mail: israelimoshe@yahoo.com; mosheis@clalit.org.il

Summary

Managing the patient's immune system after haematopoietic cell transplantation (HCT) is a challenge, mainly in the unstable period immediately after the transplant. Currently there is no standardized non-invasive diagnostic tool for the evaluation of immunological complications such as graft-versus-host disease (GVHD) and for managing the cellular immune function of the transplant recipient. The ImmuKnow assay for cellular immune function monitoring has been incorporated successfully into the clinical follow-up routine of solid organ transplant recipients. This study aims to explore the relevance and potential contribution of immune monitoring using the assay in the setting of HCT. We found that ImmuKnow-level measurement can distinguish between states of immune function quiescence and between events of acute GVHD. ImmuKnow levels were significantly higher in patients going through GVHD than the levels measured for the same patients during immunological stability. Moreover, we demonstrate a patient case where longitudinal monitoring using the ImmuKnow assay provided a trustworthy depiction of the patient's cellular immune function post-HCT. In conclusion, we provide evidence for the potential contribution of the ImmuKnow assay for longitudinal individualized cellular immune function monitoring of patients following HCT. Further studies are necessary in order to establish the optimal practice for utilizing the assay for this purpose.

Introduction

Acute graft-versus-host disease (aGVHD) is an undesirable complication which may occur following allogeneic haematopoietic cell transplantation (HCT) as a result of the interaction between the donor's immune cells, namely alloreactive T cells, and the cells of the recipient. Multiple publications have investigated and described the pathophysiology of aGVHD [1]. A noticeable consensus among the various publications indicates the essential role of the cellular immune function in the pathophysiology of aGVHD, namely alloreactive T cells [2, 3]. Therefore, in order to diagnose aGVHD in a timely manner and to manage the patient's therapy accordingly, it would be beneficial for the clinician to have a diagnostic tool for evaluation of the cellular immune function.

Currently, diagnosis of aGVHD is based mainly on subjective clinical judgement, where the clinician relies on coincidental symptoms which could provide indirect evidence of occurrence of aGVHD [4], preferably together with typical target-organ histology. There is no straightforward non-invasive diagnostic method at the disposal of the clinician which can provide a reliable evaluation of the cellular immune function of the patient.

The ImmuKnow assay (Cyclex, Inc., Columbia, MD, USA) has been used in our centre for more than 6 years for cellular immune monitoring of patients after solid organ transplantation. It identifies states of over- or underimmune suppression, thereby facilitating safer immunosuppressive patient treatment [5]. Its main utility is for monitoring the cellular immune function of an immunosuppressed patient [6], hence its potential relevance for evaluating the cellular immune function of the HCT recipient who is under threat of a potential outbreak of aGVHD. The aim of this paper is to describe our experience in using the assay for immune monitoring of patients after HCT.

Patients and methods

Patients

Seventy-eight whole blood samples were collected from 22 adult patients post-allogeneic HCT between September 2007 and November 2010. Patients who presented clinical symptoms and signs suggestive for GVHD were enrolled to this study. Patient characteristics are detailed in Table 1. ImmuKnow testing was performed once or twice to identify its correlation with the suggested clinical diagnosis of GVHD. In selected patients, further testing was carried out longitudinally in order to evaluate its correlation with the long-term clinical course. Sample collection schedule is detailed in Table 2. This study conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local Institutional Review Board (IRB) of the Rabin Medical Center. All participating patients provided their written informed consent.

Table 1. Patient and sample characteristics
Characteristics of patientsNumber of patients (%) or median (range)
  1. HLA: human leucocyte antigen; HPC: haematopoietic progenitors; HCT: haematopoietic cell transplantation; PBSC: peripheral blood stem cells.
Number of patients22
Median age (range)45 (23–69)
Male : female10:12 (45:55)
Median day of engraftment (range)12 (10–38)
Diagnosis
Acute myeloid leukaemia15 (68)
Acute lymphoblastic leukaemia3 (14)
Non-Hodgkin's lymphoma4 (18)
HLA match level
10/10 allele match16 (73)
9/10 allele match5 (22)
4/6 allele matched cord-blood1 (5)
HPC source
PBSC from sibling or related donor6 (27)
PBSC from an unrelated donor15 (68)
Unrelated double cord-blood1 (5)
Conditioning
Myeloablative conditioning20 (90)
Reduced-intensity conditioning2 (10)
Number of ImmuKnow samples per patient
17 (32)
2–37 (32)
4 or more8 (36)
Median day post-HCT of ImmuKnow test (range)48 (12–228)
Table 2. Detailed schedule of serial ImmuKnow tests
Patient no.Graft sourceDay of engraftmentDay post-HCT
ATP level category
  1. ↓↓Very low adenosine triphosphate (ATP) levels (<50 ng/ml); ↓low ATP levels (50–225 ng/ml); ↔normal ATP levels (225–525 ng/ml); ↑high ATP levels (>525 ng/ml). Retransplant. HCT: haematopoietic cell transplantation; MUD: matched unrelated donor; CB: cord blood.
 110/10 MUD121426         
         
 2Sibling12104119474        
↓↓        
 3Double CB3875          
          
 4Sibling, MUD121425334754744654   
↓↓   
 510/10 MUD144874         
         
 69/10 MUD123852         
↓↓         
 710/10 MUD1137          
          
 89/10 MUD1130          
          
 910/10 MUD1127325390102119126165196222252
↓↓↓↓
10Sibling12121825344867116167201  
↓↓  
11Sibling1320          
          
129/10 MUD1213264863151180194200   
   
1310/10 MUD111320123172228      
      
1410/10 MUD131421102151       
       
1510/10 MUD142074117123       
       
16Sibling122343         
         
179/10 related122781116130145      
      
1810/10 MUD12195368        
        
1910/10 MUD11142026        
        
2010/10 MUD1368          
          
219/10 MUD19103          
          
2210/10 MUD1012          
          

Clinical protocol

Conditioning regimens were defined as myoablative conditioning (MAC) and reduced intensity conditioning (RIC) using the criteria defined by the European Group for Blood and Marrow Transplantation, as described by Bacigalupo et al. [7]. All patients were given GVHD prophylaxis, which consisted of a calcineurin inhibitor and a short course of methotrexate. Anti-thymocyte globulin (ATG) was given to patients transplanted from an unrelated donor or patients transplanted from a mismatched related donor. Acute GVHD was diagnosed and graded according to defined criteria as described by Przepiorka et al. [8]. Wherever possible, patients presented with GVHD symptoms underwent organ-specific biopsy to confirm the diagnosis. Patients with grade II acute GVHD or higher were treated by a calcineurin inhibitor and high-dose methylprednisolone.

Cellular immune function monitoring

The ImmuKnow assay measures cellular immune function directly by quantification of intracellular adenosine triphosphate (ATP) levels in stimulated T cells.

Whole blood (100 μl of a 1:4 dilution) is tested in quadruplicate with or without phytohaemagglutinin (PHA) (2·5 μg/ml) overnight (15–18 h in a 5% CO2 incubator at 37°C). Anti-human CD4 monoclonal antibody-coated magnetic particles (Dynal, Oslo, Norway) are added to immunoselect CD4 cells from both the stimulated and non-stimulated 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 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 was within 3 standard deviations (s.d.) of the mean value of all wells.

Statistical analysis

Statistical analysis was performed using GraphPad statistical software (La Jolla, CA, USA). The two sided paired-sample t-test was used in situations where two measurements were taken for each examined factor. The two-sided independent-sample t-test was used to determine if there is a difference between two means taken from different samples, assuming unequal variance. The level of significance was set at P < 0·05.

Results

An immune response zone for HCT recipients

The median ImmuKnow level measured among 66 samples collected from HCT recipients during immunological quiescence was 183 ng ATP/ml (s.d. ± 151, range 11–651). Half the measurements were found in the range between 127 and 311 ng ATP/ml, between the 25th and 75th percentiles (Fig. 1). The median ImmuKnow level of patients who received a fully matched graft was higher than that of patients who received a non-fully matched graft: 213 ± 160 and 135 ± 75 ng ATP/ml, respectively (P < 0·05). No correlation was found between the ImmuKnow results and the white blood cell (WBC) or absolute lymphocyte counts (Fig. 2). Two events of disease progression occurred during the course of this study while the two patients were under ImmuKnow monitoring surveillance. These events were not reflected by any change in the ImmuKnow levels.

Figure 1.

ImmuKnow measurements grouped according to clinical patient status on the day of sample collection. Immune response measurements collected during events of acute graft-versus-host disease (aGVHD) were significantly higher than those collected during clinical quiescence. Horizontal line describes median ImmuKnow level in each group; vertical lines describe standard deviation and grey boxes describe the range of the central 50% of the measurement in that group. Previously established high moderate and low response zones are depicted by dashed line.

Figure 2.

Limited correlation between Immuknow levels and haematological parameters. Correlation between ImmuKnow levels and (a) white blood cell or (b) absolute lymphocyte counts was not statistically significant.

ImmuKnow measurements correlation with aGVHD episodes

Ten events of aGVHD grade II or higher were recorded. Precise GVHD clinical aspects are detailed in Table 3. The median ImmuKnow assay level measured among 10 samples collected during episodes of acute GVHD was 458 ng ATP/ml (s.d. ± 134, range 186–588). Fifty per cent of the measurements were found in the range between 388 and 524 ng ATP/ml, between the 25th and 75th percentiles. Thus, ImmuKnow levels during events of acute GVHD were significantly higher than those measured during immunological stability (P < 0·05) (Fig. 1).

Table 3. Clinical characteristics of GvHD episodes
Patient no. (event no.)GVHD gradeOrgans involved (grade)Biopsy provenDays between GVHD occurrence and Immuknow test
  1. GVHD: graft-versus-host disease.
 22Gut (2)Yes7
 4 (1st)2Skin (2)Yes1
Gut (2)
 4 (2nd)4Skin (2)Yes4
Gut (3)
 93Skin (1)Yes9
Gut (3)
10 (1st)3Gut (3)No4
10 (2nd)2Gut (2)No9
122Skin (1)Yes6
Gut (2)
132Gut (2)Yes1
153Liver (2)No5
183Gut (3)No1

During the 10 recorded episodes of acute GVHD, ImmuKnow levels were higher by an average of 258% above the most proximate ImmuKnow measurement recorded for that individual patient during immunological quiescence (range 130–455%, s.d. ± 99%) (Fig. 3).

Figure 3.

Changes in ImmuKnow levels reliably illustrate the dynamics of the immune response. ImmuKnow levels increase significantly in parallel with the manifestation of acute graft-versus-host disease (aGVHD) (P < 0·05, paired t-test). White bars indicate the median ImmuKnow levels before and during events of aGVHD. Vertical lines depict standard deviation.

Example: longitudinal immune monitoring of an individual patient

We demonstrate an example for clinical utilization of the ImmuKnow assay for longitudinal cellular immune function monitoring of an HCT recipient. A 38-year-old male with acute myeloid leukaemia (AML) was given an allograft from an unrelated fully human leucocyte antigen (HLA)-matched donor. On several occasions after the transplantation the patient experienced severe diarrhoea, thereby indicating the possible onset of GVHD. No other parameters were notable in verifying the accuracy of GVHD diagnosis. Serial ImmuKnow testing was carried out in order to evaluate the net state of cellular immune function of the patient.

Twenty-seven days post-transplant, after the appearance of severe diarrhoea, high ImmuKnow levels indicated significant undersuppression of the cellular immune function, confirming the possibility that the patient was developing GVHD. This diagnosis was not in correlation with results of blood counts or of blood drug levels. Immunosuppression dosage was increased in order to repress GVHD outbreak and, indeed, the patient's condition improved and ImmuKnow levels decreased to the ‘low’ zone below 225 ng ATP/ml. Additional events of diarrhoea were not regarded as GVHD, due to low ImmuKnow levels that accompanied these events. Some of these later incidents of diarrhoea were confirmed retrospectively as not related to GVHD after we learned that the patient's spouse had suffered from infectious diarrhoea at the same time. The only episode that was diagnosed and confirmed later by biopsy as GVHD was in accordance with the ImmuKnow indication. Furthermore, ImmuKnow measurements played a crucial part in the determination of additional events as non-GVHD, thus evading unnecessary medication (Fig. 4), in full accordance with the clinical diagnosis of infectious diarrhoea.

Figure 4.

Serial ImmuKnow immune function monitoring of an allogeneic haematopoietic cell transplantation (HCT) recipient. High ImmuKnow levels reliably distinguished an acute graft-versus-host disease (aGVHD) episode from other clinical events.

Discussion

Since the introduction of allogeneic HCT, aGVHD has been recognized as a side effect with potential undesirable outcome, presenting an immunological challenge to the transplanted patient and the transplant physician [9, 10]. Substantial research effort has been directed at enhancing the ability to predict and treat aGVHD [11, 12]. The essential role of the cellular alloimmune response in the generation of aGVHD is broadly recognized [13, 14], hence T cells and antigen-presenting cells serve as a key therapeutic target in treatment of aGVHD [15-17]. Despite comprehension of the cellular alloresponse as a crucial component of aGVHD occurrence, there are no available diagnostic tools which provide a reliable depiction of the precarious cellular immune function of the patient before and during the development of aGVHD. Various recent publications have attempted to describe a panel of proteomic biomarkers which may be predictive of an aGVHD manifestation [18, 19]. Others have sought a biomarker for reliable ‘real-time’ diagnosis and prognosis of aGVHD [20-22] or for prediction of treatment outcome [23].

Despite the promising correlative and predictive value of the proteomic biomarkers described in these recent publications, several disadvantages limit their potential to become a widespread diagnostic method for aGVHD management. The sought-after immune monitoring tool must be able to provide the clinician with a broad depiction of the patient's multi-faceted alloimmune response, which could comprise: innate immune response inducing cross-reactive T cells, cytokine secretion and activation of cytotoxic T cells. None of the proposed biomarker methods are able to meet the diagnostic requirements while answering the basic technical requirements: an assay that is standardized, reproducible, cost-effective, easy and intuitive to perform and to interpret its results.

Approximately 10 years ago the ImmuKnow assay was established for assessing the global cellular immune function in patients receiving immunosuppressive therapy [24]. It was introduced for cellular immune function monitoring in an immunosuppressed population, namely organ transplant recipients, based on a meta-analysis of over 500 solid organ transplant recipients, which proved that this assay has a substantial predictive value [25]. It has gradually become used worldwide for monitoring adult and paediatric transplant patients in diverse solid organ transplant settings; it is internally standardized among all laboratories and is simple to perform and to interpret. In our medical centre the assay has been used since 2004, and we have also published evidence of its efficacy in various clinical settings, for adult or paediatric patients [26, 27]. Our most recent data indicate that the optimal contribution of the ImmuKnow assay for patient immune function monitoring and management is reached when the assay is used for longitudinal monitoring. Personalized longitudinal follow-up based on serial immune function monitoring using the ImmuKnow assay appears to be useful for understanding the unique immune function dynamics of individual patients [28, 29]. The success in incorporating the ImmuKnow assay into the patient follow-up routine after solid-organ transplantation motivated us to investigate the relevance and potential contribution of ImmuKnow immune function monitoring in patients after HCT.

We found that the median ImmuKnow level of the HCT recipients while in a quiescent immunological state is 183 ng ATP/ml, a level which is in the ‘low zone’ which was established for organ-transplant recipients [25]. This finding was anticipated, as patients in the immediate post-HCT period are recognized as being susceptible to suffer from infections and from consequences of incompetent immune function [30, 31].

The low degree of correlation between the ImmuKnow levels and patient cell counts is in agreement with previous publications which have shown similar findings in the setting of solid-organ transplantation [28, 32].

We found that cellular immune function levels were significantly higher during events of aGVHD than during immunological quiescence. The current data presented herewith are not yet sufficient for establishing a practical scale for stratification of immune function levels that would support actual diagnosis of aGVHD based on ImmuKnow results. None the less, our data provide justification for continuing data collection in order to reach this goal in the future.

Of importance is our observation of a noticeable increase in the ImmuKnow level which accompanied the manifestation of aGVHD in a set of individual patients. The median change in ImmuKnow levels from a state of immunological quiescence to a state of aGVHD in the same patient was a 258% elevation. The extreme changes in ImmuKnow levels that accompany events of immunological destabilization, namely aGVHD, point to the benefit of serial longitudinal ImmuKnow monitoring. This ongoing monitoring can assist in comprehension of the dynamics of the cellular immune function of the individual HCT recipient.

We have presented an example of a patient who went through a non-stable post-HCT clinical course. Longitudinal immune monitoring using the ImmuKnow assay demonstrated the relevance and correlation between ImmuKnow monitoring and the adverse clinical episodes where existence or absence of aGVHD was parallel to noteworthy changes in the ImmuKnow levels. The assay provided accurate evaluation of the patient cellular immune function, and thereby guided the clinician towards the correct treatment in different events where GVHD occurrence was suspected. Thus, ImmuKnow measurements reflected reliably the cellular immune function status of the patient, and could serve as an integral contributing component in post-HCT immune monitoring.

Very limited literature is currently available about the utilization of the ImmuKnow assay in the HCT setting. Manga et al. [33] studied its relevance in the context of autologous HCT and is therefore not instructive for our study of patients going through allogeneic HCT. Thus far, only one publication has described utilization of the ImmuKnow assay for immune monitoring post-allogeneic HCT [34]. However, there are several apparent differences between the latter and the current study. In contrast to the publication by Gesundheit et al., the patient group presented herewith includes only adult patients and excludes paediatric patients. It includes only haematological malignancies and excludes solid tumour and non-malignant diseases. It includes only matched or nearly matched stem-cell donations and excludes haploidentical transplantations. It includes only measurements made in the immediate post-HCT period and excludes measurements performed years after the transplant. Overall, the patient group in our current study is more homogeneous, and thus our current findings should be regarded as more reliable than the findings in the initial pioneering study mentioned above.

In conclusion, we demonstrate the relevance and potential contribution of cellular immune function monitoring using the ImmuKnow assay in the clinical setting of HCT recipients. The ImmuKnow assay results were in correlation with events of aGVHD, expressing the elevated cellular immune response in comparison to ImmuKnow measurements performed during episodes of clinical stability. Moreover, we demonstrate a case where the ImmuKnow assay played an invaluable role in the immune monitoring and management of a patient after HCT. These data support the need to explore the method for optimal utilization of the ImmuKnow assay in the routine clinical care of patients after HCT, namely by a prospective protocol with serial prospective ImmuKnow evaluations and precise monitoring of GVHD.

Acknowledgements

The authors thank Mrs Avie Goldreich of the scientific editorial service unit of the Goodman Faculty of Life-Sciences in the Bar-Ilan University for her help in editing the manuscript.

Conflicts of interest

None.

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