Clinical radionuclide therapy dosimetry: the quest for the “Holy Gray”

Introduction Radionuclide therapy has distinct similarities to, but also profound differences from external radiotherapy.
Introduction
Radionuclide therapy (or “targeted”, “metabolic-or “molecular-radiotherapy) may be defined as a radiation therapy that uses local, loco-regional or generally administered open (i.e. “unsealed”) radionuclides to achieve a transfer of radiation energy to a pathological target tissue and by this means to exert a destructive tissue effect. This “internal-radiation therapy has distinct similarities to, but also profound differences from the more commonly used external radiotherapy (EBRT).
The tissue effect is expressed as the absorbed radiation dose in grays (Gy), i.e. the amount of transferred energy in joules per unit mass of target tissue. The fundamental use of this unit is similar to that in EBRT, and there is a similar relationship between radiation dose and response in terms of cell killing/survival. Therefore, calculation of the radiation absorbed dose to a targeted tissue makes sense at any point in treatment. In this respect, radionuclide therapy dosimetry may be considered an inherent part of radionuclide therapy in principle, as in EBRT.
In the literature, there is considerable confusion over the proper use of the term “dose”, which actually refers to the “radiation dose-in the SI unit “grays”. However, dose in the context of radionuclide therapy is frequently used when actually the “administered activity-in GBq or mCi is meant. To avoid confusion, the terms “radiation dose-and “absorbed dose-are used when indicating Gy and the term “activity dose-when indicating GBq.
Individual patient dosimetry has the following goals [1]:
To establish individual minimum effective and maximum tolerated absorbed dosesTo establish a dose-response relation to predict tumour response and normal organ toxicity on the basis of pre-therapy dosimetryTo objectively compare the dose–response results of different radionuclide therapies, either between different patients or between different radiopharmaceuticals, as well as to perform comparisons with the results routinely obtained with external radiotherapyTo increase the knowledge of clinical radionuclide radiobiology, in part with the aim of developing new approaches and regimens
In EBRT, the absorbed dose can be calculated relatively straightforwardly from the energy loss in the body from the point where the radiation enters the body to the target. Radiation in radionuclide therapy is directed to its target by a vehicle (hence “radiopharmaceutical”), which exhibits more or less specific binding to the target tissue. This is a highly dynamic metabolic process, both biochemically and physically, within the time interval of the decaying isotope, and entails a much more complex spatial and temporal radiation distribution than that in EBRT. Pharmacokinetics such as circulation, metabolisation, target expression heterogeneity and cellular uptake and release, as well as radiobiological phenomena such as biological or physical (“cross-fire”) bystander effects [2], play a determining role in the final radiation dose to the target. EBRT is typically a fractionated high dose rate radiotherapy in which episodes of radiation, aimed to cause as much lethal damage as possible, are interspersed with episodes of non-treatment, during which repair and repopulation occur. Radionuclide therapy is radiotherapy with a low and continuously decreasing dose rate, which requires a unique radiobiological approach [3, 4].
Integral activities can be determined numerically or by compartmental models [5] and presently rely heavily on the detection of the activity distribution by gamma cameras. Because the electron range is inferior to the spatial resolution of most molecular imaging devices, the “perfect-dosimetric study cannot be achieved, and calculations are always an approximation. Using the temporal and spatial radionuclide distribution data, radiation doses to target organs have generally been calculated using the MIRD formalism, formerly used for calculation of the biodistribution of diagnostic radiopharmaceuticals. Commercially available software such as MIRDOSE3 or the newer OLINDA/EXM [6, 7] is available for calculation of internal absorbed doses in organs and tumours. Although these models make important assumptions about anatomy (standard man and woman) and radiopharmaceutical distribution (uniformity of uptake in source and target) that are not necessarily valid in individual patients, they do provide a practical and standardised model for clinical end-users.
Nevertheless, to date the need for dosimetry to individually optimise the therapeutic activity to be administered has been far from self-evident. Radionuclide therapy dosimetry has not gained wide acceptance as a clinical tool among the (nuclear) medical community because of an imbalance between the accuracy and the complexity of the procedure. A number of studies have even completely discarded dosimetry, instead using fixed activities for all patients or activities based on kg or m2 body dimensions. [8-10]. To underline that this is unjustified, this review will describe the significant progress that has been achieved over recent years, especially in the fields of instrumentation (with provision of clinically useful instruments), physical modelling and radiobiology. Furthermore, the EURATOM Council Directive 97/43 stipulated that in medical exposures for radiotherapeutic purposes, including nuclear medicine, “exposures of target volumes shall be individually planned”. In this context, the nuclear medicine physician is at present confronted with a “dosimetric dilemma”, because official guidelines and recommendations for most treatments do not include advanced dosimetric calculations. Therefore, this review primarily aims to provide the nuclear medicine practitioner with an up-to-date overview of clinically applied dosimetry techniques in radionuclide therapy. For those working at a more basic level of dosimetry, information is provided on the clinical application of methods and areas of further development. Given the large size of the field of radionuclide therapy, this review is limited to those oncology indications for which there is substantial literature concerning dose calculations, with in-depth discussion thereof. As it would be impossible to cover all areas of the complex subject of dosimetry, appropriate references will be provided where necessary, e.g. regarding basic physics, (radio)biology, radiopharmaceuticals and preclinical aspects.
Radioiodine therapy in differentiated thyroid carcinoma
Radioiodine therapy has proven to be a safe and effective method in the treatment of patients with differentiated thyroid carcinoma (DTC) after total or near-total thyroidectomy [11, 12]. It has been shown to be useful not only for ablation of benign remnant thyroid tissue, facilitating subsequent diagnostic testing, but also for treatment of any remaining cancerous cells either in the thyroid bed or at metastatic sites. Dosimetry plays an important role in answering questions concerning biodistribution since the biological half-time of radioactive iodine differs substantially between individual patients and even within distinct lesions of the same patient. It has proven exceptionally helpful in clinical trials of new drugs, for example retinoids, and in assessment of the use of recombinant TSH [13], as well as in the study of other questions such as the phenomenon of “stunning”. However, its possible role in extending therapy doses to the level of individual maximum effectiveness is currently less obvious.
There is at present no consensus on the activity dose of 131I to be administered in various settings according to recent European and American guidelines [14, 15], largely because of the lack of prospective, randomised data. In the majority of cases, 1.1-3.7 GBq (30-100 mCi) is prescribed empirically for the first radioiodine therapy after thyroidectomy in newly diagnosed DTC, to ablate the remaining glandular tissue. Activity doses as low as 1 GBq are used when the size of the thyroid remnant is small, as measured by the postoperative radioiodine uptake in the neck, and/or to reduce local complications that could arise from radiation thyroiditis/oedema, which has been reported in 10-20% of cases, although usually mild. The effectiveness of this ablative approach based on standard activities is reported to be about 80%. For the treatment of residual tumour, relapses or lymph node metastases, generally higher activities of 3.7-7.4 GBq (100-200 mCi) are used.
Approaches in which quantitative dosimetry is performed to estimate the activity dose needed to deliver an effective radiation dose are scarce in the literature. Maxon and co-workers, using sequential planar scintigraphy, established in the early 1980s that an effective mean radiation dose of at least 300 Gy is required for successful remnant ablation [16]. The validity of this recommendation remains unclear as it conflicts somewhat with clinical experience. It also neglects microdosimetry of radioiodine and dose heterogeneity, which may well determine the overall response. As far as metastases are concerned, these authors concluded in the same study that a radiation dose of at least 80 Gy is associated with a significant increase in response, while a dose of less than 35 Gy offers little chance of success. In a later study [17], they found that when metastatic disease was present only in lymph nodes, a target radiation dose of at least 140 Gy was successful in 86% of patients and 90% of involved nodes. When nodal metastases were associated with either residual thyroid tissue or other metastatic foci, a single treatment calculated to deliver at least 85 Gy to the metastases proved successful in 74% of patients and 81% of nodes. The aforementioned doses were mean absorbed doses for the whole remnant or tumour; no data were provided on the dose distribution in smaller tumour parts. It is also unclear what causes the large difference between radiation dose values for remnants and metastases. This method requires sequential measurements with a scintillation camera 24, 48 and 72 h after administration of 74 MBq (2 mCi) of 131I. De Keizer et al. [18] recently calculated thyroid tumour doses with a similar methodology but with the use of recombinant TSH. A tumour dose of >80 Gy was found in only 20% of metastases visible on post-therapy 131I scanning. In 55% of patients, progressive disease was evident after 3 months and none of these patients had radiation doses to the tumour in excess of 30 Gy, confirming the existence of a clinical dose-response relation.
In cases of distant metastases, higher amounts of 131I are given in single doses and subsequent cumulative therapies. Most centres use a fixed dose of 7.4 GBq (200 mCi), but some use (much) higher activity doses. In order to avoid serious complications (bone marrow suppression, lung fibrosis), the commonly used “maximum safe-administered dose concept published by Benua et al. [19] restricts the blood absorbed dose to less than 2 Gy (200 rad) and the whole-body retention to 4.4 GBq (120 mCi) at 48 h in the absence of diffuse lung metastases. According to the protocol, activity concentration in the whole body as measured by 131I should not exceed 2,960 MBq (80 mCi) after 48 h when diffuse, functioning lung metastases are present. These measurements can be camera based without blood sampling. More recently, the blood dose formula derivation proposed by Hänscheid et al. [13] has provided a new tool for patient-specific blood dose assessment representing marrow dosimetry in DTC therapy. For more precise dosimetry, e.g. in dosimetric studies or for higher targeted blood absorbed doses, sequential blood sampling is recommended [20, 21]. The calculated radiation dose serves as a surrogate parameter for the organ at risk, the bone marrow, since to date direct determination of the bone marrow absorbed dose is not feasible. This method has been applied successfully in clinical practice. Dorn et al. [22] used a dosimetric study prior to therapy with 150-400 MBq 131I and obtained daily images up to 4-5 days. They maximised the administered activity to an absorbed dose of 3 Gy to the bone marrow or 30 Gy to the lungs, corresponding to the LD5/5 in EBRT, while aiming at >100 Gy to all known metastases. This resulted in a mean of 22.1 GBq (597 mCi) and a range of 7.4-38.5 GBq (200-1,040 mCi) activity administered per treatment. Many institutions will find it difficult to deliver these amounts owing to radioprotection restrictions, limiting the applicability of this method. To date, bone marrow depression has rarely been reported, while the rate of dose-related leukaemia has been estimated to be approximately 1% after 5-10 years. Lung fibrosis as a consequence of micronodular iodine-avid metastases has been reported in 1% of patients suffering from pulmonary metastases. The downside of these high-dose strategies may be an increase in the occurrence of these side-effects. On the other hand, assuming this “maximum safe dose-to be an optimal dosing strategy, extrapolation to a “fixed activity dose-strategy of 7.4 GBq (200 mCi), as is widely applied, would “under-treat-54% and “over-treat-3% of patients [23]. To date, there are no randomised and prospective trials that directly compare a low- and high-activity dose strategy in patients with metastases.
The treatment of DTC in childhood varies substantially from the standard approach in adults mainly owing to the different tumour biology in this age group. Usually activities of 50-100 MBq/kg are given, treatment data on children being scarce and non-systematic.
There are a number of potential drawbacks of dosimetry that may preclude its use in many centres. One problem is the uncertainty of volume determinations by neck ultrasound shortly after thyroid surgery since the differentiation between scar tissue, haematoma and thyroid remnant is often difficult. The same holds true for the use of computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) in the evaluation of distant metastatic lesions, especially in the case of diffuse lung metastases. Also, it is difficult to predict radioiodine kinetics during therapy from prior diagnostic studies owing to the large difference in administered and measured activity and potential subsequent biological effects. PET studies using the cyclotron product 124I-NaI, if possible in combination with PET/CT tomography, may prove to be particularly valuable in improving treatment planning and patient-specific dosimetry. Compared with conventional methods, including CT and 131I scanning, this technique was found to be superior in terms of lesion detection and functional assessment [24]. Using three-dimensional 124I-PET voxel-based dosimetry, Sgouros et al. [25] showed a wide range of mean absorbed dose values for individual tumours, from 1.2 to 540 Gy. Unfortunately, no correlation with response was reported in that study. However, this wide variation certainly implies that the assessment of meaningful dose-effect relationships and clinical dosimetry should include further development of a quantitative approach.
131I-mIBG therapy in neuroblastoma and phaeochromocytoma
For more than two decades, mIBG labelled with the γ/β emitter iodine-131 (131I-mIBG) has been used to treat neural crest-derived tumours, essentially neuroblastoma and phaeochromocytoma, both in relapsed and in newly diagnosed patients. In high-stage neuroblastoma, the treatment may represent (a) palliative therapy [26], (b) first-line therapy, as a single agent or combined with chemotherapy [27], (c) consolidation therapy after induction of a “good partial remission-[28] and, more recently, (d) second-line therapy after failed induction chemotherapy, combined with topotecan and stem cell rescue in children with metastatic neuroblastoma [29] or with myeloablative chemotherapy and autologous stem cell transplantation in refractory neuroblastoma [9]. In neuroblastoma, important efforts have been made to develop clinically useful dosimetry methods to predict the radiation dose to the red marrow as the critical organ for bone marrow toxicity, in order to apply optimal and individual dose maximisation in children with highly aggressive tumours. Three different methods of activity administration have evolved from the experience of treating such patients.
The first approach, developed in Amsterdam, the Netherlands, is to give fixed activity dose “fractions-of 7.4-11.1 GBq (200-300 mCi), more recently combined with hyperbaric oxygen or topotecan. This approach has been proven to have a synergistic effect on cell killing in vitro when used in combination with 131I-mIBG [30]. Intervals between fractions are determined by blood count recovery and the treatment is continued to maximum response [31]. An advantage of this method is its simplicity, which may be viewed as important in very sick children and in those who require prompt treatment, especially when receiving first-line therapy. Another important advantage of lower activity doses is the shorter isolation and hospitalisation period required for reasons of radioprotection, which may be regarded as highly significant in these usually very sick children with a grave prognosis. As whole-body and tumour radiation doses are never calculated, any kind of dose-response assessment and optimisation on the basis of absorbed radiation dose is, however, impossible. Furthermore, the relatively low dose rate of radiation compared with (ultra-)high doses may be viewed as radiobiologically suboptimal. The clinical results of this approach need to be verified in a multicentre phase III trial that should include a dosimetric assessment of the whole-body dose.
The second approach originates from the United Kingdom, where there has been a long tradition of using pre-therapy dosimetry. In a phase I/II study on advanced, chemo-refractory stage III/IV patients, Lashford et al. observed that 80% of patients developed grade 3 or 4 haematotoxicity at a whole-body absorbed dose of 2.5 Gy established from a pre-therapy 131I-mIBG scan [32]. Monsieurs et al. used 123I-mIBG prior to therapy to the same effect [33]. Previously, Tristam et al. had shown a highly skewed distribution of tumour radiation doses after a fixed, diagnostic activity [34]. Matthay et al. found a good correlation between whole-body and tumour dose [35]. As radiation dose is principally related to response, this approach may lead to a more standardised whole-body and tumour absorbed dose [36] and may offer more possibilities of dose escalation, especially in the presence of an altered biodistribution, e.g. due to partial kidney failure or high tumour load. Recently, an ESIOP experimental mIBG protocol has been proposed in which the aim is to deliver a total whole-body absorbed dose of 4.0 Gy in two fractions, in combination with topotecan, after failure of induction chemotherapy. Post-therapy dosimetry is performed after a first fixed fraction of 440 MBq/kg (12 mCi/kg), with calculation of the activity dose to be administered with the subsequent second fraction in order to arrive at the desired total whole-body absorbed dose of 4 Gy. The importance of this protocol is that whole-body and tumour absorbed dose can be estimated and the relation to both toxicity and response can be established, unlike with the non-dosimetry protocols. The schedule of intensification of mIBG therapy by dose escalation and radiosensitisation with topotecan with a haematopoietic autograft has been shown to be safe and practicable [29]. The next step will be to prove that this approach will also lead to a better outcome in neuroblastoma patients.
The third approach can be derived from recent publications from the United States, in which patients received high activity doses of mIBG on the basis of an activity dose of 550-660 MBq per kilogram body weight (15-18 mCi/kg), with stem cell support if necessary. In an early study, Sisson and co-workers examined predictors of toxicity and found the best correlation to be between whole-body absorbed dose and platelet ratio; however, comparable and statistically significant correlations were found with GBq/kg body weight and GBq/m2 body surface [37]. This observation was supported by Matthay and colleagues [35], who found the amount of 131I (GBq/kg) and whole-body dose (Gy), but not administered activity dose (GBq), to be significantly correlated with platelet and neutrophil nadir. In a recent study by Dubois et al. [38], substantial haematotoxicity was seen at 660 MBq/kg (18 mCi/kg) in a group of patients with advanced, heavily pre-treated neuroblastoma, including myeloablative chemotherapy in 81%. Of these patients, 36% required autologous haematopoietic stem cell transplantation. The resulting median whole-body absorbed dose was 2.92 Gy with a median administered activity dose of 13.5 GBq (366 mCi, range 198-895 mCi). Interestingly, whole-body radiation dose was shown to correlate with failure to engraft platelets or red cells in those patients receiving autologous stem cell transplantation. An encouraging response rate of 27% was reported, however. The same authors recently established a maximum tolerated dose (MTD) of 440 MBq/kg (12 mCi/kg) with myeloablative chemotherapy and autologous stem cell transplantation [9]. The advantage is that there is no need for planning/simulation in this high-dose protocol, which may increase in importance if multiple centres participate.
Malignant, metastatic phaeochromocytoma is a very rare disease but can be excellently treated by 131I-mIBG in both a palliative and a curative setting. Literature data on dosimetry are scarce and casuistic. In 1997, Loh et al. reviewed the literature concerning 116 previously treated cases [39]. They found a cumulative activity dose of 3.6-85.9 GBq (96-2,322 mCi, mean 490 α 350 mCi) in 1-11 (mean 3.3 α 2.2) therapies, with a mean single activity dose of 5.8 GBq (158 mCi). Dose prescriptions were as a fixed activity dose of 3.7-11.1 GBq (100-300 mCi or 3-9 mCi/kg). A recent study was performed in order to evaluate the performance of mIBG therapy with high activities after debulking surgery [40]. The median individual cumulative activity dose was 37.6 GBq (1,015 mCi), with a range of 14.3-62.5 GBq (386-1,690 mCi), in one to three consecutive therapies. Grade 3 platelet toxicity was observed after 79% of therapies, and grade 3 and 4 neutrophil toxicity after 53% and 19% of therapies, respectively. All patients had stem cells harvested before therapy, but 11 patients showed spontaneous reconstitution and only one required stem cell return. Response rate was 10/12 patients (83%).
Radiopeptide therapy for neuro-endocrine tumours
Several clinical trials have investigated the use of peptide receptor radionuclide therapy (PRRT) with a radiolabelled somatostatin analogue as one of the newly developed targeted tools for neuro-endocrine tumours [41-44]. Individual pre-therapeutic dosimetry is necessary for patient selection and therapy planning because there are huge inter-patient differences in radiopeptide uptake in normal organs and tumour tissues. This may be related to varying somatostatin receptor densities on tumour cells, as well as to factors such as tumour volume, interstitial pressure and viability. The dosimetric studies that have been performed in this treatment have been the most sophisticated in the field and exemplory for other types of treatment. Initial studies were performed with the radiopeptide used in diagnostics, [111In-DTPA0]-octreotide, given in high activities. Results were encouraging, with symptomatic and biochemical responses in a large percentage of patients, although objective responses were rare [42]. The analogue [90Y-DOTA0,Tyr3]-octreotide has been investigated in various phase I-II trials [41, 43]. 90Y-labelled lanreotide has also been investigated (in the MAURITIUS trial) [45]. For dosimetry of 90Y from post-therapeutic bremsstrahlung, images that substantially lack quality must be quantified. Therefore, two alternative approaches have been developed as a surrogate for the original radiopeptide, namely therapy simulation with either the 111In-labelled peptide or the 86Y-labelled peptide [46, 47]. These methods are intended to provide pre-therapy assessment of the optimal activity administration [48-51] (Table 1). The variation that can be appreciated among the various combinations in the table may be primarily technical/methodological in origin but also biological, based on differences in somatostatin receptors and isotope/chelator affinity. The reader is referred to the respective papers for more detailed information.
Table 1Absorbed doses to principal organs and to tumour (Gy/GBq ±SD), deriving from different radiopeptidesStabin 1997 [109], Kwekkeboom 2001 [50]Kwekkeboom 2001 [50]Cremonesi 1999 [47]Forster 2001 [110]Helisch 2004 [111]Forrer 2004 [112]Therapy[111In-DTPA0, Tyr3]-octreotide[177Lu-DOTA0, Tyr3]-octreotate[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotideDosimetry[111In-DTPA0, Tyr3]-octreotide[177Lu-DOTA0, Tyr3]-octreotate[111In-DOTA0, Tyr3]-octreotide[86Y-DOTA0, Tyr3]-octreotide[86Y-DOTA0, Tyr3]-octreotide[111In-DOTA0, Tyr3]-octreotidePatients16530385Kidneys0.52 α 0.241.65 α 0.473.9 α 1.9b
2.73 α 1.412.84 α 0.64Kidneys + protection0.88 α 0.191.71 α 0.89Liver0.065 α 0.010.21 α 0.070.72 α 0.570.66 α 0.150.72 α 0.400.92 α 0.35Spleen0.34 α 0.162.15 α 0.397.62 α 6.302.32 α 1.972.19 α 1.116.57 α 5.25Red marrow0.03 α 0.010.07 α 0.0040.03 α 0.010.49 α 0.0020.06 α 0.020.17 α 0.02Tumour (range)0.72-6.8a
3.9-37.91.4-313.21-19.582.1-29.52.4-41.7aFrom reference [50]bSeries enlarged from the original one, as in Bodei et al. [44]
Although [90Y-DOTA0,Tyr3]-octreotide (or -lanreotide) and its imageable counterpart [111In-DOTA0,Tyr3]-octreotide (or -lanreotide) are not chemically identical, the latter has been used for dosimetric simulation, based on the hypothesis that the similar physical and biological half-lives yield a comparable in vivo pharmacokinetics and biodistribution, especially concerning the renal uptake, which depends on aspecific phenomena. A drawback of this method is that the small structural modification may affect the somatostatin receptor binding affinity [52]. Regarding biodistribution, the organs receiving the highest predicted absorbed doses, in a first series of 18 patients, included the spleen (7.6 α 6.3 mGy/MBq), the kidneys (3.3 α 2.2 mGy/MBq) and the tumour (1.4-31 mGy/MBq, mean 10). When the series was enlarged to 30 patients, a slightly higher kidney absorbed dose (3.9 α 1.9 mGy/MBq) was observed [43]. Shortcut approaches involving use of the commercially available molecule [111In-DTPA0]-octreotide or OctreoScan® have been proposed [53]. Nonetheless, probably as a result of the quite different biokinetics and receptor affinity of [90Y-DOTA0,Tyr3]-octreotide and [111In-DTPA0]-octreotide, which are indeed chemically different, comparative data obtained appear not to be sufficiently overlapping for this tracer.
A thorough dosimetric study using PET with [86Y-DOTA0,Tyr3]-octreotide, biochemically identical to the therapeutic molecule, was carried out in 24 patients [54]. This offers substantial advantages in terms of spatial resolution and quantification, but the short half-life of the radionuclide leaves later phases of the biokinetics to estimates based on extrapolation.
The newest radiopeptide, [177Lu-DOTA0,Tyr3]-octreotate, offers further advantages. 177Lu has a lower energy (Emax 0.49 MeV) and penetration range (Rmax 2 mm) emission, but a longer half-life (6.7 days). The low abundance gamma emissions (113 and 208 keV) allow for dosimetry and imaging prior to as well as post therapy. Moreover, octreotate has a six- to ninefold higher affinity for somatostatin receptor 2, the somatostatin receptor most frequently expressed in neuro-endocrine tumours. Unfortunately, to date a thorough dosimetric analysis is lacking, but data deriving from a study comparing [177Lu-DOTA0,Tyr3]-octreotate with [111In-DTPA0]-octreotide indicate that, compared with [90Y-DOTA0,Tyr3]-octreotide, [177Lu-DOTA0,Tyr3]-octreotate delivers a lower burden to organs, with absorbed doses of 1.8-2.7 mGy/MBq to the spleen, 1.0-2.2 mGy/MBq to the kidneys (lowered to 0.7-1.1 mGy/MBq with protection) and 0.1-0.3 mGy/MBq to the liver. Red marrow absorbed dose, derived by the blood approach, ranged from 0.05 to 0.08 mGy/MBq [44].
Due to their marked radiosensitivity, the kidneys undoubtedly represent the critical organ, particularly after [90Y-DOTA0,Tyr3]-octreotide. Renal irradiation arises from the proximal tubular re-absorption of the radiopeptide and the resulting retention in the interstitium. According to EBRT studies, the renal maximum tolerated absorbed dose is conventionally considered to be in the range of 23-25 Gy. According to the National Council on Radiation Protection and Measurements (NCRPM) an absorbed dose of 23 Gy to the kidneys causes detrimental deterministic effects in 5% of patients within 5 years [55]. Sporadic cases of delayed renal failure, in some cases end-stage disease requiring dialysis, have been observed, especially in patients who have received an activity dose of more than 7.4 GBq/m2 [56]. Nephrotoxicity is accelerated by other risk factors, such as pre-existing hypertension or diabetes. Given the high kidney retention of radiopeptides, positively charged molecules, such as L-lysine and L-arginine, are used to competitively inhibit the proximal tubular re-absorption of the radiopeptide. This results in a reduction in the renal absorbed dose of between 9% and 53% [57]. Doses are further reduced by up to 39% by prolonging infusion over 10 h and by up to 65% by prolonging it over 2 days after radiopeptide administration, thus covering more extensively the elimination phase through the kidneys [43]. Despite kidney protection, renal function loss may become clinically evident years after PRRT. A median decline in creatinine clearance of 7.3% per year was reported in patients treated with [90Y-DOTA0,Tyr3]-octreotide and of 3.8% per year in patients treated with [177Lu-DOTA0,Tyr3]-octreotate. Cumulative and per cycle renal absorbed dose, age, hypertension and diabetes are considered factors contributing to the decline of renal function after PRRT [58]. Clinical experience and dosimetric studies clearly indicate that the renal absorbed dose estimated by conventional dosimetry does not accurately correlate with the renal toxicity observed in patients treated with [90Y-DOTA0,Tyr3]-octreotide. Consideration of additional parameters, such as patient-specific kidney volume and distribution of the radionuclide, appears to give a better correlation with the clinical effects [59]. Assessment of individual kidney volume by CT scan yields a wide variability when compared with the standardised phantom. Moreover, autoradiographic studies, performed on human kidney after in vivo injection of 111In-peptides, have demonstrated that the majority of radioactivity is deposited within the renal cortex, mainly in the juxtamedullary region. This leads to a higher deposition of energy per unit mass, compared with conventional dosimetry. Hence, calculation of the kidney absorbed dose assuming a homogeneous renal distribution of radioactivity is inadequate. New techniques accounting for the difference in radioactivity placement in the kidneys, on the basis of a CT-based volumetric analysis, appear more realistic [60].
Even if predicted absorbed doses are much lower than the threshold for toxicity, the other target organ that gives rise to concerns about acute and permanent toxicity after PRRT is the bone marrow, particularly in repeated administrations [41-43]. Acute haematological grade 3 or 4 toxicity is not uncommon, especially after [90Y-DOTA0,Tyr3]-octreotide, and sporadic cases of myelodysplastic syndromes or even overt acute myeloid leukaemia have been reported with all three therapeutic radio-compounds. Bone marrow dosimetry is usually modelled through a blood-based method, in which an equivalent distribution of the radioactivity from blood throughout the bone marrow is conservatively considered [61, 62]. Currently, the potential risk of kidney and red marrow limits the amount of radioactivity that may be administered. Indeed, when tumour masses are irradiated with suitable doses, volume reduction may be observed (Fig. 1) [63]. Tumour remission is positively correlated with high uptake during [111In-DTPA0] octreotide scintigraphy. Nevertheless, tumour radiation dose depends not only directly on the administered activity and the uptake versus time, but also on the tumour load. This is confirmed by clinical data regarding the response characteristics: patients with a limited number of liver metastases respond to PRRT, whilst patients with a high tumour load do not [45]. Mathematical models have shown that 177Lu is better in small tumours (optimal diameter 2 mm), whilst 90Y is better in larger ones (optimal diameter 34 mm): very small masses are likely not to absorb all the β-energy released in the tumour cells by 90Y, while larger tumours will suffer from lack of uniformity of activity distribution of 177Lu. Finally, differences in dose rate must be taken into account: the longer physical half-life of 177Lu means a longer period is needed to deliver the same radiation dose than when using 90Y. This may allow more time for tumour re-population [64]. Therefore, combination therapy with 90Y and 177Lu, either simultaneously or in distinct settings, has been suggested to overcome the difficulties of real clinical situations involving different sized lesions.
Fig. 1Tumour dose-response relationship in 13 patients treated with 90Y-DOTATOC. Tumour volumes were assessed by CT before and after treatment. Tumour dose estimates were derived from CT scan volume measurements and quantitative 86Y-DOTATOC imaging performed before treatment. Data were further computed using the MIRDOSE spherical model. Reprinted by permission of the Society of Nuclear Medicine from [63]
Treatment of solid tumours by radiolabelled antibodies
Most clinical radioimmunotherapy (RIT) studies have been performed in colorectal cancer, using antibodies against CEA, TAG-72, A33 and KSA. Other tumours studied have included ovarian cancer (anti-MX35, anti-folate receptor Mov18, anti-HMFG1), prostate cancer (anti-PMSA), breast cancer (anti-mucin BrE3, NR-LU-10), glioma (anti-tenascin) and renal cancer (anti-G250) [65].
Although at later time points after injection of radiolabelled monoclonal antibodies, adequate to high uptake may serve to delineate deposits of solid tumours, the relatively unfavourable therapeutic window between the anti-tumour effect and toxicity hampers the introduction of these agents in the clinic. Solid tumours are generally more radioresistant than, for example, malignant lymphoma. The absorbed doses required to achieve a response of tumour deposits are higher than those needed to obtain a response in malignant lymphoma. Activity doses leading to adequate absorbed doses in tumour deposits therefore result in significant radiation-induced toxicity, primarily of the bone marrow, as the most radiation-sensitive organ; myeloablation may be the goal as well as the result of this.
Most studies aim at administration of the MTD that results in acceptable toxicity to the bone marrow, as the organ responsible for dose-limiting toxicity. Several methods have been reported for calculation of the absorbed dose to the bone marrow, e.g. region of interest analysis of scintigraphic data or a model based on radioactivity in the blood. When radiolabelled antibodies bind to blood, bone and bone marrow components or when the radionuclide accumulates in bone or bone marrow upon metabolisation of the radionuclide-antibody complex, calculation of the red marrow dose is more complex than when using radiolabelled antibodies that lack these characteristics [66, 67]. The use of a model for bone marrow dosimetry using blood activity may result in better reproducibility of the bone marrow dosimetry. Wessels et al. [68] observed that historical variations as high as 200-700% between different institutions performing marrow absorbed dose calculations could be dramatically reduced to −29% to +20% by a central computing facility and the use of similar methodology, based on the standard American Association of Physicists in Medicine (AAPM)/Sgouros blood model [66].
In a study using 131I-labelled murine monoclonal antibody G250 in patients with metastatic renal cell cancer, haematological toxicity correlated with whole-body absorbed radiation dose [69]. In a retrospective analysis of 114 patients who underwent 131I-labelled antibody therapy, absorbed dose-based definitions of MTD and escalation variables proved to be better than activity-based methods [70]. In a study in which therapeutic doses of 131I-cG250 (the chimeric variant of G250) were administered, no correlation was found between haematological toxicity and either the radiation absorbed dose to the whole body or bone marrow or the administered activity [71]. Juweid et al. observed that besides red marrow dose, baseline blood counts, multiple bone and/or marrow metastases and recent chemotherapy are important factors related to haematological toxicity after radio-immunotherapy [72], making a dosimetric approach to identify the optimal radionuclide dose more complicated.
One way to overcome the unfavourable therapeutic index of tumour response and normal organ toxicity is to focus on treatment of patients with minimal residual disease. For example, patients with ovarian cancer in complete remission after debulking and chemotherapy have an approximately 50% chance of relapse. Compared with results in matched controls, Nicholson et al. observed an increase in 5-year survival from 55% to 80% following intraperitoneal administration of 90Y-labelled HMFG1 in patients with ovarian cancer in complete remission after chemotherapy [73]. However, a more recent randomised clinical trial in patients with ovarian cancer in complete remission after debulking and chemotherapy did not reveal a benefit in disease-free and overall survival after treatment with 90Y-labelled murine HMFG1 [74]. Introduction of 90Y-muHMFG1 did not delay the time to relapse and did not result in prolonged survival as compared with the control group. One can argue that 90Y is far from ideal for treatment of minimal residual disease owing to its high β-energy, and that radionuclides with lower range emissions, such as 177Lu, have more favourable characteristics for this purpose. Furthermore, relatively large antibody doses were used, which may have resulted in saturation of the epitopes on cancer cells and subsequently in low tumour-to-non-tumour ratios of radioactivity uptake. Obviously, clinical dosimetry could play an important role in explaining these differences in therapy results.
A very significant advance that promises to increase the radiation dose to the tumour while reducing that to the bone marrow is the development of pre-targeting strategies. In pre-targeting, the tumour is first targeted with a specific non-radioactive monoclonal antibody construct. The antibody construct is allowed to accumulate in the tumour and to clear from the blood and non-target tissues. Subsequently, a small radioactive hapten is injected which has high affinity for the antibody construct. The radioactive hapten targets the antibody construct, while demonstrating rapid renal excretion. Examples of these approaches are the use of bispecific monoclonal antibodies and the biotin-avidin system [75, 76]. As pre-targeting increases the tumour/normal organ uptake ratio [77], these approaches hold great promise for enhanced therapeutic efficacy. However, the use of multiple drugs poses the problem of fine-tuning dosing and timing of the interval between injections. Optimisation of the dosing and timing schedule is needed for optimal targeting of the tumour by the radioactive compound, while assuring low normal organ uptake. Robust dosimetric analysis of tumour and normal organ uptake of the radioactive small molecule is a requirement for the successful development and implementation of pre-targeting strategies [78, 79].
Radioimmunotherapy of B-cell lymphoma
Research in RIT in lymphoma has resulted in two FDA-approved radiopharmaceuticals, 90Y-ibritumomab or Zevalin® (IDEC Pharmaceuticals and Schering AG) and 131I-tositumomab or Bexxar® (Glaxo Smith Kline), for the treatment of B-cell lymphoma [80, 81]. Both are directed against CD20, albeit not against the same epitope. They are both approved for the treatment of relapsed or refractory follicular/low-grade or transformed B-cell lymphoma including rituximab-refractory follicular B-cell lymphoma in the US, but only Zevalin is approved in the EU and only for follicular lymphoma. Pre-treatment with unlabelled monoclonal antibody (=preload) as part of the treatment with Zevalin and Bexxar is current practice, as it leads to a more favourable biodistribution. This has been studied in animal models, as well as in the setting of myeloablative RIT [82-84]. The preload may clear peripheral B cells from the circulation, improving tumour targeting of subsequently administered radiolabelled monoclonal antibodies. This optimisation has been accomplished by diagnostic and dosimetric 111In or 131I tracer studies. On the other hand, the intrinsic therapeutic efficacy of the antibody is a confounding factor, making evaluation of the relation between absorbed dose and treatment response more difficult than for other radiopharmaceuticals [85, 86].
Bexxar has been developed using the whole-body absorbed dose as a substitute for bone marrow dosimetry by administering a trace amount of 131I-labelled CD20 antibody and determining the total body clearance prior to therapy in order to calculate the patient-specific injected activity dose that should deliver the specified absorbed dose to the whole body. The method has been further simplified to an estimation based on three points [87]. In a dose escalation study with absorbed doses to the whole body ranging in 0.1-Gy increments from 0.25 to 0.85 Gy, the MTD was found to be 0.75 Gy in patients who had not received prior high-dose chemotherapy with stem cell support and who had platelet counts of ≥150 × 109/litre. A comparison between the therapeutic activity calculated on the basis of the diagnostic pre-therapy tracer study and the amount calculated per kilogram body weight showed that, using the latter method, 50% of the patients would have been either over- or underdosed by 10% or more, and 16% of the patients by 25% or more. Interestingly, a correlation between the duration of complete remission (CR) and the absorbed dose to the whole body was found. Patients receiving a whole-body absorbed dose between 0.65 and 0.85 Gy showed longer CR than patients receiving between 0.25 and 0.55 Gy [88]. In contrast, Zevalin has been developed with the notion that dosimetry may be dispensable. In a randomised study comparing Zevalin with rituximab alone, a secondary objective was to determine whether dosimetry was required [10]. A tracer dose of 185 MBq (5 mCi) 111In was administered for dosimetric purposes with the first infusion, followed by a therapeutic activity dose of 0.4 mCi/kg (15 MBq/kg) Zevalin 7 days later. The absorbed doses to normal organs and the marrow were found to be within the specified limits of 20 Gy for solid organs and 3 Gy for red marrow in 72 patients. The median estimated absorbed dose to the tumour was 15 Gy (range 0.6-24 Gy). There was no significant correlation between dosimetric and pharmacokinetic parameters and haematological toxicity, although the correlation between nadir of the neutrophil count and the whole blood half-life of 90Y exhibited borderline statistical significance. It was concluded that dosimetry may be excluded for populations of Zevalin patients who meet certain criteria for pre-treatment platelet count and percentage of marrow involvement by tumour.
A possible explanation for this discrepancy may be found in the photon energy emitted by 131I; this may account for a large part of the cross-absorbed dose to the bone marrow, which is almost independent of the amount of bone marrow involvement. It has been reported that the self-absorbed dose accounts for 64% of the whole absorbed dose to the red marrow [89], leaving 36% as the cross-absorbed dose. Scintigraphic assessment of absorbed dose to the bone marrow has its shortcomings owing to over- and underlying tissues as well as to the local variability in bone marrow involvement by the disease targeted. It is especially difficult to use pharmacokinetic data for calculation of the absorbed bone marrow dose, since bone marrow involvement is a strong confounder. Other important issues are the bone to bone marrow ratio and variable values for the activity concentration in the blood and red marrow [90, 91]. A fixed ratio between blood and bone marrow has been used in bone marrow dosimetry based on blood radioactivity levels [10, 64]. However, it has been suggested that the red marrow to blood ratio is not fixed, but increases continuously up to 72 h post injection in both patients and rats [92]. Furthermore, haematological toxicity, like tumour response, is a deterministic effect of radiation, characterised by a sigmoid rather than by a linear dose relationship [93]. A sigmoid relationship may be discernible in a reasonably homogeneous population, but is likely to be more difficult to identify if the population is heterogeneous. The bone marrow reserve may be more relevant to the magnitude of toxicity than is the absorbed dose. Another particular challenge to haematology RIT dosimetry may be tumour regression during energy deposition, as is frequently the case in rapidly responding lymphoma. This may lead to underestimation of absorbed dose, since dose is per definition energy per unit mass [94]. The opposite will be the case if the tumour grows during energy deposition. To avoid this pitfall, repeated volume assessments during therapy would be necessary, or alternatively one could use voxel-based dosimetry, which is likely to be less sensitive to changes in mass.
Myeloablative RIT at the MTD for normal organs is by definition dependent on dosimetry. Dose-limiting organs may be the lungs, kidneys or liver. In high-dose RIT using the 131I-labelled mouse antibody tositumomab, the lungs were found to be the dose-limiting organ in 28 of 29 patients and the kidneys in the remaining patient [95]. The opposite was found when the pharmacokinetics of the chimeric 131I-labelled rituximab were studied, i.e. the kidneys were found to be the critical organ [96]. These differences may be due to the significant difference in half-life between the antibodies, the chimeric and murine antibodies having half-lives of 88 h and 56 h, respectively. The MTD for myeloablative 131I-tositumomab was established to be 25 Gy to the lungs. Studies of myeloablative RIT using 90Y are ongoing and encouraging results have been reported [97].
In several experimental studies, Auger emitters have been therapeutically superior to β emitters when taken to the MTD [98, 99]. This is likely to be due primarily to lower bone marrow toxicity but is perhaps also attributable to the deposition of more energy in single cells or small tumour cell clusters. Auger emitters would thus be advantageous in an adjuvant setting, in leukaemia, but they may also be superior where there is tumour bulk. The challenge is how to estimate absorbed dose in single cells, but one may begin in patients with a significant amount of circulating tumour cells that can be studied ex vivo [100]. Interestingly, Kaminski reported that in a number of patients who relapsed following RIT the relapse occurred only at sites previously not known to be involved with tumour [101]; this indicates a possibility that small tumour manifestations receive lower absorbed doses than expected on the basis of antigen density and tumour diameter [102].
Discussion
As stated by DeNardo [103], “claims for specific dosimetry have to demonstrate that the frequency of excess toxicity and/or tumour underdosing significantly decreases”. Dosimetry should provide a quantification procedure that is primarily of additional benefit over empirical, fixed dosing with or without visual scintigraphic assessment. In standard oncology practice, a new therapeutic agent undergoes phase I, II and III testing before becoming a standard treatment. In a phase I study, the maximum tolerated dose is established and side-effects recorded. Dosimetry should play an essential role in this phase and establish a threshold dose above which clinically significant side-effects occur. In phase II, the new radionuclide therapy is evaluated in terms of effect on tumour response and survival. Here, dosimetry should enable the determination of a clinical dose-response relationship. In phase III, the new therapy is compared with the standard one. At this stage, dosimetry helps to elucidate the clinical effects in a larger patient group, e.g. it may be observed that some subgroups have a better or worse result. Moreover, multicentre trials offer the opportunity to compare results in different institutions and countries, as well as provide the opportunity to standardise the dosimetry procedure in a larger context. Evidence-based medicine entails randomised and prospective trials. However, after 60 years of treating thyroid cancer patients, international guidelines still cannot provide a consensus on the amount of radioiodine that should be given. Undoubtedly, the need for randomised trials will increase in the coming years, as in other areas. This underlines the importance of stepping up scientific efforts to include optimal dosimetry not only as an inherent part of radionuclide therapy, but also as an inherent part of these studies. In this context, there was a remarkable recent editorial in the Journal of Clinical Oncology [104], discussing the limitations of the body surface principle that forms the basis for chemotherapy dosing. Indeed, the bioavailability of chemotherapy, and hence the dose to the target, suffers from a similar metabolic variability as is observed for radionuclides, and this variability seems to be more important than can be accounted for by the body surface in square metres as the sole parameter. Interesting parallels may be drawn for translational research, for example in pharmacokinetic modelling and molecular imaging.
Many modern gamma cameras are optimised for photons below 200 keV and are less suitable for radionuclide therapy dosimetry if higher energy photons are involved. SPECT overcomes the problem of superimposition of target and other activity, but spatial resolution at depth is always worse than in planar imaging, so quantification has to rely on more or less representative phantom models [105]. PET has a 30-40 times higher sensitivity for a given spatial resolution that is typically 5 × 5 mm. This is a major advantage over SPECT in pharmacokinetic dynamic modelling. Correction for attenuation has become relatively easy with the new PET/CT and SPECT/CT cameras, but other correction factors, e.g. for scatter, linearity and calibration, are also critical. Partial volume effects are a major issue in the quantification of small tumours and occur at below about twice the spatial resolution. This is thus more of a problem for SPECT (spatial resolution for 131I, 25-30 mm) than for PET (spatial resolution 6-8 mm). Finally, as discussed in the radiopeptide section, the radionuclides used for dosimetry must show similar biochemical and physical behaviour to those used for therapy. As previously highlighted, the use of 124I PET and 86Y-DOTATOC PET dosimetry [25, 59] has been a landmark development. PET/CT and SPECT/CT and “molecular-radiopharmaceutical developments offer major opportunities for radionuclide therapy dosimetry [1]. Further improvements in the performance of these cameras, and hence dosimetry, can be foreseen. Also, the proliferation of microsystems such as micro-PET/SPECT/CT/MRI is allowing dynamic in vivo animal research, increasing our knowledge of radiopharmaceutical biodistribution, improving quantitation and permitting early selection of therapeutic radiopharmaceuticals. Table 2 lists some of the most important methodological issues involved in performing accurate dosimetry today.
Table 2.Methodological issues in performing clinical dosimetry- Diagnostic tracer and/or therapeutic activity study- Planar and/or tomographic (SPECT and/or PET) quantification- Dynamic and/or multiple time point activity sampling- Linearity of detector response in low and/or high activity- Correction factors for attenuation, scatter and/or partial volume effects- Nuclear medicine and/or radiological volume and response- Standard (MIRD,...) and/or simulative (Monte Carlo,...) modelling- Tissue heterogeneity and/or spatial resolution limits- Treatment of minimal residual disease and/or partial volume effects- Disease-induced and/or therapy-induced changes in parameters- Macro- and/or microdosimetry techniques- Animal and/or human dosimetry data
Radiobiology is a science in itself. Nevertheless, up to now very little consideration has been given to the effects of radionuclide therapy at the cellular and molecular level [106]. Rather, extrapolations have been made from EBRT, despite the fundamental differences in radiation kinetics. The majority of observations in EBRT have been made under the condition of a high dose rate, while clinical radionuclide therapy entails a decreasing, low dose rate. The effect of fractionated EBRT is primarily influenced by the 4 R’s of radiobiology: repair of DNA damage, repopulation of tissues, re-oxygenation of tumour and redistribution in the cell cycle. In radionuclide therapy, with its decreasing dose rate, tumour DNA repair takes place simultaneously with sublethal damage. Bystander effects, i.e. radiation-like effects in unhit cells, may be of significance in low-dose, low dose rate radiotherapy [2]. Furthermore, it is becoming increasingly apparent that the physical paradigm of direct cell killing by double-strand DNA breaks is insufficient. Non-DNA targets, such as cell membrane or RNA, may also be critical to target cell death or dysfunction. Moreover, new molecular-targeted oncology treatments may not produce direct cell death, but rather alter biochemical pathways or cellular homeostasis, that may interact with classical radiation targets or produce new radiation targets [107]. In this paradigm, the concept of radiation dose may need re-definition and the future radiation dose may be determined in terms of the biological and functional changes produced and observed, such as by blood markers and by PET, SPECT and/or MRI. Biologically effective dose (BED) is a concept which has been successfully applied to radionuclide therapy through the landmarking studies of Barone et al. [59] using 86Y-labelled DOTATOC and kidney toxicity. BED is the product of the total physical dose multiplied by the “relative effectiveness”, which takes into account radiobiological parameters such as dose rate, radionuclide decay and tumour cell repair time, and allows direct quantitative comparison with EBRT. It should be noted that late-responding normal tissues and slow-growing tumours allow easier modelling than early-responding normal tissues and fast-growing tumours because of repopulation during treatment in the latter. Nevertheless, this may be regarded as a major development, stimulating further research [108] aimed towards the creation of a firm fundamental basis for radionuclide radiotoxicity and radiodosimetry. In future, it looks as if we shall no longer be looking at a “Holy Gray”, but rather at the worldly “BEGray”.
In conclusion, recent developments in molecular medicine, PET/CT and SPECT/CT cameras and radiobiology offer major scientific and clinical opportunities in radionuclide therapy dosimetry. However, only prospective, randomised trials with adequate methodology can provide the evidence that applied clinical dosimetry results in better patient outcome than is achieved with fixed activity dosing methods.

radionuclide therapy
radiotherapy
thyroid carcinoma
neuro-endocrine tumours
solid tumours
lymphoma
radio-immunotherapy
radiation dosimetry


CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.
Introduction
Our research interests have been in the purinergic modulation of vascular inflammatory and cellular immune responses in transplantation settings [1, 2]. It is generally accepted that extracellular nucleotides [e.g., ATP, uridine triphosphate (UTP), adenosine diphosphate (ADP)], and the derivative nucleosides (e.g., adenosine from ATP), are released in a regulated manner by most all cells to provide the primary components for purinergic responses [3]. High levels of ATP may be released by CD4+ and CD8+ T cells upon stimulation with Con A or anti-CD3 mAb and serve to activate cells [4]. Importantly, ATP [or uridine diphosphate (UDP)] stimulation of monocytes, lymphocytes and endothelium induces largely proinflammatory responses, such as the release of interleukin (IL)-1 (or IL-8) [4–6]. On dendritic cells (DC), exposure to extracellular ATP induces migration and differentiation to drive cellular immune responses [7]. Adenosine is also recognized as a bioactive agent in vascular inflammatory states, with effects mediated on both vascular cells and leukocytes [8]. In addition, adenosine has known anti-thrombotic effects, modulates the expression of anti-apoptotic genes and is immunosuppressive [9]. Adenosine is constitutively present in the extracellular space at low concentrations, but under metabolically stressful and hypoxic conditions, the levels rise dramatically [10]. Primary release of the mediator could occur ab initio, or this might follow conversion of released nucleotides to adenosine (see later).
The nucleotide/nucleoside mediators alluded to above bind specific purinergic receptors that comprise the second requirement for this complicated signalling network. Almost all cells carry cell-surface type 2 purinergic (P2) receptors for nucleotides and adenosine or type 1 purinergic (P1) receptors [11]. There are seven ionotropic (P2X), at least eight metabotropic (P2Y) and four adenosine receptor subtypes that have been identified and characterized to date [12]. Multiple P2X and P2Y receptor subtypes are expressed by monocytes and dendritic cells, whereas lymphocytes express only P2Y receptors [11]. These various receptors operate in both auto- and paracrine loops and are considered to play a complex, important role in the regulation of vascular and immune cell-mediated responses. Depending on the P2 or adenosine receptor subtype, the cell types and signalling pathway involved, these receptors might preferentially trigger and mediate short-term (acute) processes that affect metabolism, adhesion, activation or migration. However, purinergic signalling also has profound impacts upon other more protracted reactions, including cell proliferation, differentiation and apoptosis, such as seen in several chronic inflammatory states [12]. These mechanisms could be also implicated in immune memory [9, 13].
The third, and final, component of purinergic signalling systems comprises ecto-nucleotidases [2, 14]. These ecto-enzymes hydrolyze extracellular nucleotides to generate nucleosides that in turn activate adenosine receptors, often with opposing effects to those seen with P2-mediated effects. Within the past decade, ecto-nucleotidases belonging to several enzyme families have been discovered, cloned and functionally characterized by pharmacological means.
In this review, we will focus on CD39, the prototype of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family (EC 3.6.1.5) [15, 16]. These proteins comprise a group of ecto-enzymes that hydrolyze extracellular nucleoside tri- and diphosphates. The ecto-nucleotidase chain or cascade, as initiated by NTPDases, is terminated by ecto-5–nucleotidase (CD73; EC 3.1.3.5) [2, 17]. Together, ecto-5–nucleotidase and adenosine deaminase (ADA; EC 3.5.4.4), another ecto-enzyme that is involved in purine salvage pathways by converting adenosine to inosine, closely regulate local and pericellular extracellular concentrations of adenosine [2]. Most notably, however, in many tissues and cells, NTPDases also comprise dominant parts of a complex cell surface-located nucleotide hydrolyzing and interconverting machinery. Such ensembles also include the ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs), NAD-glycohydrolases, CD38/NADase activity, alkaline phosphatases, diadenosine polyphosphate hydrolases, adenylate kinase, nucleoside diphosphate kinase, and potentially ecto-F1-Fo ATP synthases [2].
Many aspects of these ecto-nucleotidase families and detailed expositions of structure/function relationships of the E-NTPDases and their role in the vasculature and nervous systems have been reviewed recently. The interested reader is referred to the recent special issue of Purinergic Signalling entitled ‘Ecto-nucleotidases–(PUSI 2, 2: 2006). This review will address only the immunobiology of CD39, focusing on T lymphocytes and alluding briefly to co-expression of CD73 by immunosuppressive T cell subsets.
CD39 in the immune system
Recently, almost all of the published investigative focus on CD39 has been on the thromboregulatory properties of CD39 with respect to platelet and endothelial activation [1]. However, CD39 was first described as a B lymphocyte activation marker [18]. This ecto-nucleotidase is also expressed on natural killer (NK) cells, monocytes, DC and subsets of activated T cells [19]. The relevance of the expression of CD39 by these cells is not yet clear.
Development of mutant mice either null for Cd39 or where human CD39 has been over-expressed have provided useful models to study the role of this ecto-nucleotidase in immune responses [20, 21]. In the Cd39-null mouse [20], B cell numbers [anti-CD45RA 14.8; anti-CD22.2 (Lyb-8.20; PharMingen, San Diego CA, USA)] in unstimulated blood and spleen have been noted to be normal (not shown). Somewhat surprisingly, elicited IgG xenoantibody responses to solubilized xenoantigens administered to Cd39-null mice were found to be markedly suppressed [14]. With Dr. M. Cascalho (Mayo Clinic, Rochester, MN, USA), we have investigated whether Cd39-null mice are capable of appropriate antibody responses. We have noted that Cd39-null mice exhibit impaired B cell memory responses to T-dependent antigens. Unexpectedly, however, these mutant mice show a significant increase in the frequency of somatic mutations post-immunization (not shown). These observations suggest that CD39 function may contribute to the affinity maturation of antibody responses and to facilitate post-germinal center terminal B cell differentiation (M. Cascalho et al., manuscript in preparation).
Although CD39 has been shown to be the dominant ecto-nucleotidase expressed by NK and NK-T cells and to impact upon cytokine production ([19]; G. Beldi, unpublished), the relevance of this has not yet been fully elucidated.
CD39 is also the major NTPDase expressed by monocyte-macrophages. Upregulation of tissue factor expression by these cells in vitro and alterations in splenic macrophage populations in vivo have been observed in Cd39-null mice. P2Y receptors that are impacted upon by CD39 are critically linked to monocyte and endothelial cell responsiveness [22, 23]. P2Y receptors on monocytes could be also subject to desensitization, comparable to what we have observed in platelet P2Y1 receptors in Cd39-null mice [20]. Therefore, we were interested to observe major defects in monocycle entry and migration into the substance of Matrigel plugs injected into the subcutaneous tissue of Cd39-null mice. In parallel, we also evaluated parameters of monocyte transendothelial migration influenced by ATP in vitro and noted failure of Cd39-null cells to migrate in response to exogenous nucleotides. This defect could be overcome by co-stimulation with serotonin, suggesting a degree of P2Y receptor desensitization in Cd39-null monocytes [14, 24].
Ecto-enzymes, specifically ecto-nucleotidases, are known to play an important role in leukocyte trafficking but this complex area will not be dealt with here (for an excellent review on this topic, see [25]). However, CD39 has been recognized as a critical control point in the regulation of leukocyte accumulation within hypoxic tissues and a sixfold increase in CD39/CD73 tandem functioning in the setting of hypoxia has been demonstrated [26, 27].
Langerhans cells (LC) are members of the DC family of antigen-presenting cells residing in the skin. NTPDase1 enzymatic function on antigen presenting DC is involved in the recruitment, activation and polarization of naïve T cells. Mizumoto et al. established that LC from the Cd39-null mice do not hydrolyze ATP and ADP, unlike wild-type cells [28]. Cd39-null DC are also markedly unresponsive to ATP and are susceptible to cell death, but only after prolonged exposure to nucleotides [28].
Mutant mice null for Cd39 have amplified inflammatory responses to irritant chemicals. In these Cd39-null mice, there are major defects in dendritic cell formation, antigen presentation and T cell responses to haptens. These result in markedly attenuated responses to contact allergens in type IV hypersensitivity cutaneous responses that are also seen in inflammatory bowel disease models following haptenic stimulation (not shown).
These data suggest that Cd39 expression is required for optimal stimulation of hapten-reactive T cells in mice [28]. Somewhat paradoxically, CD39 also appears to function as an additional recognition structure on haptenated target immunocytes for HLA-A1-restricted, hapten-specific cytotoxic T cells [29]. Curiously, following deletion of Cd39, LC are fully functional with respect to homing and phenotypic maturation. However, the major defect is that these cells appear less able to stimulate T cells. These abnormalities are potentially also relevant to allograft rejection processes [14]. Sequelae of these putative immune abnormalities include the relative failure of Cd39-null mice to reject allografts under limited co-stimulation blockade [30]. These data indicate a previously unrecognized role of CD39 and the effects on nucleotide-mediated signalling in immunological responses [28].
Localization of CD39 within lipid rafts implies that this ecto-enzyme may be involved in cell-cell contacts and signalling [31]. Such membrane micro-domains serve as the ‘message center–in which numerous signalling molecules are concentrated and scaffolding developed [32]. The Cd39-null DC might exhibit defective functions because of putative defects in initiating and maintaining cell-cell contacts. This mechanism speculatively involves facilitating integrin associations by purinergic signals within the ‘immunological synapse–and would have parallels to the way chemokines have been implicated in this process [33–36]. CD39 on DC and/or T cells may be likewise translocated to the immunological synapse during antigen presentation to facilitate intercellular signalling. More typically, CD39 is considered to play a cellular immunoregulatory role by hydrolyzing ATP (and perhaps ADP) released by T cells during antigen presentation and thereby generating adenosine, a known immunosuppressive molecule.
Recent work has also indicated that regulatory CD4+ve CD25+ve T cells (Treg cells) play important roles in the suppression of immunological reactivity and maintenance of tolerance [37, 38]. Patterns of expression of CD39 by Treg and the possibility that the balance of extracellular nucleotides/nucleosides influence(s) the function of these interesting cells have been a major focus of investigation in our laboratories of late.
CD39 expression by immunosuppressive regulatory T cells
Treg are central to the acquisition of immunological tolerance. Here the immune system does not mount cellular responses against specific antigens while reactivity towards other antigens is maintained. The precise mechanisms underlying the acquisition of tolerance are not fully understood. Transplanted graft outcomes in the absence of heavy immunosuppression depend on consistent balances between cytopathic effector cells and Treg [39]. Depletion of the cytopathic T cell clone has been proposed as one mechanism for the induction of tolerance. However, it is now clear that the long-term maintenance of tolerance is also dependent on self-perpetuating immunoregulatory mechanisms that limit or constrain alloresponses. The suppressive Treg populations are considered key to the development and maintenance of peripheral tolerance [33, 40].
Significant deficiencies are apparent in identifying Treg. Although traditionally defined by CD4+CD25+ expression, the latter marker along with other membrane proteins such as GITR and CTLA4 becomes non-specific and somewhat redundant following activation. Such markers are widely upregulated on other cells thereby losing specificity for the Treg population. The forkhead winged transcription factor FoxP3 is specific for Treg; however, its intracellular location limits its usefulness in the study of this population. Moreover, it is now apparent that CD25 does not encompass all Treg as defined by FoxP3+ expression [41].
Similarly, the mechanisms of Treg action are poorly defined. Putative mechanisms of suppression by Treg include cell-to-cell contact predominant in vitro and the release of soluble mediators that may predominate in vivo. As an example, IL-10 and transforming growth factor β have been identified as soluble factors that may mediate Treg suppression. Our recent studies and data from other groups have indicated that adenosine is an important mediator generated by Treg cells, and appears responsible for, at least in part, their functions [42].
Patterns of immune expression of CD39 by Treg were determined using standard techniques; using the Cd39-null mouse cells as negative controls. CD3+, CD8+, NK1.1+, B220+, CD11b+ and CD11c+ cells were positively selected from spleens and lymph nodes of 8- to 10-week-old C57BL6 mice through the use of MACS Sort magnetic beads in MACS LS Separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells enriched for CD4 were obtained from lymph node and spleen preparations using CD4 T cell columns (R&D Systems, Minneapolis, MN, USA). CD4+/CD25+ and CD4+/CD39+ were positively selected using the relevant antibody and MACS Sort magnetic beads in MACS MS Separation columns (Miltenyi Biotech, Bergisch Gladbach, Germany). Where indicated, the same subsets were also sorted after staining the purified CD4+ cells with CD25 or CD39, using the MoFlo cell sorter (BD Biosciences, San Jose, CA, USA). In selected experiments, CD4+/CD25+ cells were purified using the murine CD4+CD25+ T regulatory cell isolation kit from Miltenyi Biotech (Bergisch Gladbach, Germany). The purity of the different cell populations was verified by flow cytometry on FACSort (BD Biosciences, San Jose, CA, USA). Anti-mouse and anti-human CD4, CD8, CD19, B220, NK1.1 and CD25 were from eBiosciences (San Diego, CA, USA), as were anti-mouse CD5, CD62L, CD45RB, interferon (IFN)-γ and IL-4. Purified anti-mouse CD3 and CD28 were from PharMingen (BD Biosciences,San Diego CA, USA). Rabbit anti-mouse CD39 polyclonal antibody was used to stain cells purified from wild-type lymph nodes and from spleen, as described [43].
Using cells harvested from naïve C57BL6 mice, CD39 was found to be expressed by the majority of monocytes and by subsets of lymphocytes, also inferred by gating on forward scatter (FSC) and side scatter (SSC; not shown). Among node-derived lymphocytes, the majority of CD39+ cells are found in CD19+ (or B220+) B cells. The remaining CD39+ cells were shown to reside almost totally within the CD4+ subset (Fig. 1a). There, they consistently range from 8 to 12% of all CD4+ cells in lymph node (and spleen). Further characterization of resting C57BL6 lymphocytes revealed that CD39 is selectively expressed on CD4+/CD25+ T cells (Fig. 1a). CD39 is consistently and abundantly expressed in CD4+/CD25high T cells, while the CD4+/CD25dim populations show a dichotomic expression pattern of CD39, with ~50% of the cells positive. Less than 1% of CD39+ cells were found in the CD4+/CD25− compartment. The majority of peripheral CD4+/CD39+ cells are also CD45RBlow and mostly CD62Llow, with similar expression patterns observed in BALB/c mice (not shown).
Fig. 1
a Expression of CD39 on mouse lymphoid cells. Lymph node cell suspensions were prepared from 8-week-old C57/BL6 mice and were gated based on FSC and SSC parameters (not shown). CD4+ cells were gated based on CD25 expression. CD39 expression on the different subsets is shown in the histograms (open profiles) plotted against an irrelevant control (gray profiles). Differential expression of CD39 on CD4+ cells can be shown to be closely associated with CD25. b RT-PCR analysis of foxp3 mRNA expression. This was done in sorted cells with the following markers: Foxp3+/CD39+, Foxp3+/CD39-, Foxp3-/CD39+ and Foxp3-/CD39-. CD4+ cells obtained from the Foxp3-GFP ‘knockin–animals that had been generated by Mohamed Oukka and Vijay K. Kuchroo with colleagues (Ref). B lymphocytes were used as CD39+ control and included for comparison (striped bars). c RT-PCR analysis of CD73 mRNA expression. This was done in sorted cells with the following markers: Foxp3+/CD39+, Foxp3+/CD39-, Foxp3-/CD39+ and Foxp3-/CD39-. B lymphocytes were used as CD39+ control and included for comparison (striped bars). CD73 is also a useful immunophenotypic marker for Treg cells (not shown); and when combined with CD39 provides near-concordance with foxp3 expression. The Foxp3-/CD39+ subset does not express CD73 and these cells resemble the memory phenotype (not shown)
These results were confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis of selected cell populations. Both CD11b+ and CD11c+ cells expressed high levels of CD39 mRNA, indicating that macrophages and dendritic cells constitutively express CD39. Within the T cell compartment, the highest expression levels of CD39 transcripts were found in CD4+ cells. Gene expression profiling of CD4+ T cells, sorted on the basis of CD39 cell surface expression, indicated that the CD4+/CD39+, but not CD4+/CD39−, T cells robustly express markers of Treg cells [40], viz. Foxp3, CD25, GITR and CTLA-4. Further, CD4+/CD39+ cells are anergic in the absence of IL-2 (not shown) and suppress T effector (CD4+/CD25−) proliferation with an efficacy similar to that observed with classic CD4+/CD25+ Treg (S. Deaglio et al., submitted manuscript).
We have also confirmed the utility of CD39 in defining human Treg populations as comparable patterns of CD39 antigen expression in human cells are observed. The majority of human CD19+ B cells express CD39; however, within the T cell compartment CD39+ cells are present in the CD4+ subset (not shown). As in the mouse, CD39 is highly expressed in the CD4+/CD25high T cells, with only ~50% of cells CD39+ within the CD4+/CD25dim population. Negligible CD39 expression is found in the CD4+/CD25− population (not shown). Gene expression profiling of human CD4+/CD39+ T cells also confirms that these cells express Foxp3, GITR and CD25 in a pattern analogous to that of traditional CD4+/CD25+ Treg (K. Dwyer et al., submitted). Our analysis demonstrates that CD39 expression in both murine and human T cells is restricted to a subpopulation of CD4+/CD25+ cells that expresses markers associated with T regulatory function.
We have further examined the exact relationship between CD39 expression and the regulatory phenotype by using T cells from mutant mice with the green fluorescent protein (GFP) reporter gene introduced into the endogenous Foxp3 locus [designated as ‘Foxp3+(GFP+) knockin–cells; kindly provided for these experiments by M. Oukka and V. K. Kuchroo, Boston, MA, USA] [44]. Four populations could be defined by differential CD39 and Foxp3 expression: Foxp3+/CD39+, Foxp3+/CD39− (minor population grouping), Foxp3−/CD39+ and Foxp3−/CD39−. These populations were sorted and gene expression profiles determined. The GFP+/CD39+ fraction was shown to mirror the genetic profile of Treg, as defined by the presence of Foxp3 transcripts in this positive control (Fig. 1b). Interestingly, the next major subpopulation Foxp3–/CD39+ cells contain T lymphocytes that are not classic Treg and yet appear to be associated with the memory compartment (W. Gao, manuscript in preparation; Fig. 1b).
Foxp3+(GFP+) Treg however were also found to co-express the ecto-nucleotidase CD73, a unique situation amongst T lymphocytes (Fig. 1c). Consistent with other phenotypic data (not shown), RT-PCR analysis confirms that Foxp3+/CD39+ cells have high levels of gene expression of both CD39 and CD73 (Fig. 1b). CD73, which converts AMP to adenosine downstream of CD39, has been independently identified on CD25+ (FoxP3+) Treg and CD25− uncommitted primed precursor Th cells [42]. This recent work further supports our observations that the expression of ecto-nucleotidases and consequent adenosine generation play a role in the mediation of some of the suppressive capabilities of Treg cells. Hence, CD73 is co-expressed with CD39 as a cell surface marker of murine Tregs (Fig. 1c).
Adenosine as a Treg effector molecule
Tregs from mutant mice deficient in CD39 have impaired regulatory function manifesting as a 50% decrease in the ability of Cd39-null Tregs to modulate effector T cell function in vitro and in vivo. These results indicate that CD39 expressed by Treg is the major and rate limiting ecto-nucleotidase responsible for the generation of adenosine and suggest that a putative CD39/CD73-adenosinergic axis (i.e., generating adenosine) may contribute to the immunoregulatory function of Treg (S. Deaglio et al., manuscript submitted; also Fig. 2).
Fig. 2Schematic representation of Treg markers. The cellular phenotype of these suppressive T cells can be defined by FoxP3+/CD39+/CD73+ expression. Phosphohydrolysis of extracellular nucleotides by CD39 and CD73 generates adenosine, which exerts a component of the immunosuppressive effect
Adenosine plays a central and direct role in the regulation of inflammatory responses and limiting inflammatory tissue destruction [9, 10, 45]. Potentially, close cell-cell contacts with pericellular generation of adenosine and regulated expression of adenosine receptors may be important modulatory factors directly suppressing T cell responses. Early in the immune response, adenosine favors recruitment of DC, which initiates specific immune responses [9, 10, 28]. The immunosuppressive effects of adenosine on T cells are thought to be mediated primarily through the A2A receptor. Adenosine inhibits the production of proinflammatory cytokines and superoxide anions. Adenosine activation of A2A receptor also induces heterologous desensitization of chemokine receptors, which are critical in leukocyte trafficking, through the activation of protein kinase A [46].
It is also feasible that the suppression mediated via Treg-generated adenosine could be exerted indirectly through downregulation of co-stimulatory molecules on DC or by competition for other non-characterized signal molecules [47]. Irrespective of the mechanism of action, we have shown that FoxP3+ Tregs are the only T cells that contain the full enzymatic machinery necessary to generate adenosine. This effect, in conjunction with the expression of the adenosine A2A receptor on effector (CD4+/CD25−) T cells, generates immunosuppressive loops limiting effector cell proliferation both in vitro and in vivo (S. Deaglio et al., submitted).
Immune deviation and autoimmune diathesis in Cd39-null mice
The pathophysiological relevance of the CD39-adenosinergic loop was further tested by examining effects of exogenous, pharmacological adenosine A2A receptor agonists on T cells, committed to either Th1 or Th2 lineages [48]. Adenosine might contribute to the resolution of inflammation by facilitating Th2 pathways by the inhibition of Th1 cell functions [49]; this property has been addressed further in the Cd39-null mice. Subsets of naïve CD4+ T cells were polarized to a Th1 or Th2 phenotype through the addition of IL-12 (10 ng/ml) and anti-IL-4 (10 μg/ml) or IL-4 (10 ng/ml) and anti-IFN-γ (10 μg/ml), respectively, following stimulation with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml). The polarization of cells was confirmed by intracellular cytokine staining and gene profiling via RT-PCR analysis.
Heightened expression of the A2A receptor, as measured by RT-PCR, could be detected in CD4+/CD25− cells polarized in vitro to Th1, but not Th2, phenotypes at day 3 (not shown). In keeping with this observation, the addition of the adenosine A2A receptor agonist, ATL146e (kind gift from J. Linden, Adenosine Therapeutics), at day 3 of culture, when all cells are already committed to either a Th1 or Th2 phenotype, selectively inhibits Th1 proliferation. This suggests once more that the A2A receptor is the critical immunomodulatory adenosine receptor [9]. No inhibition of the Th2 proliferation response by ATL146e [50] could be noted (Fig. 3a).
Fig. 3CD39 generation of adenosine preferentially regulates Th1 immune responses. a 3H-thymidine incorporation of CD4+ T cells purified from WT mice as polarized towards a Th1 or a Th2 phenotype for 5 days. The selective adenosine A2A agonist ATL146e was added after 3 days of culture. Data are expressed as % of inhibition and are the mean of duplicates; error bars represent the SEM of three independent experiments. b CD4+ T cells were purified from Cd39-null (filled histograms) or WT (open histograms) mice and polarized towards a Th1 phenotype. The mRNA was extracted at day 3 and assayed for IFN-g (left panel) by RT-PCR. Data are representative of more than four independent experiments. c CD4+ T cells were purified from Cd39-null (filled histograms) or WT (open histograms) mice and polarized towards a Th1 phenotype. The indicated adenosine receptor agonists and antagonists were added at the beginning of the experiment (left panel). d This panel shows the effects of apyrase on IFN-g production by Cd39-null CD4+ T cells polarized towards a Th1 phenotype. For these experiments, mRNA was extracted at day 3 and assayed for IFN-g (RT-PCR). Representative data are shown from three independent experiments for each. e Representative images of Cd39-null mouse on C57BL/6/129 SVJ (upper panel) or BALB/c backgrounds (lower panel) manifesting alopecia. Histology of skin samples obtained from Cd39-null mice affected by alopecia. Uninvolved skin areas from the same animals as well as samples from WT mice were used for comparison. Biopsies were fixed and stained for CD4 cells. Original magnifications: ×10 for the upper middle panel, ×40 for the upper right and ×20 for the lower panels. f Skin samples were obtained from Cd39-null mice affected by alopecia, the tissue homogenized and mRNA extracted. Cytokine profiling was performed by RT-PCR, using a preamplification technique. Uninvolved skin from the same animal or a matched WT mouse used as controls. Representative data are from three animals
Consistent with these results, in vitro polarization of CD4+ T cells towards a Th1 phenotype results in the increased production of IFN-γ by Cd39-null T cells, with a Th1-deviated phenotype (Fig. 3b). Addition of ATL146e [50], or soluble NTPDases (apyrase), strongly inhibits the Th1 response in both the wild-type and Cd39-null polarized cells confirming that A2A receptors are present and functional in both groups. These data suggest lack of substrate in the Cd39-null cells to be responsible for the observed Th1 bias (Fig. 3c). The A2A antagonist 8-(3-chloro-styryl) caffeine was shown to augment IFN-γ production in wild-type cells polarized to a Th1 phenotype (Fig. 3c), further supporting this hypothesis. In addition, reconstitution of Cd39-null cells with apyrase restores the catalytic potential of the cells, producing adenosine and inhibiting IFN-γ production in a dose-dependent manner, effects similar to that of ATL146e (Fig. 3d).
Additional evidence that the perturbation in adenosine generation causes Th1 deviation, is that Cd39-null mice spontaneously develop autoimmune alopecia. Fifteen per cent (9/59) of designated Cd39-null animals develop skin lesions characterized by extensive and well-demarcated hair loss (Fig. 3e). No lesions are observed in age-matched WT controls (0/28). Similar lesions were observed in Cd39-null mice on the BALB/c background suggesting that the phenotype is strain independent (Fig. 3e). The alopecia typically appears at ~20–0 weeks of age in the facial region and thereafter extends out to include the trunk. The skin lesions are characterized by the presence of a population of CD4+ and CD8+ lymphocytes within the damaged hair follicles (Fig. 3e). Moreover, sixfold increases in IFN-γ transcript levels are noted in areas of skin with active disease, when compared to areas of uninvolved skin from the same animal or from wild type controls (Fig. 3f). In addition, there is increased expression of IL-2, Foxp3 and CTLA-4, consistent with the cellular infiltration. IL-4 and IL-10 were not detected (Fig. 3f). Autoimmune manifestations impacting other organ systems and on renal function are under evaluation (D. Friedman, not shown).
These data validate the importance of adenosine in directing T cell subset differentiation and support a role for CD39 in orchestrating Treg cell suppressive responses under both in vitro and in vivo conditions.
Summation
This review summarizes components of extracellular nucleotide-mediated signalling pathway in T cells that are impacted upon largely by CD39, the prototypic member of the E-NTPDase family of ecto-nucleotidases. Modulated, distinct NTPDase expression appears to regulate nucleotide- and nucleoside-mediated signalling in the immune system. As the vasculature uses similar mediators to regulate blood fluidity and hemostasis, expression of CD39 on either endothelial or immune cells might allow for full integration of vascular inflammatory and immune cell reactions at sites of injury.
There is a wide field for future investigations of the role of nucleotides, nucleosides and ecto-nucleotidases in immune-mediated diseases. Increasing interest in this field may open up new avenues for investigation and the development of new treatment modalities for a large variety of illnesses, including atherosclerosis and the vascular or immune inflammation seen in transplant-related diseases.

cd39
dendritic cells
t cells
ntpdase
vasculature
platelet
b cells
immunology
apyrase
ecto-atpase
e-ntpd
kidney
liver