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Imaging the in vivo fate of human T cells following transplantation in immunoincompetent mice — Implications for clinical cell therapy trials
Transplant Immunology 29 (2013) 105–108
Contents lists available at ScienceDirect
Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Brief communication
Imaging the in vivo fate of human T cells following transplantation in immunoincompetent mice — Implications for clinical cell therapy trials David Berglund a,b,⁎, Marie Karlsson a, Senthilkumar Palanisamy c, Björn Carlsson a, Olle Korsgren a, Olof Eriksson c a b c
Department of Immunology, Genetics and Pathology, Section of Clinical Immunology, Uppsala University, Sweden Department of Surgical Sciences, Section of Transplantation Surgery, Uppsala University, Sweden Department of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Sweden
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 23 September 2013 Accepted 24 September 2013 Keywords: T cell In vivo imaging Cell therapy SPECT/CT
a b s t r a c t Many forms of adoptive T cell therapy are on the verge of being translated to the clinic. To gain further insight in their immunomodulating functions and to optimize future clinical trials it is essential to develop techniques to study their homing capacity. CD4+ T cells were labeled using [111In]oxine, and the radioactive uptake was determined in vitro before intravenous injection in immunodeficient mice. In vivo biodistribution of [111In] oxine-labeled cells or tracer alone was subsequently measured by μSPECT/CT and organ distribution. CD4+ T cells incorporated [111In]oxine with higher labeling yield using Ringer-Acetate compared to 0.9% NaCl. Cellular viability after labeling with [111In]oxine was not compromised using less than 0.4 MBq/million cells. After intravenous infusion CD4+ T cells preferentially homed to the liver (p b 0.01) and spleen (p b 0.05). This study presents a protocol for labeling of T cells by [111In]oxine with preserved viability and in vivo tracking by SPECT for up to 8 days, which can easily be translated to clinical cell therapy trials. © 2013 Elsevier B.V. All rights reserved.
T cells play a crucial role in controlling the immune system and have been proposed for the use as cellular therapies in several clinical settings including malignancies [1] (effector T cells) and autoimmune/inflammatory conditions [2] (regulatory T cells). It is valuable to develop tools for tracking of T cells in vivo to evaluate their safety and efficacy. Several different approaches have been described for imaging of cells following infusion, including pre-labeling of cells with radioactive tracers [3] as well as in situ targeting of cell specific reporter genes [4]. However, there is insufficient data on clinically viable approaches to specifically label T cells. A number of radioactive isotopes are used routinely in patient care with extensive safety data available. Out of these, [111In]oxine was assessed to hold most promise for rapid translation to the clinic. [111In]oxine is a gamma-emitting SPECT isotope with a half-life of 3 days which has been extensively used for labeling of leukocytes (peripheral blood mononuclear cells) for molecular imaging of their homing to sites of infections and inflammation [5]. More recently, [111In]oxine-labeling was used to image regulatory macrophages after intravenous infusion in a renal transplant recipient [6]. [111In]oxine has a relatively long half-life and is readily taken up and trapped in Abbreviations: CT, computed tomography; PET, positron emission tomography; SPECT, single photon emission computed tomography; MACS, magnetic activated cell sorting; CM, complete media. ⁎ Corresponding author at: Department of Surgical Sciences, Section of Transplantation Surgery, Uppsala University, Akademiska sjukhuset, Sweden. Tel.: +46 708 20 07 22; fax: +46 18 50 52 61. E-mail addresses: [email protected], [email protected] (D. Berglund). 0966-3274/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.trim.2013.09.009
most cell-types, allowing imaging for several days. Furthermore, functional imaging modalities such as single photon emission tomography (SPECT) in combination with computed tomography (CT) are readily accessible within Nuclear Medicine Departments. We therefore set out to refine a protocol using labeling with [111In]oxine in clinically relevant doses with preserved cell viability, to track T cells in vivo after intravenous injection. This protocol can be translated to clinical cell therapy trials with only minor modifications. CD4+ T cells from healthy blood donors were pre-enriched using magnetic-activated cells sorting (MACS, Miltenyi Biotec) at purities of N90% (results not shown). Cells were cultured over night in complete media (CM) consisting of RPMI-1640 (Gibco, Invitrogen) with 1% penicillin–streptomycin, 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.5% L-glutamine, 0.04% β-mercaptoethanol and supplemented with 10% pooled human AB serum (pooled, sterile filtered, and heat inactivated AB serum from 15 to 20 healthy blood donors; tested for pathogenic contamination according to hospital standards). 37 MBq GMP grade [111In]oxine (Covidien Sweden AB, Solna, Sweden) was obtained in the morning of each experiment. 0.29–2.05 million CD4+ cells were washed in incubation buffer to remove culturing media. The cells were then incubated together with [111In]oxine for 30–90 min in 1 ml Ringer-Acetate + 11 mM glucose, 0.2 M TRIS or 0.9% NaCl + 11 mM glucose. After incubation, excess non-bound radiotracer was removed by washing the cells 3 times by centrifugation followed by re-suspension in 1 ml buffer. Finally, the radioactivity associated to the cells was measured by a well-counter. The uptake was
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normalized to the amount of radiotracer in the incubation buffer as well as the corresponding uptake in 1 million cells to allow for comparison between different batches and incubation conditions. The uptake is reported as MBq/million cells or % incorporated radioactivity per million cells. To assess cellular viability after radiolabeling with [111In]oxine different numbers of T cells were incubated with the same amount of radioactivity (2 or 5 MBq), so that each cell received 1/15–10 Bq/cell. Viability was assessed 48 h after labeling by microscopy using trypan blue exclusion and photographs were obtained of cultured cells (Infinity capture, Lumenera Corp). Unlabeled cells were used as controls. The LD50 of the cells, i.e. the amount of internalized radiation needed to kill 50% of the cells, was assessed by a Variable slope dose–response curve (GraphPad Prism 5.04). All cells were cultured in CM as described above and supplemented with 10% pooled human AB serum and lowdose IL-2 (Proleukin, 30 U/ml). Nu/nu Balb/c mice (n = 10, 18–23 g, Taconic, Denmark) were housed under standard laboratory conditions with free access to food and water. All handling and experiments were carried out in accordance with the guidelines of the Uppsala University and were approved by the local ethics committee for animal research (C223/11). CD4+ T cells from human donors were incubated with [111In]oxine in 1 ml Ringer Acetate + 11 mM glucose for 30 min (Table 1), followed by washing to remove excess non-incorporated radiotracer as described above. Animals were administered [111In]oxine labeled CD4+ T cells (n = 5) or [111In]oxine alone (n = 3) through the tail vein in a maximum volume of 100 μl using an INSUMED Ultrafin 30G 0.3 ml syringe (Artsana, Grandate, Italy). The radioactivity in each syringe was measured both before and after administration to calculate the correct administered dose (net amount of administered [111In]oxine, Table 1). On days 1, 4, 6 and 8 following administration each mouse was anesthetized by 3.0% isoflurane. The animal was placed on a heated gantry bed of a Triumph Trimodality System (GammaMedica ideas Ltd., Northridge, CA, USA), and wrapped in a cloth to preserve body heat. Anesthesia (2.0–2.5% isoflurane in 400 ml/min 50% O2/50% medical air) was administered continuously through a nose cone. Breathing rate was continuously monitored by an integrated sensor. A CT examination was performed using an 8 cm Field of View (FOV) for anatomical correlation and to confirm animal position in the scanner. Next, a tomographical SPECT examination was performed over the same position (75A10 collimators, acquisition over 200–250 keV, 64 projections, 20 s/projection; total examination time: 22 min). After imaging examinations on days 1, 4 and 6 anesthesia was discontinued and the animal was returned to its cage. Image analysis was performed using PMOD v3.13 (PMOD Technologies Ltd., Zurich, Switzerland) software. Volumes of interest (VOIs) were created by drawing regions of interest (ROIs) delineating the heart, liver, lungs, spleen, kidneys and muscle on consecutive slices. All organs except the kidneys and spleen were readily seen on the CT images and could be delineated on morphological images. The kidneys and spleen could however not be resolved on the CT image because of poor soft tissue contrast. VOIs constituting these organs were instead drawn by using the SPECT images as a reference since a high uptake was noted in these organs from the organ distribution experiments. Uptake in tissue decay was corrected to the time of tracer administration to allow for between and within individual comparisons. On day 8, directly following the μSPECT/CT examination, the animals were euthanized by CO2 and excised tissues (blood, heart, lung, spleen, pancreas, kidneys, muscle, bone and injection site) were measured for tissue
weight and for radioactivity by a well-counter. The uptake in each tissue was expressed with muscle as a reference tissue. Data were analyzed using GraphPad Prism 5 (GraphPad, La Jolla, CA, USA) unless otherwise stated. Results at group levels are reported as means ± standard deviation, and statistical difference between groups was assessed by one-way ANOVA at p b 0.05, followed by Bonferroni's Multiple Comparison test, or in the case of cellular viability by Mann Whitney test. The uptake of [111In]oxine by CD4+ T cells was assessed in different incubation buffers (Fig. 1). Highest yield was achieved in RingerAcetate + 11 mM glucose followed by 0.2 M TRIS, where N 45% (corresponding to N 1.2 MBq/million cells) of the incubation dose was associated per million cells after 30 min. No increase was seen when increasing the incubation time to 60 min. The uptake of [111In]oxine was lower in saline (15% of incubated dose/million cells), but increased with longer incubation. Based on the these results, 30 minute incubation in Ringer-Acetate + 11 mM glucose was chosen as incubation conditions for the in vivo studies over 0.2 M TRIS, due to its established use as cellular media during clinical cell transplantation. Viability of CD4+ cells labeled with [111In]oxine was evaluated 48 h after radioactive incubation. Cellular viability was related to the amount of radioactivity per cell and not to the total amount of radioactivity in the incubation media, i.e. 25 million CD4+ cells incubated with 5 MBq [111In]oxine had better viability than 1 million CD4 + cells incubated with 5 MBq. Viability decreased substantially beyond a radioactive load of 0.4 MBq/millon cells (p b 0.0001, Fig. 2). The LD50 of the cells was 0.446 +/− 0.045 MBq/million cells (R2 = 0.9165). Upon intravenous injection [111In]oxine administered alone did not accumulate appreciably in any tissue except the kidneys, which represent renal excretion (Fig. 3). By contrast, after infusion of [111In]oxine labeled CD4+ T cells tracer accumulation was observed in the liver and the spleen as measured by μSPECT/CT imaging with no markedly increased accumulation in any other tissue. Hepatic and splenic uptake reached a maximum on day 1, followed by a gradual decrease to reach a plateau during days 4–8. This is not likely to indicate redistribution of viable cells as no corresponding increase of uptake is seen in other tissues. Instead, this probably reflects loss of viability and cellular membrane integrity in a subpopulation of CD4+ T cells, followed by renal excretion of the released Indium-111. In some mice, due to the fragility of the tail vein, parts of the injected cells were deposited in the soft tissue of the tail. No redistribution of these cells were noted over time indicating that cells not injected intravasally are unable migrate from their original location (Fig. 3, top panels). μSPECT imaging was sensitive enough to detect cells in the liver and spleen up to 8 days, even when administering doses as low as 3.3 MBq. Considering the radionuclide decay over approximately 3 half-lives, less than 0.5 MBq remained at this time. Ex vivo organ distribution following the SPECT examination on day 8 verified the imaging results. Increased uptake of [111In]oxine labeled CD4 + T cells was found in liver (p b 0.01) and spleen (p b 0.05) but not in any other tissues (Fig. 4). Some accumulation was found in kidneys, lungs and bone but this was not significantly higher as compared to administration of [111In]oxine alone. Here, we report a protocol for imaging of the in vivo fate of T cells following intravenous injection. The protocol was developed with the intent on translation to clinical cell therapy trials. The labeling yield using Ringer-Acetate + 11 mM glucose as incubation buffer was
Table 1 Labeling of CD4+ T cells with [111In]oxine for intravenous injection and in vivo imaging, free tracer was used as control. CD4+
Total amount of incorporated [111In]oxine in the labeling procedure Radioactive load per million labeled cells Net amount of administered [111In]oxine
Control
Unit
1
2
3
4
5
1
2
3
MBq MBq/millon cells MBq
9.0 0.35 8.9
5.4 0.27 5.3
4 0.17 3.3
4 0.17 3.4
6.4 0.32 6.3
N/A N/A 6.8
N/A N/A 7.1
N/A N/A 4.6
D. Berglund et al. / Transplant Immunology 29 (2013) 105–108
Fig. 1. In vitro labeling of CD4+ T cells by [111In]oxine. Labeling yield expressed as actual MBq incorporated per million CD4+ cells (left axis) or %ID/million CD4+ cells (right axis) after incubation with 2.6 MBq [111In]Oxine in different incubation media for 30, 60 or 90 min. Stars indicate significant difference between groups.
superior to 0.9% NaCl + 11 mM glucose, suggesting that the former should be used to optimize in vivo follow-up time. This finding may be explained by the differences in osmolality, where Ringer-Acetate (and TRIS 0.2 M) are hypotonic compared to 0.9% NaCl and may result in a slight net influx of liquid into the cells. Next, we investigated the
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effect on cellular viability imposed by the radiation burden from [111In]oxine. We found that the total amount of radioactivity does not affect cellular viability to the same extent as the radioactive load per cell. In particular, the viability was compromised when the incorporated radioactivity exceeded 0.4 MBq/million cells. Using a maximum radioactivity of less than 0.4 MBq/million cells is therefore recommended in future clinical trials. This finding is crucial, as it sets a biological limit for the amount of radioactivity that can be utilized for the in vivo molecular imaging for a particular cell dose. Assuming a cell dose of approximately 1–4 million T cells/kg body weight in a 70 kg individual and a radioactive load of 0.1 MBq/million cells, gives a total radioactive dose in the order of 7–28 MBq [111In]oxine. This dose is well within the range of routine clinical SPECT examinations of infection using labeled leukocytes. Administration of higher cell doses per kg would allow even lower radioactive loads per cell while preserving the imaging potential. We subsequently investigated the possibilities for tracking the distribution of radiolabeled T cells in vivo and could detect hepatic and splenic accumulation of [111In]oxine-labeled cells up to 8 days after administration, both by μSPECT/CT imaging and by ex vivo organ distribution. Uptake in the kidney cortex was also elevated, but this likely represents renal excretion of free tracer as the accumulation was similar to that observed after administration of tracer alone. Accumulation of tracer after injection of labeled T cells to spleen and liver reached a maximum at the first SPECT scan after 24 h (approximately 25 times the uptake in reference tissue muscle), followed by a slight decrease to a plateau between 10 and 20 times above the reference tissue. These results indicate that the majority of the cellular redistribution in
Fig. 2. Viability after radiolabeling with [111In]oxine. Different numbers of CD4+ T cells from three blood donors (each denoted by one of three squares) were incubated with the same amount of total radioactivity (2 or 5 MBq). Viability was assessed with trypan blue exclusion after 48 h. Photographs were obtained in a light microscope. There is a relationship between cell viability and the level of radioactivity per cell, where the frequency of dead cells is increased beyond 0.4 MBq/million cells (dotted line, p b 0.0001). However, cells denoted by black squares were incubated with a total dose of 2 MBq and were not viable to a greater extent compared to cells incubated with a higher amount of total radioactivity 5 MBq (white and dotted squares). It therefore seems like the ratio of total radioactivity:number of T cells, i.e. radioactive uptake per cell, determines the toxic effects more than total radioactivity alone. The mean viability of non-labeled control cells was 5.6% and is visualized by the green horizontal line, this viability is not different compared to cells labeled with b0.4 MBq/million cells (p = 0.26).
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Day 4
Day 6
Day 8
Tracer only
CD4+ T cells
Day 1
Fig. 3. In vivo imaging of labeled CD4+ T cells. Representative SPECT/CT images following administration of CD4+ T cells (top panels) and [111In]oxine (bottom panels) in nu/nu mice. SPECT imaging shows hepatic (white arrows) and splenic (red arrows) accumulation of CD4+ cells. The uptake in the tail in the animal transplanted with CD4+ cells represents residual tracer at injection site.
this model occurs during the first day after infusion. Studies of cellular homing often show considerable accumulation of transplanted cells in lung tissue, shortly after intravenous administration [7]. This likely reflects a first-pass effect of cells trapped in alveolar sinusoids during the initial biodistribution phase, as it can be avoided by intraarterial administration in the ascending aorta. Trapped cells are usually slowly released from the lungs during the first hours following trapping, and in this study, no significant uptake resulting from a potential first-pass effect in lungs is seen after 24 h. To conclude, the present study shows that [111In]oxine and SPECT can be used to label T cells without compromising their viability and track their in vivo fate in clinically relevant doses. This provides a tool to gain further insight in the
immunomodulating properties of T cells as they are introduced in clinical cell therapy trials. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by grants from the Swedish Medical Research Council (65X-12219-15-6), JDRF, the Tommy and Gösta Andersson Memorial Foundation, the Professor Lars-Erik Gelin Memorial Foundation and Barndiabetesfonden. OK's position is supported by the National Institutes of Health (2U01AI065192-06) and OE's position is supported by EXODIAB (Excellence of Diabetes Research in Sweden). References
Fig. 4. Biodistribution of radiolabeled CD4+ T cells. Biodistribution of radiolabeled CD4+ T cells (n = 5) 8 days after transplantation as measured by organ distribution, compared to the tissue distribution of [111In]oxine administered alone (n = 3). CD4+ T cells accumulated in the liver (p b 0.01) and spleen (p b 0.05). Stars indicate a significantly different tissue uptake compared to [111In]oxine administered alone.
[1] Kalos M, June CH. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 2013;39:49–60. [2] Riley JL, June CH, Blazar BR. Human T regulatory cell therapy: take a billion or so and call me in the morning. Immunity 2009;30:656–65. [3] Rini JN, Bhargava KK, Tronco GG, Singer C, Caprioli R, Marwin SE, et al. PET with FDGlabeled leukocytes versus scintigraphy with 111In-oxine-labeled leukocytes for detection of infection. Radiology 2006;238:978–87. [4] Sharif-Paghaleh E, Sunassee K, Tavare R, Ratnasothy K, Koers A, Ali N, et al. In vivo SPECT reporter gene imaging of regulatory T cells. PLoS One 2011;6:e25857. [5] Roca M, de Vries EF, Jamar F, Israel O, Signore A. Guidelines for the labelling of leucocytes with (111)In-oxine. Inflammation/Infection Taskgroup of the European Association of Nuclear Medicine. Eur J Nucl Med Mol Imaging 2010;37:835–41. [6] Hutchinson JA, Riquelme P, Sawitzki B, Tomiuk S, Miqueu P, Zuhayra M, et al. Cutting Edge: immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients. J Immunol 2011;187:2072–8. [7] Eriksson O, Sadeghi A, Carlsson B, Eich T, Lundgren T, Nilsson B, et al. Distribution of adoptively transferred porcine T-lymphoblasts tracked by (18)F-2-fluoro-2-deoxy-Dglucose and positron emission tomography. Nucl Med Biol 2011;38:827–33.
Contents lists available at ScienceDirect
Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Brief communication
Imaging the in vivo fate of human T cells following transplantation in immunoincompetent mice — Implications for clinical cell therapy trials David Berglund a,b,⁎, Marie Karlsson a, Senthilkumar Palanisamy c, Björn Carlsson a, Olle Korsgren a, Olof Eriksson c a b c
Department of Immunology, Genetics and Pathology, Section of Clinical Immunology, Uppsala University, Sweden Department of Surgical Sciences, Section of Transplantation Surgery, Uppsala University, Sweden Department of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Sweden
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 23 September 2013 Accepted 24 September 2013 Keywords: T cell In vivo imaging Cell therapy SPECT/CT
a b s t r a c t Many forms of adoptive T cell therapy are on the verge of being translated to the clinic. To gain further insight in their immunomodulating functions and to optimize future clinical trials it is essential to develop techniques to study their homing capacity. CD4+ T cells were labeled using [111In]oxine, and the radioactive uptake was determined in vitro before intravenous injection in immunodeficient mice. In vivo biodistribution of [111In] oxine-labeled cells or tracer alone was subsequently measured by μSPECT/CT and organ distribution. CD4+ T cells incorporated [111In]oxine with higher labeling yield using Ringer-Acetate compared to 0.9% NaCl. Cellular viability after labeling with [111In]oxine was not compromised using less than 0.4 MBq/million cells. After intravenous infusion CD4+ T cells preferentially homed to the liver (p b 0.01) and spleen (p b 0.05). This study presents a protocol for labeling of T cells by [111In]oxine with preserved viability and in vivo tracking by SPECT for up to 8 days, which can easily be translated to clinical cell therapy trials. © 2013 Elsevier B.V. All rights reserved.
T cells play a crucial role in controlling the immune system and have been proposed for the use as cellular therapies in several clinical settings including malignancies [1] (effector T cells) and autoimmune/inflammatory conditions [2] (regulatory T cells). It is valuable to develop tools for tracking of T cells in vivo to evaluate their safety and efficacy. Several different approaches have been described for imaging of cells following infusion, including pre-labeling of cells with radioactive tracers [3] as well as in situ targeting of cell specific reporter genes [4]. However, there is insufficient data on clinically viable approaches to specifically label T cells. A number of radioactive isotopes are used routinely in patient care with extensive safety data available. Out of these, [111In]oxine was assessed to hold most promise for rapid translation to the clinic. [111In]oxine is a gamma-emitting SPECT isotope with a half-life of 3 days which has been extensively used for labeling of leukocytes (peripheral blood mononuclear cells) for molecular imaging of their homing to sites of infections and inflammation [5]. More recently, [111In]oxine-labeling was used to image regulatory macrophages after intravenous infusion in a renal transplant recipient [6]. [111In]oxine has a relatively long half-life and is readily taken up and trapped in Abbreviations: CT, computed tomography; PET, positron emission tomography; SPECT, single photon emission computed tomography; MACS, magnetic activated cell sorting; CM, complete media. ⁎ Corresponding author at: Department of Surgical Sciences, Section of Transplantation Surgery, Uppsala University, Akademiska sjukhuset, Sweden. Tel.: +46 708 20 07 22; fax: +46 18 50 52 61. E-mail addresses: [email protected], [email protected] (D. Berglund). 0966-3274/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.trim.2013.09.009
most cell-types, allowing imaging for several days. Furthermore, functional imaging modalities such as single photon emission tomography (SPECT) in combination with computed tomography (CT) are readily accessible within Nuclear Medicine Departments. We therefore set out to refine a protocol using labeling with [111In]oxine in clinically relevant doses with preserved cell viability, to track T cells in vivo after intravenous injection. This protocol can be translated to clinical cell therapy trials with only minor modifications. CD4+ T cells from healthy blood donors were pre-enriched using magnetic-activated cells sorting (MACS, Miltenyi Biotec) at purities of N90% (results not shown). Cells were cultured over night in complete media (CM) consisting of RPMI-1640 (Gibco, Invitrogen) with 1% penicillin–streptomycin, 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.5% L-glutamine, 0.04% β-mercaptoethanol and supplemented with 10% pooled human AB serum (pooled, sterile filtered, and heat inactivated AB serum from 15 to 20 healthy blood donors; tested for pathogenic contamination according to hospital standards). 37 MBq GMP grade [111In]oxine (Covidien Sweden AB, Solna, Sweden) was obtained in the morning of each experiment. 0.29–2.05 million CD4+ cells were washed in incubation buffer to remove culturing media. The cells were then incubated together with [111In]oxine for 30–90 min in 1 ml Ringer-Acetate + 11 mM glucose, 0.2 M TRIS or 0.9% NaCl + 11 mM glucose. After incubation, excess non-bound radiotracer was removed by washing the cells 3 times by centrifugation followed by re-suspension in 1 ml buffer. Finally, the radioactivity associated to the cells was measured by a well-counter. The uptake was
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D. Berglund et al. / Transplant Immunology 29 (2013) 105–108
normalized to the amount of radiotracer in the incubation buffer as well as the corresponding uptake in 1 million cells to allow for comparison between different batches and incubation conditions. The uptake is reported as MBq/million cells or % incorporated radioactivity per million cells. To assess cellular viability after radiolabeling with [111In]oxine different numbers of T cells were incubated with the same amount of radioactivity (2 or 5 MBq), so that each cell received 1/15–10 Bq/cell. Viability was assessed 48 h after labeling by microscopy using trypan blue exclusion and photographs were obtained of cultured cells (Infinity capture, Lumenera Corp). Unlabeled cells were used as controls. The LD50 of the cells, i.e. the amount of internalized radiation needed to kill 50% of the cells, was assessed by a Variable slope dose–response curve (GraphPad Prism 5.04). All cells were cultured in CM as described above and supplemented with 10% pooled human AB serum and lowdose IL-2 (Proleukin, 30 U/ml). Nu/nu Balb/c mice (n = 10, 18–23 g, Taconic, Denmark) were housed under standard laboratory conditions with free access to food and water. All handling and experiments were carried out in accordance with the guidelines of the Uppsala University and were approved by the local ethics committee for animal research (C223/11). CD4+ T cells from human donors were incubated with [111In]oxine in 1 ml Ringer Acetate + 11 mM glucose for 30 min (Table 1), followed by washing to remove excess non-incorporated radiotracer as described above. Animals were administered [111In]oxine labeled CD4+ T cells (n = 5) or [111In]oxine alone (n = 3) through the tail vein in a maximum volume of 100 μl using an INSUMED Ultrafin 30G 0.3 ml syringe (Artsana, Grandate, Italy). The radioactivity in each syringe was measured both before and after administration to calculate the correct administered dose (net amount of administered [111In]oxine, Table 1). On days 1, 4, 6 and 8 following administration each mouse was anesthetized by 3.0% isoflurane. The animal was placed on a heated gantry bed of a Triumph Trimodality System (GammaMedica ideas Ltd., Northridge, CA, USA), and wrapped in a cloth to preserve body heat. Anesthesia (2.0–2.5% isoflurane in 400 ml/min 50% O2/50% medical air) was administered continuously through a nose cone. Breathing rate was continuously monitored by an integrated sensor. A CT examination was performed using an 8 cm Field of View (FOV) for anatomical correlation and to confirm animal position in the scanner. Next, a tomographical SPECT examination was performed over the same position (75A10 collimators, acquisition over 200–250 keV, 64 projections, 20 s/projection; total examination time: 22 min). After imaging examinations on days 1, 4 and 6 anesthesia was discontinued and the animal was returned to its cage. Image analysis was performed using PMOD v3.13 (PMOD Technologies Ltd., Zurich, Switzerland) software. Volumes of interest (VOIs) were created by drawing regions of interest (ROIs) delineating the heart, liver, lungs, spleen, kidneys and muscle on consecutive slices. All organs except the kidneys and spleen were readily seen on the CT images and could be delineated on morphological images. The kidneys and spleen could however not be resolved on the CT image because of poor soft tissue contrast. VOIs constituting these organs were instead drawn by using the SPECT images as a reference since a high uptake was noted in these organs from the organ distribution experiments. Uptake in tissue decay was corrected to the time of tracer administration to allow for between and within individual comparisons. On day 8, directly following the μSPECT/CT examination, the animals were euthanized by CO2 and excised tissues (blood, heart, lung, spleen, pancreas, kidneys, muscle, bone and injection site) were measured for tissue
weight and for radioactivity by a well-counter. The uptake in each tissue was expressed with muscle as a reference tissue. Data were analyzed using GraphPad Prism 5 (GraphPad, La Jolla, CA, USA) unless otherwise stated. Results at group levels are reported as means ± standard deviation, and statistical difference between groups was assessed by one-way ANOVA at p b 0.05, followed by Bonferroni's Multiple Comparison test, or in the case of cellular viability by Mann Whitney test. The uptake of [111In]oxine by CD4+ T cells was assessed in different incubation buffers (Fig. 1). Highest yield was achieved in RingerAcetate + 11 mM glucose followed by 0.2 M TRIS, where N 45% (corresponding to N 1.2 MBq/million cells) of the incubation dose was associated per million cells after 30 min. No increase was seen when increasing the incubation time to 60 min. The uptake of [111In]oxine was lower in saline (15% of incubated dose/million cells), but increased with longer incubation. Based on the these results, 30 minute incubation in Ringer-Acetate + 11 mM glucose was chosen as incubation conditions for the in vivo studies over 0.2 M TRIS, due to its established use as cellular media during clinical cell transplantation. Viability of CD4+ cells labeled with [111In]oxine was evaluated 48 h after radioactive incubation. Cellular viability was related to the amount of radioactivity per cell and not to the total amount of radioactivity in the incubation media, i.e. 25 million CD4+ cells incubated with 5 MBq [111In]oxine had better viability than 1 million CD4 + cells incubated with 5 MBq. Viability decreased substantially beyond a radioactive load of 0.4 MBq/millon cells (p b 0.0001, Fig. 2). The LD50 of the cells was 0.446 +/− 0.045 MBq/million cells (R2 = 0.9165). Upon intravenous injection [111In]oxine administered alone did not accumulate appreciably in any tissue except the kidneys, which represent renal excretion (Fig. 3). By contrast, after infusion of [111In]oxine labeled CD4+ T cells tracer accumulation was observed in the liver and the spleen as measured by μSPECT/CT imaging with no markedly increased accumulation in any other tissue. Hepatic and splenic uptake reached a maximum on day 1, followed by a gradual decrease to reach a plateau during days 4–8. This is not likely to indicate redistribution of viable cells as no corresponding increase of uptake is seen in other tissues. Instead, this probably reflects loss of viability and cellular membrane integrity in a subpopulation of CD4+ T cells, followed by renal excretion of the released Indium-111. In some mice, due to the fragility of the tail vein, parts of the injected cells were deposited in the soft tissue of the tail. No redistribution of these cells were noted over time indicating that cells not injected intravasally are unable migrate from their original location (Fig. 3, top panels). μSPECT imaging was sensitive enough to detect cells in the liver and spleen up to 8 days, even when administering doses as low as 3.3 MBq. Considering the radionuclide decay over approximately 3 half-lives, less than 0.5 MBq remained at this time. Ex vivo organ distribution following the SPECT examination on day 8 verified the imaging results. Increased uptake of [111In]oxine labeled CD4 + T cells was found in liver (p b 0.01) and spleen (p b 0.05) but not in any other tissues (Fig. 4). Some accumulation was found in kidneys, lungs and bone but this was not significantly higher as compared to administration of [111In]oxine alone. Here, we report a protocol for imaging of the in vivo fate of T cells following intravenous injection. The protocol was developed with the intent on translation to clinical cell therapy trials. The labeling yield using Ringer-Acetate + 11 mM glucose as incubation buffer was
Table 1 Labeling of CD4+ T cells with [111In]oxine for intravenous injection and in vivo imaging, free tracer was used as control. CD4+
Total amount of incorporated [111In]oxine in the labeling procedure Radioactive load per million labeled cells Net amount of administered [111In]oxine
Control
Unit
1
2
3
4
5
1
2
3
MBq MBq/millon cells MBq
9.0 0.35 8.9
5.4 0.27 5.3
4 0.17 3.3
4 0.17 3.4
6.4 0.32 6.3
N/A N/A 6.8
N/A N/A 7.1
N/A N/A 4.6
D. Berglund et al. / Transplant Immunology 29 (2013) 105–108
Fig. 1. In vitro labeling of CD4+ T cells by [111In]oxine. Labeling yield expressed as actual MBq incorporated per million CD4+ cells (left axis) or %ID/million CD4+ cells (right axis) after incubation with 2.6 MBq [111In]Oxine in different incubation media for 30, 60 or 90 min. Stars indicate significant difference between groups.
superior to 0.9% NaCl + 11 mM glucose, suggesting that the former should be used to optimize in vivo follow-up time. This finding may be explained by the differences in osmolality, where Ringer-Acetate (and TRIS 0.2 M) are hypotonic compared to 0.9% NaCl and may result in a slight net influx of liquid into the cells. Next, we investigated the
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effect on cellular viability imposed by the radiation burden from [111In]oxine. We found that the total amount of radioactivity does not affect cellular viability to the same extent as the radioactive load per cell. In particular, the viability was compromised when the incorporated radioactivity exceeded 0.4 MBq/million cells. Using a maximum radioactivity of less than 0.4 MBq/million cells is therefore recommended in future clinical trials. This finding is crucial, as it sets a biological limit for the amount of radioactivity that can be utilized for the in vivo molecular imaging for a particular cell dose. Assuming a cell dose of approximately 1–4 million T cells/kg body weight in a 70 kg individual and a radioactive load of 0.1 MBq/million cells, gives a total radioactive dose in the order of 7–28 MBq [111In]oxine. This dose is well within the range of routine clinical SPECT examinations of infection using labeled leukocytes. Administration of higher cell doses per kg would allow even lower radioactive loads per cell while preserving the imaging potential. We subsequently investigated the possibilities for tracking the distribution of radiolabeled T cells in vivo and could detect hepatic and splenic accumulation of [111In]oxine-labeled cells up to 8 days after administration, both by μSPECT/CT imaging and by ex vivo organ distribution. Uptake in the kidney cortex was also elevated, but this likely represents renal excretion of free tracer as the accumulation was similar to that observed after administration of tracer alone. Accumulation of tracer after injection of labeled T cells to spleen and liver reached a maximum at the first SPECT scan after 24 h (approximately 25 times the uptake in reference tissue muscle), followed by a slight decrease to a plateau between 10 and 20 times above the reference tissue. These results indicate that the majority of the cellular redistribution in
Fig. 2. Viability after radiolabeling with [111In]oxine. Different numbers of CD4+ T cells from three blood donors (each denoted by one of three squares) were incubated with the same amount of total radioactivity (2 or 5 MBq). Viability was assessed with trypan blue exclusion after 48 h. Photographs were obtained in a light microscope. There is a relationship between cell viability and the level of radioactivity per cell, where the frequency of dead cells is increased beyond 0.4 MBq/million cells (dotted line, p b 0.0001). However, cells denoted by black squares were incubated with a total dose of 2 MBq and were not viable to a greater extent compared to cells incubated with a higher amount of total radioactivity 5 MBq (white and dotted squares). It therefore seems like the ratio of total radioactivity:number of T cells, i.e. radioactive uptake per cell, determines the toxic effects more than total radioactivity alone. The mean viability of non-labeled control cells was 5.6% and is visualized by the green horizontal line, this viability is not different compared to cells labeled with b0.4 MBq/million cells (p = 0.26).
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D. Berglund et al. / Transplant Immunology 29 (2013) 105–108
Day 4
Day 6
Day 8
Tracer only
CD4+ T cells
Day 1
Fig. 3. In vivo imaging of labeled CD4+ T cells. Representative SPECT/CT images following administration of CD4+ T cells (top panels) and [111In]oxine (bottom panels) in nu/nu mice. SPECT imaging shows hepatic (white arrows) and splenic (red arrows) accumulation of CD4+ cells. The uptake in the tail in the animal transplanted with CD4+ cells represents residual tracer at injection site.
this model occurs during the first day after infusion. Studies of cellular homing often show considerable accumulation of transplanted cells in lung tissue, shortly after intravenous administration [7]. This likely reflects a first-pass effect of cells trapped in alveolar sinusoids during the initial biodistribution phase, as it can be avoided by intraarterial administration in the ascending aorta. Trapped cells are usually slowly released from the lungs during the first hours following trapping, and in this study, no significant uptake resulting from a potential first-pass effect in lungs is seen after 24 h. To conclude, the present study shows that [111In]oxine and SPECT can be used to label T cells without compromising their viability and track their in vivo fate in clinically relevant doses. This provides a tool to gain further insight in the
immunomodulating properties of T cells as they are introduced in clinical cell therapy trials. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by grants from the Swedish Medical Research Council (65X-12219-15-6), JDRF, the Tommy and Gösta Andersson Memorial Foundation, the Professor Lars-Erik Gelin Memorial Foundation and Barndiabetesfonden. OK's position is supported by the National Institutes of Health (2U01AI065192-06) and OE's position is supported by EXODIAB (Excellence of Diabetes Research in Sweden). References
Fig. 4. Biodistribution of radiolabeled CD4+ T cells. Biodistribution of radiolabeled CD4+ T cells (n = 5) 8 days after transplantation as measured by organ distribution, compared to the tissue distribution of [111In]oxine administered alone (n = 3). CD4+ T cells accumulated in the liver (p b 0.01) and spleen (p b 0.05). Stars indicate a significantly different tissue uptake compared to [111In]oxine administered alone.
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