Journal of Immunological Methods 314 (2006) 123–133www.elsevier.com/locate/jim

Research paper

A rapid method for labelling CD4+ T cells with ultrasmallparamagnetic iron oxide nanoparticles for magneticresonance imaging that preserves proliferative,regulatory and migratory behaviour in vitro

O.A. Garden a,b,1, P.R. Reynolds a,c,1, J. Yates a, D.J. Larkman d, F.M. Marelli-Berg a,D.O. Haskard e, A.D. Edwards c,d, A.J.T. George a,⁎

a Department of Immunology, Imperial College London, Hammersmith Campus, Du Cane Road, W12 ONN, UKb Department of Veterinary Clinical Sciences, The Royal Veterinary College, Hawkshead Campus, North Mymms,

Hatfield, Hertfordshire, AL9 7TA, UKc Department of Paediatrics, Imperial College London, Hammersmith Campus, Du Cane Road, W12 ONN, UK

d Department of Imaging Sciences, Imperial College London and MRC Clinical Sciences Centre, Hammersmith Campus,Du Cane Road, W12 ONN, UK

e BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College London, Hammersmith Campus,Du Cane Road, W12 ONN, UK

Received 19 May 2006; accepted 7 June 2006Available online 12 July 2006

Abstract

A number of techniques have been developed to track the migration of T cells in vivo, but they all suffer significantshortcomings, including the examination of selected organs rather than the organism as a whole — thus precluding longitudinalstudies — or limitations imposed by poor spatial resolution and the application of ionizing radiation. By conjugating the HIV tatpeptide to ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles in a reaction yielding a mean valence of 45, a magneticresonance (MR) contrast agent was synthesised that allowed T cells to be efficiently labelled within just 5 min. The USPIOnanoparticles were incorporated into both the cytoplasm and nucleus of labelled cells, which retained normal in vitro proliferativeresponses to a polyclonal stimulus; suppressive responses mediated by labelled CD4+ CD25+ regulatory T cells; chemotacticresponses to the chemokine CXCL-12; and transmigration of an activated endothelial monolayer. We believe that this rapid,efficient and essentially non-toxic approach to labelling both murine and human T cells for MRI holds considerable promise,paving the way for the wider immunological application of this exciting technology.© 2006 Elsevier B.V. All rights reserved.

Keywords: CD4+ T cell; Magnetic resonance; Contrast agent; Regulatory T cell

⁎ Corresponding author. Tel.: +44 20 8383 1604; fax: +44 20 83832788.

E-mail address: a.george@imperial.ac.uk (A.J.T. George).1 These authors contributed equally to this work and share dual first

authorship.

0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2006.06.010

1. Introduction

A number of techniques have been developed to trackthe migration of T cells in animal models, including thehistological examination of tissue sections by light or

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confocal laser microscopy, intravital microscopy, wholeorgan or body scintigraphy (Iparraguirre and Weninger,2003), and two photon (2P) microscopy (Denk et al.,1990; Scheinecker, 2005). However, these methodssuffer various shortcomings, including the examinationof selected organs rather than the organism as a whole,and the inability to perform longitudinal studies. Anumber of whole-animal approaches have therefore beendeveloped to allow non-invasive, repetitive monitoringof T cell migration, including bioluminescence imaging(BLI), micro-positron emission tomography (PET),single-photon emission computed tomography(SPECT), and magnetic resonance (MR) imaging(Iparraguirre and Weninger, 2003). While highly sensi-tive, BLI, PET and SPECT are limited by relatively poorspatial resolution, short label half-lives, limited tissuepenetration (BLI) and the application of ionizingradiation (PET; SPECT).

Achieving high-resolution images with MRI — to thelevel of tens of microns — can require long acquisitiontimes. This is a significant limitation for in vivo studies,particularly when small quantities of contrast agent arepresent in the target tissue. Much of the published literatureon imaging the transmigration of T cells from the vascularcompartment has therefore used MRmicroscopy, in whichthe subject is perfusion fixed and whole body imaging, ororgan imaging ex vivo, is performed using acquisition timesof several hours (Gimi, 2006). While this strategy achievesthe desired resolution, it precludes dynamic longitudinalstudies. An alternative approach that allows serial MRimaging is to optimize contrast by the local or intraperi-toneal injection of labelled T cells (Moore et al., 2002;Kircher et al., 2003).

In the current study, we have employed ultrasmallsuperparamagnetic iron oxide (USPIO) nanoparticles as aT2 contrast agent for both murine and human CD4+Tcells.Previous MRI studies using iron oxide nanoparticles tolabel cells have employed various methods to internalizecontrast agent, including fluid phase endocytosis (pinocy-tosis), receptor-mediated endocytosis and phagocytosis(Yeh et al., 1993; Josephson et al., 1999; de Vries et al.,2005). However, both the lack of internalizing receptorsexpressed by lymphocytes and the inefficiency of the fluidphase pathway in general have prompted the developmentof alternative strategies to label T cells. Of the variousmembrane-translocating peptides described, the humanimmunodeficiency virus-1 (HIV-1) tat peptide — whichfunctions in viral replication (Green and Loewenstein,1988) — is one of the most widely used (Tung andWeissleder, 2003). We have synthesized a multivalent tatpeptide-coatedUSPIOnanoparticle to label CD4+ Tcells ina five-minute reaction that leaves the cells functionally

intact. This reagent represents a significant advance onpreviously engineered particles that all require considerablylonger reaction times to achieve comparable labellingefficiency (Josephson et al., 1999;Moore et al., 2002; Zhaoet al., 2002), and thus paves the way for longitudinal MRIstudies of CD4+ T cell migration in vivo.

2. Materials and methods

2.1. Synthesis of tat-labelled ultrasmall superparamag-netic iron oxide nanoparticles

USPIO nanoparticles, comprising an iron oxide coreof approximate diameter 5.0 nm, enveloped by a lowmolecular weight dextran cage to confer aqueous solu-bility with a total mean diameter of 25.7±6.1 nm aspreviously used (Reynolds et al., in press), were preparedfor conjugation of the tat peptide by amination of thedextran and activation with N-succinimidyl 3-(2-pyr-idyldithio) propionate (SPDP; Sigma–Aldrich)(Josephson et al., 1999). Tat peptide was synthesizedby a 9-fluorenylmethoxycarbonyl (Fmoc)-based solidphase technique, with purification by high-pressure liq-uid chromatography (Dr. I. Moss, Imperial CollegeLondon Peptide Synthesis Service) (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Lys-Cys-CONH2); aportion of the sample was labelled with FITC. The tatpeptide was incubated with SPDP-USPIO nanoparticlesovernight, before purification by dialysis using a 10 kDamolecular weight cut-off (Visking tubing; MedicellInternational Ltd, London, UK). The efficiency of theconjugation reaction was verified by rapid labelling ofChinese hamster ovary cells. The number of tat peptideresidues conjugated per USPIO nanoparticle (valence)was estimated by measuring the number of availableSPDP residues — assuming 1:1 binding — using theabsorbance of pyridine-2-thione (P2T). This estimate wasconfirmed by cleavage of FITC-labelled tat from USPIOnanoparticles by reduction with dithiothreitol (DTT),quantitating the free peptide by measurement of FITCabsorbance (A494). Tat-USPIO nanoparticles remainedstable for at least 4monthswhen stored at 4 °C in the dark.

Two batches of tat-USPIO nanoparticles were prepared,differing in the conditions of the cross-linking andamination reactions. In the first (‘standard’), a 15.4% v/vconcentration of epichlorohydrin was used in the cross-linking reaction solution of 38.5% v/v strong sodiumhydroxide; crosslinking was allowed to proceed overnight,before extensive dialysis against phosphate buffered saline(PBS); and amination was performed by the addition ofconcentrated ammonia solution (18.8%v/v) and incubationfor 24 h. The mean number of tat molecules per μg iron

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was 3.75×1013; given the concentration of iron (20mg/ml)and thus the nominal concentration ofUSPIOnanoparticles(5.0×1016/ml), the mean valence of the particles wasestimated to be 15. The technique was subsequentlyimproved to increase the mean valence of the nanoparticlesto 45 by reducing the concentration of epichlorohydrin to7.7% v/v; decreasing the crosslinking reaction time to 4 h;and increasing the amination reaction time to 36 h. Thesecond, improved preparation was used in all experimentsunless otherwise indicated.

2.2. Selection of CD4+ T cells

Animals were maintained according to UK HomeOffice regulations and institutional animal welfareguidelines. All murine assays were performed inRPMI-1640 (Life Technologies; Paisley, UK) supple-mented with 100 U/ml penicillin/streptomycin (GIBCO;Paisley, UK), 2 mM L-glutamine (GIBCO), 10 mMHEPES (GIBCO) and 10% v/v heat-inactivated fetal calfserum (FCS; Helena BioSciences, Sunderland, UK).Spleens and selected lymph nodes were maceratedthrough a 70 μm cell strainer, before treatment withRed Cell Lysis Buffer® (Sigma–Aldrich; Dorset, UK).Negative magnetic selection of CD4+ T cells wasperformed with sheep anti-rat DynaBeads® (DynalInvitrogen; Oslo, Norway) to capture cells labelledwith anti-class II (M5/114.15.2), CD8 (53.6.72) andCD32 (2.4.G2) monoclonal antibodies (mAbs). Purity ofCD4+ T cells selected in this way was >90%.

Human T cells were purified from either buffy coatpreparations (Blood Transfusion Service, London, UK)or freshly isolated blood after venesection of healthyvolunteers. Human CD4+ T cells were cultured in RPMI1640 medium supplemented with 2 mM L-glutamine,100 U/ml penicillin/streptomycin and 10% v/v humanAB serum (HS; Biowest, Ringmer, UK). Negative mag-netic selection of CD4+ T cells from peripheral bloodmononuclear cells (PBMCs) — prepared by densitygradient centrifugation—was performed with goat anti-mouse Fc-coated Biomag® beads (Qiagen; MetachemDiagnostics, Northampton, UK) to capture cells labelledwith anti-CD8 (B-H7), CD14 (B-A8), CD16 (B-E16) (allDiaclone; Boldon, UK), CD19 (HD37), CD56(MEM188), glycophorin A (BRIC 256) (all Chemicon;Chandlers Ford, UK), and CD33 (P67.6) and TCR γδ(11F2) mAbs (both Becton Dickinson; Cowley, UK).

2.3. Flow cytometry and PERL staining

Chinese hamster ovary (CHO) cells (Puck et al.,1958) were incubated with fluoresceinated tat-USPIO

nanoparticles (100 μg Fe/ml/107 cells) with agitation forvarying periods of time, before washing in PBS cont-aining 1% v/v FCS, 0.1% w/v sodium azide and 5 mMEDTA (FACS™ buffer). Flow cytometry was thenperformed using a FACSCalibur™ Flow Cytometer(Becton–Dickinson). In separate experiments, humanCD4+ T cells were incubated with tat-USPIO nanopar-ticles (100 μg Fe/ml/107 cells) for varying periods oftime, before preparing cytospins that were subsequentlyfixed in methanol/acetone (1:1 v/v) for 5 min. PERLstaining was then performed by incubating the cytospinswith an equal volume of 2% w/v potassium ferrocyanideand 2% w/v hydrochloric acid for up to 20 min, beforemounting in DPX medium (BDH Ltd).

2.4. Inductively coupled plasma optical emissionspectrometry (ICP-OES)

Iron was quantified using an inductively coupledplasma optical emission spectrometer (Optima™ 3300RL, SoftwareWinlab 32 v2.2; Perkin-Elmer, Cambridge).Cells were suspended in 10% v/v nitric acid, beforesonication (MSE; Soniprep 150) for 30 s and 1/10 v/vdilution in sterile water. All readings were performed intriplicate.

2.5. Selection of CD4+ CD25+ regulatory T cells andco-culture with CD25- T cells

CD4+ CD25+ T cells derived from naïve BALB/cmice were positively selected on MiniMACS® columns(Miltenyi; Bergish Gladbach, Germany), using Strepta-vidin MicroBeads® (Miltenyi) to capture CD4+ cellslabeled with biotinylated anti-CD25 mAb (clone 7D4;BD). Purity of both CD25+ and CD25- T cells was>93%. Purified CD4+ CD25+ T cells (1×105/well) werecultured with CD25- T cells in the presence of EpoxyDynaBeads® (Dynal Invitrogen) coated with anti-CD3and anti-CD28 mAbs, within round-bottom 96-wellplates. After 3 days, proliferation was measured by theincorporation of 3H-methyl thymidine (3H-TdR; Amer-sham Biosciences) over 16 h.

Human CD4+ CD25+ T cells were positively selectedusing anti-CD25-coated Dynabeads®, detaching thecells with DETACHabead® (Dynal Invitrogen). Cellsrecovered in the supernatants were CD4+ CD25-. Purityof bothCD25+ andCD25- Tcells was >90%; the numberof CD25+ T cells isolated was typically 1–3% of totalCD4+ cells. Purified CD4+ CD25+ T cells (2.5×104/well) were cultured with CD25− T cells (2.5×104/well)in the presence of irradiated allogeneic PBMCs (2.5×104/well) in a mixed leucocyte reaction. Proliferation

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was measured by the incorporation of 3H-TdR within thefinal 16 h of a five-day culture period. The Cytotoxic TLymphocyte Leukaemic (CTLL) cell line assay, per-formed in routine fashion, was used to measure IL-2 insupernatants recovered from the cultures on day 3 (Mills,2000).

2.6. Chemotaxis and transmigration assays

Splenic CD4+ T cells selected from naïve C57BL/6mice were cultured in complete medium containingrecombinant human IL-2 (10 IU/ml; ROCHE,Mannheim,Germany) and anti-mouse CD3ε mAb (10 μg/ml 2C11;BD Biosciences) for 10 days to generate blasts, prior tolabelling with the USPIO nanoparticles. Labelled orcontrol, unlabelled CD4+ T cell blasts were seeded intothe upper chambers of 5 μm-pore polycarbonate Trans-wells (1×106/chamber; Costar Ltd, High Wycombe,UK), containing chemotaxis medium in the lowerchambers (CXCL-12 (SDF-1α, 50 ng/ml; PeproTech) inRPMI-1640 with 2% v/v FCS). Cells undergoingchemotaxis to the lower chamber were counted over24 h. BALB/c microvascular endothelial cells (ECs) ofheart and lung origin were isolated according to anestablished protocol, before serial subculture (Marelli-Berg et al., 2000). At confluence, the ECs were activatedwith 600 U/ml IFNγ (Pepro Tech) for 72 h, beforedetachment by trypsinisation. Washed ECs were layeredonto gelatin-coated (Sigma–Aldrich) 3 μm-pore polycar-bonate Transwell inserts (2×104 / chamber; Costar Ltd),for overnight culture in RPMI-1640 containing 10% v/vFCS. Non-adherent ECs were washed off the inserts,before seeding 1×106 labelled or control, unlabellednaïve BALB/c peripheral lymphoid CD4+ T cells into theupper chambers. Cells undergoing transmigration to thelower chamber were counted over 24 h.

2.7. Magnetic resonance imaging

Cells were suspended (10×106/ml) in sterile phos-phate-buffered saline (PBS), before the addition of7.5 μg Fe in the form of tat-USPIO nanoparticles permillion cells. The cell-nanoparticle suspension wasgently shaken at 37 °C in 5% CO2 for 5 min, beforewashing twice in PBS. Aliquots of 1×106 cells wereprepared for transmission electron microscopy inroutine fashion. Cells for imaging were dispensed intoa mould of 2% w/v agarose in 1×Tris-borate-EDTA,before aspirating off the supernatant PBS to leave avolume of 20 μl/well and adding sufficient molten 2%w/v agarose to seal the wells. The phantoms were storedin a sealed container at 4 °C and wrapped in cling film

during imaging to minimise dehydration. Imaging wasperformed using a horizontal bore 9.4 Tesla MRI system(Varian Inc; Palo Alto, CA, USA). A standardised T2measurement protocol was applied, using a spin-echosequence with five slices of 1 mm, four non-arrayedecho times (TE=5.5, 15, 25 and 50 ms) and a fixedrepetition time (TR=1500 ms) collected consecutively(two averages per echo time: matrix 128×128, field ofview 25×25 mm, acquisition time 25 min). The exactspatial resolution achieved was 0.2×0.2 mm2 in plane.A customised program was written using ‘InteractiveData Language' (IDL; Research Systems Incorporated,Colorado, USA) to fit the data — pixel by pixel — to astandard T2 decay model.

3. Results

3.1. Crosslinked USPIO: physical characteristics

The USPIO nanoparticles remained homogeneousafter amination using the improved conjugation tech-nique,with amean (±SD) hydrodiameter of 31.3±8.5 nm(Zetasizer®; Malvern Instruments, UK), compared to25.7±6.1 nm for unmodified nanoparticles. The numberof SPDP groups per nanoparticle (45±5) was inferred byspectrophotometric analysis of the product of thereduction reaction (P2T), using an extinction coefficientat 343 nm of 8.08×103 M−1cm−1. With the tat peptidepresent in excess, each SPDP molecule was assumed tocarry one tat residue. This estimate of valence wasconfirmed by measuring absorbance of FITC-labelled tatreleased by DTT, using an extinction coefficient at494 nm of 73.0×103 M−1cm−1.

3.2. Tat-derivatized USPIO nanoparticles show rapidinternalisation into CHO cells and both murine andhuman CD4+ T cells

As proof of concept, the uptake of FITC-labelled tat-USPIO nanoparticles was initially examined using CHOcells, since they represented a readily accessible, robustlaboratory cell line that would yield some generalconclusions about the handling of the nanoparticles bymammalian cells. Rapid internalisation of labelledUSPIO nanoparticles was observed at both 4 °C and37 °C, with significant labelling after just 10 min (Fig. 1).Longer incubation times resulted in greater uptake of theUSPIO nanoparticles, and uptake was increased from4 °C to 37 °C. No uptake of unconjugated USPIO nano-particles was observed (Fig. 1).

Following these encouraging results in CHO cells, wenext examined the uptake of the USPIO nanoparticles

Fig. 1. Tat-USPIO nanoparticles show rapid internalisation into Chinese hampster ovary (CHO) cells. CHO cells were incubated with fluoresceinatedtat-USPIO nanoparticles (100 μg iron/ml/107 cells) for the durations indicated at either 4 °C or 37 °C. Flow cytometry was performed after washing,using unlabelled cells as a negative control population. Uptake of the tat-USPIO nanoparticles was rapid and was greater at 37 °C than 4 °C;appreciable internalisation was observed after only 10 min at both 4 °C and 37 °C.

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in human and then murine CD4+ T cells, whichrepresented our focus of interest. Loading of theUSPIO nanoparticles into human CD4+ T cells wasexamined using ICP-OES to measure the iron contentof treated cells. A concentration-dependent increase incell labelling was observed when the tat-derivatizednanoparticles were employed in a five-minute reaction,with no uptake of iron when unconjugated USPIO

Fig. 2. Tat derivatization of USPIO nanoparticles allows concentration-depenincubated with tat-USPIO nanoparticles at room temperature (21 °C) for 5 mininductively coupled plasma optical emission spectrometry. Results are expresconcentrations of tat-USPIO nanoparticles yielded increasing cellular uptakverifying that passive uptake is negligible. This experiment is representativetriplicate wells.

nanoparticles were used (Fig. 2). However, cellviability was reduced at the higher concentrations oftat-USPIO nanoparticles: for example, >50% cell deathwas visible by light microscopy 1 h after incubationwith the highest concentration (10 μg Fe/106 cells) —associated with a shrunken appearance of remainingintact cells — and all cells had died by the followingday.

dent uptake by human CD4+ T cells in vitro. Human CD4+ T cells wereat increasing concentrations. The iron content of cells was analysed bysed as iron internalised per million cells, demonstrating that increasinge of iron. Uptake for unmodified USPIO nanoparticles was minimal,of a total of three. Error bars represent standard errors of the mean of

Table 1Improved tat derivatization of USPIO nanoparticles expedites theiruptake by human CD4+T cells in vitro

USPIO preparation Incubation (min) Death (%) Loading (%)

Unmodified 5 <5 <110 <5 <130 <5 <160 <5 <1

Standard tat-USPIO 5 5–10 20–4010 5–10 30–5030 50–70 >9560 >95 >95

High valence tat-USPIO 5 5–10 >9510 30–40 >9530 50–70 >9560 >95 >95

Human CD4+ T cells were incubated with unmodified USPIOnanoparticles or those derivatized with the tat peptide (100 μg Fe/ml/107 cells) at room temperature (21 °C). Two derivatizedpreparations were compared — the improved version with a valenceof 45 and a standard version with a valence of 15. The proportion ofCD4+ T cells labelled with USPIO nanoparticles was determined byPERL staining; cell death was measured by Trypan blue staining.Almost complete internalisation of the optimised tat-USPIO nanopar-ticles occurred within 5 min, versus 30 min for the standardpreparation. Longer incubation times offered no further increase inproportional internalisation for the optimised preparation, butincreased cell death. Internalisation was negligible when unmodifiedUSPIO nanoparticles were used.

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Loading of human CD4+ T cells with tat-derivatizedor unmodified USPIO nanoparticles as a function ofincubation time was examined, using PERL staining todetermine the proportion of T cells containing particles(Table 1). The nanoparticles with increased valence

Fig. 3. Tat-USPIO nanoparticles localise to the cytoplasmic and nuclear comincubated with HIV transactivator peptide-derived USPIO nanoparticles at rtransmission electron microscopy in routine fashion. Uniform cytoplasmic locis apparent after only 5 min incubation with the T cells; nanoparticles are als

prepared in the current study labelled >95% of T cellswithin 5 min, over which time only 5–10% death wasobserved. This performance contrasted with only 20–40% labelling efficiency when the ‘standard’ tat-derivatized USPIO nanoparticles — with their muchlower valence (∼ 15) — were examined over the sameperiod of incubation (Table 1). Increasing the incuba-tion time increased the toxicity of both tat-USPIOpreparations. Cell death was thought to reflect acumulative effect of nanoparticle uptake rather than adelayed effect of cell labelling, since negligibleadditional death was observed over a 1 h periodfollowing each of the shorter labelling reactions (datanot shown). Unmodified USPIO nanoparticles showedonly negligible loading into human CD4+ T cells(Table 1).

We then investigated whether murine T cells could besimilarly labelled with the improved tat-USPIO nano-particles. Over 95% of murine CD4+ T cells showeduptake of the improved tat-USPIO preparation in just5 min, with negligible cell death as assessed by Trypanblue staining. However, longer reaction times resulted insignificant cell death, in similar fashion to the humanCD4+ T cells; once again, this was thought to reflectcumulative uptake of the nanoparticles rather than adelayed effect of cell labelling, since incubation inmedium alone for 1 h after the labelling reaction did notelicit any additional death with time (data not shown).Electron microscopy of murine CD4+ T cells labelledwith tat-USPIO nanoparticles in a five-minute reactionclearly demonstrated cytoplasmic and nuclear distribu-tion of the contrast agent (Fig. 3).

partments of labelled murine CD4+ T cells. Murine CD4+ T cells wereoom temperature (21 °C) for 5 min, before fixation and processing foralization of the nanoparticles, visible as circular black inclusion bodies,o observed within the nucleus. Scale bar=1 μm.

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3.3. Labelled CD4+ T cells show normal proliferative,regulatory, chemotactic and transmigratory function invitro

Following optimisation of the labelling reaction inboth human and murine CD4+ T cells, we next

Fig. 4. Labelled human CD4+ CD25+ Tregs mediate suppression ofCD25- T cells in vitro. Labelled (L) and unlabelled CD25+ and CD25−

CD4+ T cells were cultured either alone or together at ratios of 1:1, inthe presence of irradiated allogeneic peripheral blood mononuclearcells for 120 h. Supernatants were collected at 72 h for IL-2 bioassay(CTLL) and the cells were pulsed with tritiated thymidine for the last16 h of culture. Unlabelled CD25+ Tregs potently suppressedproliferation (A) and IL-2 synthesis (B); labelled CD25+ Tregsdemonstrated equipotent suppression of labelled or unlabelled CD25-

T cells. Error bars represent standard errors of the mean (proliferation)of triplicate cultures; standard deviations for the CTLL assays are notshown since they were all <15% of the respective mean values. Theseresults are representative of two independent experiments.

Fig. 5. Labelled murine CD4+ CD25+ Tregs mediate titratablesuppression of CD25- T cells in vitro. Labelled (L) and unlabelledCD25+ and CD25−CD4+ T cells were cultured either alone or togetherat ratios of 1:1 to 1:32, in the presence of anti-CD3:CD28 DynaBeads®for 88 h. The cells were pulsed with tritiated thymidine for the last 16 hof culture. Unlabelled CD25+ Tregs potently suppressed proliferation;labelled CD25+ Tregs also demonstrated suppression of proliferation,but were less potent. Error bars represent standard errors of the mean oftriplicate cultures. These results are representative of two independentexperiments.

examined various functions of these cells after treatmentwith the improved tat-USPIO nanoparticles in a five-minute reaction. In these experiments, selections wereperformed to isolate the CD25+ (regulatory) and CD25−

(helper) T cells within the CD4+ population. First, weexamined the proliferative potential of labelled humanCD4+ T cells. In common with unlabelled cells, labelledCD25+ T cells were anergic, while labelled CD25− Tcells showed robust proliferation and IL-2 synthesis inresponse to a polyclonal stimulus consisting of anti-CD3/CD28-coated beads (Fig. 4). When co-cultured,labelled CD25+ T cells demonstrated potent suppres-sion of both proliferation and IL-2 synthesis of labelledor unlabelled CD25-cells (Fig. 4).

We next extended our studies to murine CD4+ T cells.In common with the human T cells, labelled murineCD25+ T cells remained anergic and labelled CD25− Tcells showed robust proliferation in response to thepolyclonal anti-CD3/CD28 bead stimulus (Fig. 5). Wecultured labelled murine CD25+ with unlabelled CD25−

T cells in the presence of the polyclonal anti-CD3/CD28bead stimulus; comparisons were made with co-culturescontaining unlabelled CD25+ T cells (Fig. 5). UnlabelledCD25+ T cells showed potent, titratable suppression ofproliferation that was apparent even at a ratio (CD25+:CD25−) of 1:32. Labelled murine CD25+ T cells were

Fig. 6. Labelled murine CD4+ T cells show a normal chemotactic response to CXCL-12 in vitro. Murine CD4+ T cell blasts, either unlabelled orlabelled with tat-USPIO nanoparticles following a five-minute incubation reaction, were seeded into the upper chambers of 5 μm-pore Transwellscontaining CXCL-12 (SDF-α; 50 ng/ml) in the lower chambers, or medium alone (control). Cells present in the lower chambers of the Transwellswere counted over 24 h. Labelled cells showed equivalent chemotactic responses to unlabelled. Error bars represent standard errors of the mean oftriplicate cultures.

Fig. 7. Labelled murine CD4+ T cells undergo normal transmigrationof an endothelial monolayer in vitro. Naïve murine CD4+ T cells,either unlabelled or labelled with tat-USPIO nanoparticles following afive-minute incubation reaction, were seeded into the upper chambersof gelatin-coated 3 μm-pore polycarbonate Transwell inserts, ontowhich IFNγ-activated microvascular endothelial cells had beenlayered overnight. Cells present in the lower chambers of theTranswells were counted over 24 h. No significant differences intransmigration between the two populations were observed (p>0.05;repeated measures ANOVA). Error bars represent standard errors ofthe mean of triplicate cultures.

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also able to suppress proliferation, though their regulatoryinfluencewasweaker, with loss of suppression at a ratio of1:8. The regulatory function ofmurine CD25+ Tcells wasabolished completely by incubation with tat-USPIOnanoparticles for 10 or 15 min (data not shown).

Having established that labelled CD4+ T cellsretained proliferative and regulatory function in vitro,we next examined the chemotactic and transmigratorybehaviour of these cells in vitro as surrogate markers oftheir likely trafficking behaviour in vivo. We focused onmurine CD4+ T cells in these experiments. Similarchemotactic responses to the chemokine CXCL-12 wereobserved among labelled and control, unlabelled murineCD4+ Tcell blasts (Fig. 6). Thus, approximately 16% ofcells labelled with tat-USPIO nanoparticles werecounted in the bottom chambers of Transwells 24 hafter the addition of CXCL-12, comparable to experi-ments performed with unlabelled cells. Similarly, nodifferences were observed in the transmigratory behav-iour of labelled and unlabelled naïve CD4+ T cells: inboth cases, approximately 5% of cells seeded in theupper chambers traversed the endothelial monolayerover a 24 h period (Fig. 7). We therefore reasoned thatthe labelling process had not affected the migratoryfunction of murine CD4+ T cells in vitro, either inresponse to a chemokine (chemotaxis) or followingengagement of an activated endothelial monolayer(transmigration).

3.4. Rapid labelling of CD4+ T cells provides effectiveMRI contrast enhancement

Having established that the five-minute tat-USPIOnanoparticle labelling reaction preserved the prolifera-

tive, regulatory, chemotactic and transmigratory behav-iour of CD4+ T cells in vitro, we then examined theimaging potential of this contrast agent. Human CD25+

and CD25−CD4+ T cells were incubated with the tat-USPIO nanoparticles for 5 min and then dispensed intoan agarose mould. Magnetic resonance imaging wasperformed using a standardised T2 measurement pro-tocol, yielding effective contrast enhancement of thephantoms containing labelled, but not unlabelled, cells.Thus, the spin–spin (T2) relaxation times of the labelledT cells were appreciably reduced, thus darkening thecorresponding areas of the phantoms (Fig. 8). Similar

Fig. 8. Rapid labelling of CD4+ T cells with tat-USPIO nanoparticles provides effective MRI contrast enhancement. Aliquots of 0.9×106 CD25+ orCD25−CD4+ T cells were incubated with tat-USPIO nanoparticles for 5 min, before being dispensed into wells stencilled into a 2% w/v agarosemould. The wells were sealed with 2% w/v agarose and imaged with a standardised T2 measurement protocol, using a spin-echo sequence with fiveslices of 1 mm, four non-arrayed echo times (TE=5.5, 15, 25 and 50 ms) and a fixed repetition time (TR=1500 ms) collected consecutively (twoaverages per echo time: matrix 128×128, field of view 25×25 mm, acquisition time 25 min). A customised program was written using ‘InteractiveData Language’ (IDL; Research Systems Incorporated, Colorado, USA) to fit the data — pixel by pixel — to a standard T2 decay model. Labelledcells are seen as darkened areas; unlabelled cells do not yield any contrast.

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results were obtained when labelled murine CD4+ Tcells were imaged (data not shown).

4. Discussion

The current study describes a rapid, highly efficienttechnique for labelling murine CD4+ T cells for MRI,thus contributing to recent developments in magneticcellular labelling (Ahrens et al., 2005; de Vries et al.,2005). This technique, which we believe represents asignificant advance on previously published protocols inthe speed and efficiency of labelling, preserved chemo-tactic, transmigratory and regulatory functions of thecells in vitro when used appropriately. Various strategiesto label T cells for MRI have been described, promptedby the non-phagocytic nature of these cells (Dodd et al.,1999; Lewin et al., 2000; Frank et al., 2004). Forexample, modification of the nanoparticles with theHIV-1 tat peptide(48–57) increases the efficiency of theirtranslocation into murine lymphocytes by 100-fold ormore (Josephson et al., 1999). Of the various factorsinfluencing labelling efficiency, the number of tat pep-tides conjugated to each nanoparticle and the duration ofthe labelling reaction are among the most important(Bhorade et al., 2000; Zhao et al., 2002; Zhao andWeissleder, 2004). In the current study, optimisation ofthe amination step yielded a high number of functionalgroups per nanoparticle for peptide conjugation (45±5),facilitating rapid labelling. Two key strategies allowedthis improvement in valence of the nanoparticles. Thefirst involved the cross-linking reaction, in which freedextran hydroxyl groups formed carbonyl linkages withadjacent molecules to confer stability of the dextrancage. Thus, the extent of cross-linking was reduced tothe minimum possible to retain stability in the face ofthermal and oxidative stress — induced by heating to95 °C for 20 min and treatment with 100 mM sodiummetaperiodate — by reducing both the concentration ofepichlorohydrin by 50% and the duration of the reaction

to 4 h. This modification offered the advantage ofmaximising the number of available hydroxyl groups foramination. The second strategy capitalised on this ben-efit by optimising the subsequent amination step, byincreasing its duration from 24 to 36 h.

To our knowledge, the valence of our functionalizednanoparticles was higher than previously reportedvalues in the literature, which have ranged from 6.7(Josephson et al., 1999) to 23 (Kircher et al., 2003).However, our calculation of valence was liable to acertain degree of inaccuracy owing to our inability tocalculate exactly the concentration of nanoparticles insuspension, precluding definitive statements of superi-ority over other preparations cited in the literature.Nevertheless, both light and electron microscopyrevealed that efficient cytoplasmic and nuclear labellingwas achieved in a five-minute incubation step, whichrepresents a significant advance on previously docu-mented reaction durations of 1 h or longer (Josephson etal., 1999; Moore et al., 2002; Zhao et al., 2002). Thoughthe detailed intracellular fate of the nanoparticles wasnot investigated in the current study, tat-mediated cel-lular uptake is generally thought to occur by adsorptiveendocytosis (Fawell et al., 1994; Nagahara et al., 1998).From the images obtained, we were unable to excludethe possibility that the nanoparticles were internalisedinto endosomes, as described for several superparamag-netic iron oxide preparations (Bulte, 2006). Furthercharacterisation of the intracellular localisation of thenanoparticles would require fluorescence microscopy ofthe internalised compound, counter-staining with mar-kers for the various cellular compartments for detailedanalysis.

The functional properties of labelled CD4+ T cellswere assessed in vitro, in anticipation of their ultimateuse to track cells in vivo. In every labelling reaction,>95% of the CD4T cells had internalised USPIOnanoparticles and >90% excluded Trypan blue, verify-ing viability. Both the murine and human CD4+ CD25−

132 O.A. Garden et al. / Journal of Immunological Methods 314 (2006) 123–133

T cells proliferated as robustly when labelled as they didwhen unlabelled; furthermore, synthesis of bioactive IL-2 was not impaired in the human CD25− T cellsfollowing labelling, suggesting that this agent isunlikely to abolish their helper cell function in vivowhen labelling reactions are restricted to 5 min. Theresults of these experiments also demonstrated thatlabelled CD4+ T cells survived for at least 88 h in themurine, and 120 h in the human, assay system in vitro,ruling out delayed cell death over the time period of theculture.

While labelled murine CD4+ CD25+ T cells wereable to suppress the proliferation of CD4+ CD25− cells,their regulatory function was somewhat attenuated bythe USPIO nanoparticles. Though this attenuation offunction was minimal, it nevertheless prompted consid-eration of possible sources of toxicity. While the full-length tat protein induces apoptosis of CD4+ T cells byactivation of the caspase 8 pathway (Bartz and Emer-man, 1999), the smaller tat peptide has minimal toxicityin cell culture (Hallbrink et al., 2001). Its hydrophilicnature causes minimal perturbation of the plasma mem-brane and pulse-labelling experiments indicate that it ismetabolised within the cell to yield innocuous break-down products (Zhao and Weissleder, 2004). Anotherpotential source of toxicity was the USPIO nanoparticleitself, including the iron oxide core and the surroundingdextran cage. While all current reports suggest a lack oftoxicity of USPIO nanoparticles (Wang et al., 2001), weobserved overt toxicity — cell death and completeabrogation of regulatory activity — of the derivatizednanoparticles for both human and murine CD4+ T cellswhen labelling reactions of longer than 5 min wereemployed, suggesting that there is a critical period ofoptimal loading for our labelling reagent. The possibilityremained that high intracellular concentrations of ironwere able to catalyse the formation of toxic reactiveoxygen intermediates (Nappi and Vass, 2000), despitethe protective dextran coating. The labelled humanCD4+ CD25+ T cells mediated equally potent suppres-sion of proliferation and IL-2 synthesis as the unlabelledat a ratio (CD25+:CD25−) of 1:1, though a full titrationof cells — which could have disclosed a more subtleeffect — was not performed. The relative resilience ofhuman CD4+ regulatory Tcells to the influence of the tat-USPIO nanoparticles accords with the generally morerobust nature of human versus murine T cells in vitro.

Following these tests of proliferative and regulatorybehaviour, we went on to examine the chemotactic andtransmigratory function of labelled murine CD4+ T cellsin vitro. Labelled cells exhibited similar CXCL-12 che-motactic responses to unlabelled cells, and underwent

normal transmigration of an activated endothelial mono-layer. These tests were anticipated to predict themigratorybehaviour of labelled cells in vivo in response to aninflammatory stimulus. Similarly, (Dodd et al., 2001)demonstrated normal proliferation, activation-inducedcell death, and up-regulation of CD69, CD54, CD62Land CD95 (Fas) by labelled, unfractionatedmurine Tcellsexposed to a polyclonal stimulus; while Kircher andothers (Kircher et al., 2003) showed normal proliferationand cytotoxic function of labelled murine CD8+ Tcells inresponse to stimulation by a cognate antigen. Further-more, both labelled and unlabelled CD8+ T cells showedsimilar transmigration of an endothelial monolayer withina parallel plate flow chamber (Kircher et al., 2003).

Clear MR images of the labelled human CD25+ andCD25−CD4+ T cells were obtained from the phantoms,demonstrating the ability of our reagent to providecontrast enhancement and its potential to facilitate thetracking of T cells in vivo. We measured T2 because itrepresents a robust and reproducible physical parameterthat reflects the magnetic characteristics of the cells in amanner independent of coil registration, unlike measuresof signal intensity that are vulnerable to system stability.One of the principal drawbacks of MRI for the study ofcell migration remains the relative insensitivity to con-trast detection when compared to techniques such as PETimaging, which is several orders of magnitude moresensitive (Phelps, 2000). In this work, optimisation of thetat-mediated incorporation of USPIO nanoparticles intoCD4+T cells is likely to ensure a maximal contrast-to-noise ratio within the target region. However, the reali-sation of this prediction awaits the rigorous trial of thiscontrast agent in suitable animal models in vivo and —ultimately — human patients in the clinic.

Though the use of high field MR imaging in thedissection of Tcell migration is still in its infancy (Dodd etal., 2001; Moore et al., 2002; Kircher et al., 2003;Delikatny and Poptani, 2005), the current study representsa significant advance towards the realisation of thisaspiration. The main challenges of cell labelling appear tohave been overcome. The next challenge will be toincrease the sensitivity and spatial resolution of imagingsystems, to accommodate the limited amounts of contrastagent that can be loaded into cells before experiencingtoxicity; we anticipate that such advances will come asnew engineering and software solutions are introduced.Many questions pertaining to T cell migration remain tobe answered in the contexts of autoimmune disease,neoplasia, transplant rejection and infectious disease. Webelieve that this approach to labelling T cells for MRIholds considerable promise, paving the way for the widerimmunological application of this exciting technology.

133O.A. Garden et al. / Journal of Immunological Methods 314 (2006) 123–133

Acknowledgements

The authors would like to acknowledge the access to theICP-OES facilities at the Department of Geology, RoyalHolloway University, kindly arranged by Professor N.Walsh. Algorithms to measure iron in samples weredevised by Dr S. James, Department of Geology, RoyalHolloway University. This work was funded by the BritishHeart Foundation, Wellcome Trust and Medical ResearchCouncil. OAG was the recipient of a Wellcome TrustAdvanced Fellowship at the time of this work; JY was arecipient of a Medical Research Council Clinical Fellow-ship; andAJTG is aBiotechnology andBiological SciencesResearch Council Research Development Fellow.

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