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In Vivo High Resolution Three-Dimensional Imaging of Antigen

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					[CANCER RESEARCH 63, 6838 – 6846, October 15, 2003]


In Vivo High Resolution Three-Dimensional Imaging of Antigen-Specific Cytotoxic
T-Lymphocyte Trafficking to Tumors1,2
Moritz F. Kircher, Jennifer R. Allport, Edward E. Graves, Victoria Love, Lee Josephson, Andrew H. Lichtman, and
Ralph Weissleder3
Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129 [M. F. K., J. R. A., E. E. E., L. J.,
R. W.], and Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115 [V. L.,
A. H. L.]


ABSTRACT                                                                                    track these CTLs4 in vivo at sufficiently high spatial and temporal
                                                                                            resolutions (28). The majority of imaging approaches available have
   Magnetic resonance imaging (MRI) allows noninvasive and three-
                                                                                            involved either mass distribution analysis of radiolabeled cells (29,
dimensional visualization of whole organisms over time, and, therefore,
would be ideally suited to monitor cell trafficking in vivo. Until now,
                                                                                            30) or bioluminescence imaging of cells stably transfected with lu-
systemically injected cells had been difficult to visualize by MRI because                  ciferase (31) at relatively low spatial resolution, or for greater spatial
of relatively inefficient labeling methods. We developed a novel, biocom-                   resolution, invasive intravital microscopy analysis of small, relatively
patible, and physiologically inert nanoparticle (highly derivatized cross-                  superficial areas of the tumor (32). For an imaging method to ulti-
linked iron oxide nanoparticle; CLIO-HD) for highly efficient intracellu-                   mately be clinically viable and allow the evaluation of both cell
lar labeling of a variety of cell types that now allows in vivo MRI tracking                delivery and therapeutic effectiveness in patients, it must be nonin-
of systemically injected cells at near single-cell resolution. CD8 cytotoxic                vasive, nontoxic, and allow an accurate and quantitative determination
T lymphocytes labeled with CLIO-HD were detectable via MRI with a
                                                                                            of the cell-based therapy. Both MRI and nuclear imaging (positron
detection threshold of 2 cells/voxel in vitro and 3 cells/voxel in vivo in live
                                                                                            emission tomography and single-photon emission computed tomog-
mice. Using B16-OVA melanoma and CLIO-HD-labeled OVA-specific
CD8 T cells, we have demonstrated for the first time high resolution                        raphy) are in routine clinical use, but nuclear imaging has limited
imaging of T-cell recruitment to intact tumors in vivo. We have revealed                    spatial resolution, often requires genetic modification of the admin-
the extensive three-dimensional spatial heterogeneity of T-cell recruitment                 istered cells (33), and most radiochemicals have significant cellular
to target tumors and demonstrated a temporal regulation of T-cell re-                       toxicity and short half-lives that allow imaging for only 24 – 48 h. In
cruitment within the tumor. Significantly, our data indicate that serial                    contrast, MRI is both noninvasive and provides high spatial resolution
administrations of CD8 T cells appear to home to different intratumoral                     in vivo. However, until now, adoptively transferred cells have been
locations, and may, therefore, provide a more effective treatment regimen                   extremely difficult to visualize via MRI because of a combination of
than a single bolus administration. Together, these results demonstrate
                                                                                            relatively inefficient labeling methods and the dilution of systemically
that CLIO-HD is uniquely suited for quantitative repetitive MRI of
adoptively transferred cells and that this approach may be particularly                     injected cells in vivo. Most published reports have followed the
useful for evaluating novel cell-based therapies in vivo.                                   migration of either locally injected cells labeled with different super-
                                                                                            paramagnetic iron oxide nanoparticles into the adjacent parenchyma
INTRODUCTION                                                                                (34, 35) or have examined nonspecific accumulation of magnetically
                                                                                            labeled cells in whole organs at short time points (36); however,
   Cell-based therapies (1) have received much attention as novel                           labeling and imaging of immune-specific cells by MRI has not yet
therapeutics for the treatment of cancer (2, 3), autoimmune (4, 5),                         been described. Indeed, at present there exists no high-resolution,
cardiovascular (6), inflammatory (7), and degenerative diseases (8 –                        three-dimensional method to visualize the immune-specific recruit-
10). A number of native cells (4, 7, 11), antigen-specific T-lympho-                        ment of systemically administered cells in vivo over time.
cytes (12, 13), or, more recently, stem and progenitor cells have been                         Initially, we developed an HIV Tat peptide derivatized magnetic
used for these approaches. These cells alone (14, 15) or armed with                         nanoparticle that allows efficient intracellular labeling for MRI (37).
additional transgenes (16 –18) have been effective in mediating tumor                       Recently, by exploiting principles of multivalency (38) and by im-
regression in vivo. Recent advances allowing more efficient ex vivo                         proving conjugation strategies, peptide sequences and labeling proto-
expansion of such cells could have far reaching implications in a                           cols we developed a nanoparticle (CLIO-HD) that is 200-fold more
number of therapeutic paradigms; however, there remains a need to                           efficient in lymphocyte labeling. Here, using a model antigen-express-
better understand the in vivo fate of injected cells, including their                       ing tumor, we demonstrate quantitative, high-resolution in vivo
distribution, migration, and homing to targeted sites.                                      imaging of CLIO-HD-labeled antigen-specific CTL recruitment to
   Tumor antigen-specific lymphocytes in particular have been used                          tumors in live animals. Using high resolution MRI and three-dimen-
for adoptive transfer and treatment in lymphoma, melanoma, and                              sional reconstructions, we have revealed the heterogeneous three-
other malignancies (19 –27). A major obstacle to accurate evaluation                        dimensional distribution of the recruited cells within the tumor. In
of treatment efficacy and antitumor effects has been the inability to
                                                                                            addition, we have observed that repeated injections of CTL were
                                                                                            apparently recruited to different anatomical regions of the tumor,
   Received 3/10/03; revised 7/9/03; accepted 8/4/03.
   The costs of publication of this article were defrayed in part by the payment of page
                                                                                            suggesting that multiple dosing strategies may allow attack of the
charges. This article must therefore be hereby marked advertisement in accordance with      tumor from multiple fronts simultaneously. Together, these results
18 U.S.C. Section 1734 solely to indicate this fact.                                        illustrate that the developed nanoparticle enables quantitative high-
   1
     Supplementary data for this article are available at Cancer Research Online (http://
cancerres.aacrjournals.org).                                                                resolution repetitive MRI of adoptively transferred cells, and may be
   2
     Supported by NIH Grants AI86782-02 (to R. W., M. F. K., and J. R. A.),                 particularly useful for tailoring cell-based anticancer therapies and
CA96978-01 (to J. R. A.), HL36028 (to A. H. L.), GM64931 (to V. L.), and a grant from
the Charles A. Dana Foundation (to J. R. A.). M. F. K. was supported by a fellowship
                                                                                               4
from the Deutsche Forschungsgemeinschaft (DFG).                                                  The abbreviations used are: CTLs, cytotoxic T lymphocytes; MRI, magnetic reso-
   3
     To whom requests for reprints should be addressed, at Center for Molecular Imaging     nance imaging; CLIO-HD, highly derivatized cross-linked iron oxide nanoparticles; OT-I,
Research, Massachusetts General Hospital, 13th Street, Building 149, Room 5403,             ovalbumin-specific, MHC class I-restricted T-cell receptor transgenic; DPBS, Dulbecco’s
Charlestown, MA 02129. Phone: (617) 726-8226; Fax: (617) 726-5708; E-mail:                  PBS; IL, interleukin; OVA, ovalbumin; ROI, region of interest; MR, magnetic resonance;
weissleder@helix.mgh.harvard.edu.                                                           FACS, fluorescence-activated cell sorter.
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                                                   IN VIVO TRACKING OF CTL RECRUITMENT TO TUMORS BY MRI


evaluating therapeutic effectiveness in both experimental and clinical           1 h at 37°C and washed. CLIO-HD-labeled (300 g/ml for 4 h) or vehicle-
settings.                                                                        labeled OT-I CD8 T cells were titrated in 2-fold dilutions in 96-well
                                                                                 flat-bottomed plates (100 l volume), mixed with 100 l of target cells, and
                                                                                 incubated for 4 h at 37°C. Total 51Cr release was obtained by adding 0.25%
MATERIALS AND METHODS                                                             Triton X-100 (final concentration) to the wells. Supernatants were collected
                                                                                  and radioactivity quantitated using a gamma counter. Percentage of specific
   Materials. DMEM, RPMI 1640, HBSS, and fetal bovine serum (for culture lysis was calculated according to the following formula: percentage of specific
of tumor cells) were purchased from Cellgro (Herndon, VA). FCS (CD8 T lysis                [(experimental cpm      spontaneous cpm)/(total cpm       spontaneous
cells) was purchased from Sigma (St. Louis, MO). DPBS, DPBS with Ca2             cpm)]        100 (45). Freshly isolated OT-I CD8 T cells were labeled as
and Mg2 , and PBS were obtained from BioWhittaker (Walkersville, MD). described above with either CLIO-HD or vehicle control and then perfused
                                                                                                                                               6
Recombinant murine IL-2 was purchased from R&D (Minneapolis, MN). The across 4 h mTNF- activated murine heart endothelium (10 cells/ml in flow
OVA-derived MHC class I immunogenic peptide OVA257–264 (SIINFEKL)                buffer 1:1 DPBS with Ca2 and Mg2 :DPBS containing 0.1% BSA) for
was synthesized by Analytical Biotechnology Services (Boston, MA).               10 min at 0.52 ml/min (estimated wall shear stress of 1.0 dynes/cm2) using a
   Antibodies. Anti-CD3 (clone 145-2C11), anti-CD28 (clone 37.51), FITC- parallel plate flow chamber (42). T cell:endothelial interactions were recorded
anti-CD8b (clone 53-5.8), phycoerythrin-anti-CD3 (clone 17A2), rat anti- using video-linked phase contrast microscopy and assessment of attachment,
mouse CD8a (clone 53-6.7, IgG2a, ), and purified rat IgG2a were purchased rolling, adhesion, and migration determined offline from four to eight high
from BD PharMingen (San Diego, California). Biotinylated goat antirat IgG power fields per coverslip. A minimum of three coverslips per condition were
monoclonal antibody was obtained from Jackson Immunoresearch Labs (West quantified in each experiment.
Grove, Pennsylvania).                                                                Flow Cytometry. Purity of OT-I CD8 T cells was determined by staining
   Animals. C57Bl/6 (female, 10 –14 weeks) were purchased from the Na- with anti-CD8-FITC and anti-CD3-PE using standard protocols (46) and
tional Cancer Institute (Bethesda, MD), OT-I mice were kindly provided by analysis using a FACScalibur (Becton Dickinson, Mountain View, CA). To
William R. Heath and Francis Carbone (Walter and Eliza Hall Institute of determine the efficiency of CLIO-HD uptake, labeled cells were analyzed for
Medical Research, Melbourne, Australia; Ref. 39). All of the animals were fluorescence in the FITC channel and compared with an unlabeled control. A
maintained in our pathogen-free institutional facilities.                         minimum of 10,000 cells/sample was analyzed.
   Cells. OVA-transfected B16 melanoma cell line (B16-OVA) and B16                   Animal Model. C57Bl/6 mice were injected s.c. with 5 106 B16-OVA or
melanoma cell line (B16F0) were provided by Drs. Edith Lord and John             B16F0 in the right or left flank, respectively, and 10–12 days later, 1–3     107
Frelinger (University of Rochester, Rochester, New York; Ref. 40). Both cell CLIO-HD-labeled OT-I CD8 T cells were adoptively transferred via i.p. injec-
lines were cultured in DMEM containing 10% FCS, 0.075% sodium bicar- tion. Distribution of CLIO-HD-labeled cells over time was assessed via repetitive
bonate, and antibiotics, and serially passaged (1:10 split ratio) as required. MRI. In some experiments, tumors were subsequently excised and used for
CD8 T cells were isolated from the spleens and lymph nodes of mice using histological analysis. In additional experiments, tumor-bearing C57Bl/6 mice were
                                                                                                                        111                          8
the MACS separation system (Miltenyi Biotec, Auburn, CA) and were cultured adoptively transferred with CLIO-HD/ In-oxine (600 Ci/5 10 cells/2 ml)
in DMEM containing 10% FCS, nonessential amino acids, 1 mM sodium dual-labeled OT-I CD8 T cells and sacrificed after 12 or 36 h. For biodistribution
pyruvate, 0.075% sodium bicarbonate, 50 M 2-mercaptoethanol, and antibi- studies, the organs were excised, weighed, and radioactivity counted to determine
otics. For selected in vitro experiments, wild-type CD8 T cells were prolif- the percentage of injected dose/g of organ tissue. For autoradiography studies,
erated in plates precoated with anti-CD3 (10 g/ml), supplemented with 2.4 tumors were excised from the 36-h animals, fixed in 4% paraformaldehyde, and
ng/ml IL-2 and 2 g/ml anti-CD28 for two rounds of 3 days (41). For imaged by MRI (500 m slice thickness). Subsequently, tumors were cut into
functional assays and in vivo experiments, OT-I CD8 T cells were stimulated slices (same orientation and slice thickness as MR images) and exposed on a
with 0.7 g/ml SIINFEKL-peptide and 10-fold mitomycin C (50 g/ml)- phosphorimager (Molecular Dynamics, Sunnyvale, CA) to reveal the distribution
treated syngeneic antigen presenting cells (APC) fed on day 3 with 0.8 ng/ml of radiolabeled OT-I CD8 T cells.
IL-2 and harvested on day 5. Murine heart endothelium was isolated and               MRI and Three-Dimensional Reconstructions. Under general isoflurane
cultured as described previously (42). Endothelium was plated at confluence anesthesia (0.5–1.5% at 2 liter/min), mice were imaged at 0, 12, 16, and 36 h after
on 25-mm diameter glass coverslips precoated with 1 g/cm2 human fibronec- adoptive transfer of CLIO-HD-labeled CD8 cells (Bruker DRX 360, 8.5 T
tin, activated for 4 h with 120 ng/ml murine tumor necrosis factor (mTNF)- , magnet, 2-cm diameter birdcage-coil, T2-weighted spin-echo sequences;
and then used in experiments.                                                     TR 3000 ms; TE 15– 60 ms; matrix size 256 256; in-plane resolution 75
   Synthesis of CLIO-HD, Cell Labeling, and Uptake Studies. The CLIO                m; slice thickness 500 m; NEX 2; imaging time 25 min). Phantoms were
nanoparticle has been described previously (43). CLIO-SC-Tat preparations imaged with identical parameters, except TE              10 –240 ms in 10 ms intervals
were synthesized as described (38) with modifications, with Tat:CLIO ratios Carr-Purcell-Meiboom-Gill sequence (CPMG). Distribution of 111In-oxine/CLIO-
(6.4, 7.5, 11.73, 19.5, 22.95, and 23.24 Tat/CLIO), and routinely used with HD dual-labeled OT-I CD8 T cells was determined by MRI, and correlated with
23 Tat/CLIO (CLIO-HD). CD8 T cells were incubated with CLIO-HD autoradiography of identical tumor sections. Image segmentation, T2 analysis and
(2–300 g Fe/ml/10 106 cells) for 4 h at 37°C and washed three times by            three-dimensional volume rendering were performed using CMIR-Image (devel-
centrifugation through 40% Histopaque-1077. CLIO-HD-labeled OT-I CD8              oped in Interactive Data Language; Research Systems Inc., Boulder, CO). ROIs
T cells were fixed in 1% paraformaldehyde in PBS for 30 min at 4°C, washed were defined manually, and serial images of a subject were registered using a rigid
in HBSS, and permeabilized in 0.1% Triton X-100. Cells were then washed body algorithm to generate optimal coincidence of the tumor ROIs for each
and resuspended in 1 mg/ml RNase in PBS for 15 min incubated in PBS- dataset. T2 maps were constructed by performing fits of a standard exponential
propidium iodide solution (10 g/ml) for 15 min at room temperature, washed relaxation model to the data on a pixel-by-pixel basis within the ROIs. Only pixels
in HBSS, and mounted using Vectorshield (Vector Labs, Burlingame, CA). with intensity greater than a threshold level (mean background 5 SD) were
Dual-channel laser scanning confocal microscopy was performed using a Zeiss considered during the fitting process. Registered T2 maps of each mouse were
LSM 5 PASCAL (Zeiss, Thornwood, NY). Quantification of CLIO-HD uptake color mapped and rendered using blending, and then superimposed on trans-
was performed as described previously (38).                                      parent outlines of the mouse surface and the segmented tumors. For data in Fig. 6,
   CD8 T-Cell Functional Assays. Toxic effects of CLIO-HD labeling tumor volumes with T2 changes of 20 ms (corresponding to 2 cells/voxel) were
were assessed by functional assays of proliferation. OT-I CD8 T cells at day extracted from the registered T2 maps of the mouse after each injection of
5 of culture were labeled with 75 g, 150 g, or 300 g/ml CLIO-HD or CLIO-HD-labeled CD8 T cells. These volumes were rendered as red, green, and
vehicle control for 4 h. Cells were then proliferated as described above in blue, respectively, and superimposed on a transparent outline of the tumor surface.
96-well plates (5      104 cells/well). Positive control wells received 10 l/ml Renderings were performed at multiple angles to highlight the three-dimensional
phytohemagglutinin (Life Technologies, Inc.). After 48 h, all of the wells were nature of the calculated T2 maps.
pulsed for 18 h with 1 Ci of [3H]thymidine (Perkin-Elmer, Boston, MA) and            Histology. After MRI, animals were sacrificed, and B16-OVA and B16F0
then harvested onto filter mats. [3H]Thymidine incorporation was determined tumors excised preserving the capsule, fixed for 2 h in 2% paraformaldehyde,
by liquid scintillation counting (44). Target cells (B16F0 and SIINFEKL- and equilibrated in 18% sucrose, embedded in OCT (Sakura, Torrance, CA),
coated B16-OVA cells) were labeled with 20 Ci of 51Cr (Perkin-Elmer) for snap-frozen in liquid N2, and sectioned in 10 m slices. Tissue sections were
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                                                             IN VIVO TRACKING OF CTL RECRUITMENT TO TUMORS BY MRI


incubated with antimouse CD8a or isotype-matched control monoclonal anti-               after 15 min of perfusion, compared with an accumulation of
body. Intrinsic tissue avidin or biotin was blocked (avidin/biotin blocking kit;        217 46 cells/mm2 for unlabeled cells, and 6.0 0.6% of CLIO-HD
Vector Labs), and biotinylated goat antirat-IgG was used to detect the 1°               labeled cells underwent transmigration compared with 5.2          0.9%
antibody, and visualized using Vector ABC Elite and Vector NovaRed (Vector              unlabeled cells.
Labs). Sections were counterstained with Gills No.2 Hematoxylin (Sigma),
                                                                                           In Vitro Detection Threshold of CLIO-HD-Labeled OT-I CD8
dehydrated, mounted with Vectashield, and examined via bright field (Nikon)
with 20 magnification. Serial sections were viewed unstained in the FITC                T Cells and Stability of Intracellular Labeling. To determine the
channel using an inverted epifluorescence microscope (Zeiss Axiovert). For              threshold of detection for CLIO-HD-labeled OT-I CD8 T cells and
Prussian Blue staining, the same sections were then incubated for 30 min with           to create a T2 map standard curve, we next performed MRI phantom
2% potassium ferricyanide (Perls’ reagent) in 2% HCl, washed, and counter-              in vitro experiments. CD8 T cells were labeled with CLIO-HD (300
stained with Eosin.                                                                       g Fe/ml/106 cells for 4 h) and mixed with unlabeled CD8 T cells
                                                                                        in the ratios described (Fig. 2A). Cell pellets were implanted in agar
RESULTS                                                                                 plugs and imaged by MRI using the identical sequences used for in
                                                                                        vivo experiments. MRI signal intensity reduction induced by
   Labeling of OT-I CD8 T Cells with CLIO-HD Does Not
                                                                                        CLIO-HD was observed with as few as 3           104 labeled cells/50 l
Influence Cell Behavior in Vitro. To optimize the labeling protocol
                                                                                        (Fig. 2A), representing an in vitro detection threshold of 2 cells/
for CD8 T cells, we examined the kinetics of CLIO-HD uptake in
                                                                                        voxel. These data were used to prepare a standard curve of 1/T2
these cells. The uptake of CLIO-HD by CD8 T cells was propor-
                                                                                        against Fe concentration (Fig. 2B) that would be used later to quan-
tional to both the incubation time and CLIO-Tat concentration (Fig.
                                                                                        titate cell numbers within MRI slices. In additional in vitro experi-
1B). Using the FITC label incorporated into the Tat peptide, we were
                                                                                        ments we examined the stability of CLIO-HD-mediated signal reduc-
able to additionally validate the correlation between incubation time
                                                                                        tion over time in CD8 T cells. Isolated CD8 T cells were labeled
and labeling efficiency via FACS analysis (Fig. 1C), confirming that
the cells were uniformly labeled by CLIO-HD. When we examined                           with CLIO-HD (300 g Fe/ml/106 cells for 4 h), and 8           105 cells
the intracellular distribution of CLIO-HD via laser-scanning confocal                   placed into each well of a 24-well plate and cultured as described in
microscopy, we determined that the particle localized to both the                       “Materials and Methods.” At the intervals indicated (Fig. 2C), total
nucleus and cytoplasm of CD8 T cells (Fig. 1D). Intracellular                           cells from each well were washed, counted, and implanted in agar
labeling of CD8 T cells with CLIO-HD was not cytotoxic (Trypan                          plugs with unlabeled cells (8 105) used as a control. Cells were then
Blue exclusion) in concentration ranges of 50 –300 g Fe/ml/106 cells                    imaged by MRI using identical sequences as those described above.
(data not shown), with cells retaining 95% viability. In additional                     The signal reduction caused by the presence of CLIO-HD is similar in
experiments, we examined the effects of CLIO-HD labeling on OT-I                        each of the wells (Fig. 2C), indicating that intracellular labeling of
CD8 T cell functions of in vitro tumor cell killing (Fig. 1E) and                       OT-I CD8 T cells with CLIO-HD was maintained for at least 120 h.
proliferation (Fig. 1F). Treatment of OT-I CD8 T cells with up to                       Furthermore, when resting OT-I CD8 T cells and activated OT-I
300 g Fe/ml/106 cells did not affect their ability to kill target cells or              CD8 T cells were labeled with CLIO-HD, there was no detectable
to proliferate in vitro. In addition, we examined the interactions of                   difference in the degree of MR signal reduction induced by CLIO-HD
OT-I CD8 T cells with 4 h mTNF- activated murine heart endo-                            (Fig. 2D), confirming that this technique is equally applicable to the
thelium under in vitro flow conditions, and did not observe any                         study of either resting or activated T-cell trafficking.
significant difference in their initial rate of attachment, rolling frac-                  Recruitment of OT-I CD8 T Cells to B16-OVA in Vivo Can
tion, stable arrest, or transmigration (Table 1). CLIO-HD-labeled                       Be Visualized by MRI. We next performed in vivo experiments in
CD8 T cells exhibited a total accumulation of 217 26 cells/mm2                          which C57Bl/6 mice were implanted with both control B16F0 (left


    Fig. 1. CLIO-HD is internalized by OT-I CD8 T
cells and does not influence cell function. A, molec-
ular rendering of CLIO-HD; B, OT-I CD8 T-cell
uptake of 125I-CLIO-HD is proportional to both the
incubation time (mean; bars, SD; n            3) and the
125
   I-CLIO-HD concentration (inset; mean; bars,
  SD; n 3); C, FACS analysis of CLIO-HD-labeled
CD8 cells after incubation with CLIO-HD for 0 h
(open graph), 1 h (yellow), and 4 h (red). Data are
representative of two experiments; D, dual-channel
confocal micrographs of a CLIO-HD labeled CD8
T cell demonstrating uptake of CLIO-HD into both
the cytoplasm and the nucleus: A, CLIO-HD (FITC
visualization); B, nuclear staining (propidium iodide);
C, Nomarski of OT-I CD8 T cell, D, overlay of
Nomarski and CLIO-HD. CLIO-HD colocalizes with
propidium iodide staining indicating that CLIO-HD is
taken up by the nucleus; E, CLIO-HD does not affect
the ability of OT-I CD8 T cells to kill target tumor
cells. There was no significant difference in the ability
of either CLIO-HD-labeled CD8 T cells (open sym-
bols) or unlabeled CD8 T cells (closed symbols) to
kill either the target tumor cells, B16-OVA (squares),
or the control tumor cells, B16F0 (diamonds). Data
are mean; bars, SD; F, CLIO-HD does not affect
the proliferation of OT-I CD8 T cells. There was no
significant difference in the proliferation rate of either
vehicle-labeled (open bars) or CLIO-HD-labeled
(closed bars) OT-I CD8 T cells in the absence or
presence of the antigen SIINFEKL, or the absence or
presence of phytohemagglutinin. Data are mean;
bars, SD.
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                                                            IN VIVO TRACKING OF CTL RECRUITMENT TO TUMORS BY MRI


                                                   Table 1 OT-I CD8 T cell interactions with murine heart endothelium under flow
    OT-I CD8 T cells were labeled for 4 h with either 300 g/ml CLIO-HD or vehicle alone (unlabeled) and then perfused across 4 h mTNF- activated murine heart endothelial
monolayers for 15 min at 0.52 ml/min (estimated wall shear stress, 1.0 dynes/cm2). T cell:endothelial cell interactions were recorded via video-linked phase contrast microscopy, and
interactions were determined later off line from four to eight high power fields. Data are the mean SD of three identical experiments. Student’s t test was used to calculate significance.
No significant difference was observed between the two groups for any parameter measured.
                                                                                                                                                          Initial rate of attachment
                                        Accumulation cells/mm2                   Rolling fraction (%)              Transmigrated cells (%)                      cells/min/mm2
       CLIO-HD labeled                          217    26                             1.6   1.5                           6.0    0.6                              47    9
       Unlabeled                                217    46                             1.7   1.2                           5.2    0.9                              60    18



side) and the antigen-presenting B16-OVA (right side) melanoma                                    significant signal reduction (dark areas) was observed in some
in the thigh so that each animal served as its own control (n 8).                                 regions of the B16-OVA tumor at 12 h after adoptive transfer. At
After 10 –12 days, when the tumors had reached 5–10 mm diam-                                      later times, a significantly greater signal reduction was observed
eter, serial MR image slices were taken of the animals before                                     (Fig. 3, C, D, G, and H), reflecting the increase of CD8 T-cell
adoptive transfer (Fig. 3A). OT-I CD8 T cells were then labeled                                   recruitment across the tumor. The heterogeneous nature of the
with CLIO-HD as described, and 3         107 cells were injected i.p.                             T-cell recruitment is especially noticeable in the three-dimensional
into the recipients. Animals were imaged again at 12, 16, and 36 h                                reconstructions (Fig. 3, E–I) of the MRI slices, and in the addi-
after adoptive transfer (Fig. 3, B–D), and the T2 data used to create                             tional axial, sagittal, and coronal slices presented (Fig. 3, J–L). The
three-dimensional reconstructions of the images (Fig. 3, E–L). In                                 mean MRI signal intensity also decreased with time after adoptive
one representative example shown in Fig. 3, very little change in                                 transfer, indicating the continued recruitment of CLIO-HD-labeled
signal intensity was observed in the control B16F0 tumor, indicat-                                OT-I CD8 T cells to the tumor. For a 360° animation of the data
ing that few OT-I CD8 T cells had been recruited. In contrast, a                                  see web-movie 1 in Supplemental Data.




    Fig. 2. In vitro MRI detection threshold for CLIO-HD-labeled OT-I
CD8 T cells and retention of CLIO-HD detection. A, each well contains
3 106 OT-I CD8 T cells, with an increasing ratio of labeled to unlabeled
cells (0.03–3.0     106, left to right, corresponding to 1.7, 5.6, 17, 56, and
170 cells/voxel); A, raw MRI data; B, T2-color-map of A; B, correlation of
Fe concentration in CLIO-HD-labeled cells and T2 relaxation rate; C, OT-I
CD8 T cells (8          105) labeled with either CLIO-HD (300 g/ml) or
vehicle control for 4 h and then placed in culture. At the times indicated,
total cells were pelleted in agar and imaged via MRI; D, comparison of MR
signal reduction induced by CLIO-HD labeling (300 g/ml) of equal
numbers of activated (center) or resting (right) OT-I CD8 T cells.
Unlabeled cells were incubated with vehicle alone (left).




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                                                         IN VIVO TRACKING OF CTL RECRUITMENT TO TUMORS BY MRI




   Fig. 3. Time course of CLIO-HD OT-I CD8 T cell homing to B16-OVA tumor. Serial MR imaging was performed after adoptive transfer into a mouse carrying both B16F0 (left)
and B16-OVA (right) melanomas. A–D, axial slices through the mouse thighs at A, before adoptive transfer; B, 12 h; C, 16 h; D, 36 h after adoptive transfer of CLIO-HD-labeled OT-I
CD8 T cells. E–H, three-dimensional color-scaled reconstructions of B16F0 (left) and B16-OVA (right) melanomas at E, 0 h; F, 12 h; G, 16 h; H and I, 36 h after adoptive transfer.
Numbers of cells/voxel are color-coded as shown in scale, J, axial; K, sagittal; L, coronal plane slices through the three-dimensional reconstruction shown in I. For a 360° animation
of the data see Supplemental Data web-movie 1. Data are representative of 8 individual animals.



   To control for signal intensity reduction caused by tumor necrosis,                       (Fig. 4I). There was no visible FITC staining present within the
we performed parallel experiments with unlabeled OT-I CD8 T                                  B16F0 tumor (Fig. 4F), but the presence of CLIO-HD within the
cells and saw no significant change in signal intensity within the time                      B16-OVA tumor (Fig. 4, G and H) correlated with both CD8a staining
frame of the experiment (data not shown). Recruitment of unlabeled                           and the MRI signal reduction observed. In a parallel experiment,
cells to B16-OVA tumors was confirmed by histology (data not                                 CLIO-HD/111In-oxine dual-labeled OT-I CD8 T cells were adop-
shown). When we performed biodistribution studies of CLIO-HD/                                tively transferred into a C57Bl/6 mouse carrying B16F0 and B16-
111
    In-oxine dual-labeled OT-I CD8 T cells at 12 and 36 h after                              OVA tumors. After 36 h, the tumors were excised, sectioned, and
injection, we determined that 25% of T cells accumulated in the                              imaged via MRI. The identical sections were then exposed for auto-
spleen, 7% in the liver, and 2% in the lung, with 6% in the B16-OVA                          radiography. As can be seen in Fig. 4, J and K, the regions of MRI
tumors, compared with 2% in the B16F0 tumors (expressed as %                                 signal reduction present within the B16-OVA tumor correlated with
injected dose/g of tissue). Therefore, OT-I CD8 T cells demon-                               the darkened areas observed via autoradiography, indicating that the
strated a 3:1 difference in recruitment to the target tumor as compared                      recruited OT-I CD8 T cells were responsible for the MRI signal
with the null tumor.                                                                         reduction observed. Using quantitative autoradiography we also val-
   Signal Reduction by MRI Correlates with Recruitment of                                    idated MRI measurements of accumulated cells and determined the in
CLIO-HD-Labeled OT-I CD8 Cells. To confirm that the signal                                   vivo detection threshold to be 3 cells/voxel.
reduction within the B16-OVA tumor was because of the specific                                  To determine the in vivo longitudinal limit of detection for CLIO-
recruitment of CLIO-HD-labeled OT-I CD8 T cells, we performed                                HD-labeled OT-I CD8 T cells within the target tumor, we per-
MR imaging of recipient mice carrying both B16F0 and B16-OVA                                 formed an additional series of experiments. We adoptively transferred
tumors, 12 h after adoptive transfer of labeled T cells. Subsequently,                       CLIO-HD-labeled OT-I CD8 T cells into recipient tumor-bearing
both tumors were excised, sectioned, and used for histological anal-                         mice and continued to image these animals up to 60 h after adminis-
ysis. A representative axial MRI slice (Fig. 4A) was correlated with                         tration (Fig. 5). We observed that the changes in signal intensity
serial sections of the same region of each tumor, staining for the                           observed via MRI persisted for 48 h (Fig. 5A) and then receded by
presence of CD8 T cells (CD8a, Fig. 4, B–D), CLIO-HD (FITC                                   60 h (Fig. 5B), suggesting that CLIO-HD-labeled OT-I CD8 T cells
visualization, Fig. 4, F–H), and Fe content (Fig. 4I). In the control                        could be detected in the tumor for a maximum of 48 h. The loss of MR
B16F0 tumor, very few CD8 T cells could be visualized (Fig. 4B);                             detectability by 60 h in vivo could be because of several factors,
however, there were numerous CD8 T cells present within the                                  including: (a) dilution because of rapid cell division; (b) emigration of
B16-OVA tumor (Fig. 4, C and D), the density of which correlated                             OT-I cells from the tumor; (c) OT-I cell apoptosis and removal; or (d)
with the degree of signal reduction observed via MRI. Because CD8a                           intracellular biodegradation of the superparamagnetic iron oxide core.
antibody is not specific for OT-I CD8 T cells, but will also stain host                      From our in vitro experiments (Fig. 2C) we had observed that CLIO-
CD8 T cells, we confirmed the presence of adoptively transferred                             HD-induced MRI signal reduction persisted for at least 120 h in
OT-I CD8 T cells by examining the presence of CLIO-HD via                                    actively proliferating cells; however, OT-I CD8 T cells divided only
fluorescence microscopy (Fig. 4, F–H) and Prussian Blue staining                             once every 44.5 h in vitro, compared with division of up to once every
                                                                                        6842
                                                        IN VIVO TRACKING OF CTL RECRUITMENT TO TUMORS BY MRI




    Fig. 4. Correlation of MR imaging with histology and autoradiography. Twelve h after adoptive transfer of CLIO-HD-labeled OT-I CD8 T cells into a mouse carrying B16F0
(left) and B16-OVA (right) melanomas, the animal was imaged by MRI, and the tumors subsequently excised and analyzed via immunohistochemistry; A, axial MRI slice through the
thighs of the animal, indicating significant signal reduction in B16-OVA because of recruitment of CLIO-HD-labeled OT-I CD8 T cells; B–I, histology of serial sections of B16F0
(B and F) and B16-OVA (C–E and G–I) tumors excised from the same mouse; B–D, CD8a staining identifying OT-I CD8 T cells in the B16F0-OVA tumor, correlating with MRI
signal reduction; E, negative control for CD8a staining of D; I, Prussian Blue staining to indicate the presence of Fe, correlating with the location of CLIO-HD-labeled OT-I CD8
T cells; F–H, fluorescence microscopy of FITC-CLIO-HD-labeled OT-I CD8 T cells, indicating the correlation of CLIO-HD with both CD8a staining and Fe content in B16-OVA.
Data are representative of 6 animals. J and K, in a separate experiment, CLIO-HD/111In-oxine colabeled OT-I CD8 T cells were adoptively transferred into recipient animal. Thirty-six
h later the tumors were excised and sectioned. The same section was imaged via MRI and then exposed for autoradiography; J, MR imaging; K, autoradiography. Note the correlation
between MRI signal reduction and radioactivity. Data are representative of 4 animals.


4 – 6 h in vivo. This difference in proliferation rate could explain the                    each administration color coded (Fig. 6, E–H) in red (0 h), green (48
discrepancy between the in vitro and in vivo temporal detection limits.                     h), and blue (96 h). When the three reconstructions were merged (Fig.
   Serial Administrations of Effector Cells Are Recruited Heter-                            6E) there was very little overlap between the color-coded maps,
ogeneously into the Target Tumor. We hypothesized that serial
doses of adoptively transferred cells might provide a better therapeutic
approach than a single bolus, but because of the dynamic environment
within the tumor we aimed at investigating the intratumoral recruit-
ment of these cells with each administration. In the next experiments
we used the same tumor model with C57Bl/6 mice (n 3) carrying
both B16F0 and B16-OVA tumors and performed serial injections of
107 CLIO-HD-labeled OT-I CD8 T cells into the same animal at
different times (0, 48, and 96 h). Mice were imaged via MRI before
adoptive transfer, and at 12, 60, and 108 h (12 h after each cell
administration). The selection of these time points allowed the signal
changes induced by the previous injection to return to baseline before
the next injection. Representative axial MR images are shown in Fig.                           Fig. 5. Temporal detection limits for MR imaging of recruitment to tumors. Axial slices
6, A–D, indicating a signal reduction in the B16-OVA tumor because                          of serial MR images of mice carrying B16F0 (left) and B16-OVA (right) at A, 48 h after
of recruitment of CLIO-HD-labeled T cells, but not in the B16F0                             administration, demonstrating the MRI signal reduction within the B16-OVA tumor
                                                                                            because of the recruitment of CLIO-HD-labeled OT-I CD8 T cells; B, the same animal
tumor. The image slices for each time point were then reconstructed                         at 60 h after adoptive transfer, showing no signal reduction via MR imaging and
into three-dimensional volumes, and the signal intensity reduction for                      demonstrating a return to baseline signal intensity by 60 h.
                                                                                       6843
                                                          IN VIVO TRACKING OF CTL RECRUITMENT TO TUMORS BY MRI




    Fig. 6. Intratumoral distribution of sequentially administered OT-I CD8 T cells. A C57Bl/6 mouse was implanted with both B16F0 (left) and B16-OVA (right) melanomas. Ten
days later, 107 OT-I CD8 T cells were labeled with CLIO-HD and adoptively transferred into the recipient (0 h). Sequential adoptive transfers of 107 CLIO-HD-labeled OT-I CD8
T cells were administered to the same mouse at 48 and 96 h. MR imaging was performed 12h after each adoptive transfer of CLIO-HD-labeled OT-I CD8 T cells; A–D, axial slices
through the mouse thighs at A, before adoptive transfer; B, 12 h after first adoptive transfer; C, 12 h after second adoptive transfer (60 h); D, 12 h after third adoptive transfer (108
h); indicating the heterogeneity of OT-I CD8 T cell recruitment to the B16-OVA tumor with sequential injections. E–H, three-dimensional-rendering of the B16-OVA tumor at F,
16 h (red, first injection; G, 60 h (green, second injection); H, 108 h (blue, third injection). E, color-coded compilation of F–H. The data indicate that there is very little overlap in
the recruitment of CD8 T cells to the same tumor with separate injections. For a 360° animation of the data see Supplemental Data web-movie 2.


indicating that the cells in each administration had been recruited to                         as unlabeled cells. In addition, their interactions with activated endo-
different regions of the tumor mass. A 360° animation of this data is                          thelial monolayers in an in vitro flow model were identical to those
shown in the Supplemental Data web-movie 2.                                                    observed for unlabeled cells, indicating that CLIO-HD does not in-
                                                                                               fluence expression of, or the activation of, cell surface adhesion
DISCUSSION                                                                                     molecules or cytokine receptors required for CD8 T-cell recruit-
                                                                                               ment. CLIO-HD-labeled CD8 T cells retained the ability to kill
   Previous reports have demonstrated the feasibility of intracellular                         target cells in vitro and in vivo, indicating no impairment of their
labeling with iron oxide particles (37) and imaging of locally injected                        cytotoxic capacity by CLIO-HD labeling.
cells (34). However, systemically administered cells undergo huge
                                                                                                  The present study has indicated significant intratumoral heteroge-
dilutions, and existing intracellular magnetic labels and protocols have
                                                                                               neity in the recruitment of CTL from the systemic circulation. In our
not allowed us to track cell recruitment in live animals. In our own
                                                                                               antigen-specific model at least, OT-I CD8 T cells were recruited
experience, previous studies have achieved limited success and have
required a combination of high field strengths (9 –14 T), long imaging                         primarily to focal regions within the tumor and areas of the tumor
times ( 8 h), and ex vivo imaging of resected tissues (37, 47).                                capsule, with some areas of the tumor apparently spared of OT-I
Currently there exists no high-resolution three-dimensional method to                          CD8 T-cell accumulation. Currently very little is known regarding
quantitate the recruitment of systemically administered cells over                             the intratumoral distribution of infiltrating cytolytic cells. In experi-
time. In the present study, we have used an improved superparamag-                             mental studies in rats carrying mammary tumors, CD8 T cells were
netic particle (CLIO-HD) and optimized the labeling protocol to                                observed largely in the tumor periphery (48), with limited infiltration
efficiently label lymphocytes, at levels that can be detected in vivo via                      into the tumor mass. A similar pattern was observed in human tissue
MRI, that are not cytotoxic and that do not influence cell behavior or                         sections from B-cell non-Hodgkin’s lymphoma patients (49). How-
effector function. CLIO-HD-labeled OT-I CD8 T cells remained                                   ever, these studies use histological analyses of limited serial sections,
  95% viable and exhibited the same profile of proliferation in vitro                          and do not describe the true three-dimensional distribution of CD8


    Fig. 7. Imaging of antitumor effects of serial OT-I CD8 T
cell injections. Axial slices of serial MR images of mice
carrying B16F0 (left) and B16-OVA (right) at A, 12 h after
initial adoptive transfer of 107 OT-I CD8 T cells; B, 12 h
after second adoptive transfer; and C, 72 h after third adoptive
transfer (7 days after initial administration). Both B16F0 and
B16-OVA tumors continue to grow at equal rates up until 60 h
(B, 12 h after second dose), but by 7 days the B16F0 tumor has
clearly continued to grow (C, left), whereas the B16-OVA
tumor has been reduced significantly (C, right). Data are
representative of 3 animals.


                                                                                          6844
                                                IN VIVO TRACKING OF CTL RECRUITMENT TO TUMORS BY MRI


T cells within the whole tumor. One of the most powerful aspects of           acknowledge Michael Delfs and Nir Grabie for helpful discussions, Deborah
our approach is the ability to describe the distribution of infiltrating      Burstein, Jeeva Munasinghe, and Alik Petrovsky for assistance with MR
CD8 T cells in three dimensions, across the entire tumor simulta-             imaging, and G. Stavrakis for advice with immunohistochemistry.
neously, and in a quantitative and repetitive manner in the same
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