Kinetic phases of distribution and tumor targeting by
T cell receptor engineered lymphocytes inducing
robust antitumor responses
Richard C. Koyaa,1, Stephen Moka, Begoña Comin-Anduixa, Thinle Chodonb, Caius G. Raduc, Michael I. Nishimurad,
Owen N. Wittec,e,f,g,1, and Antoni Ribasa,b,g,h,1
aDepartment of Surgery, Division of Surgical Oncology,bDepartment of Medicine, Division of Hematology/Oncology,cDepartment of Molecular and Medical
Pharmacology,eDepartment of Microbiology, Immunology and Molecular Genetics,fHoward Hughes Medical Institute,gBroad Stem Cell Research Center, and
hJonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095; anddDepartment of Surgery, Medical University of South Carolina,
Charleston, SC 29403
Contributed by Owen N. Witte, June 18, 2010 (sent for review March 3, 2010)
receptor (TCR) engineered lymphocytes for cancer therapy is demon-
strating how TCR transgenic cells repopulate lymphopenic hosts and
target tumors in anantigen-specific fashion.ACT ofsplenocytesfrom
murine/human TCR specific for tyrosinase, together with lymphode-
pletion conditioning, dendritic cell (DC)-based vaccination, and high-
dose interleukin-2 (IL-2), had profound antitumor activity against
with bioluminescence imaging (BLI) and positron emitting tomogra-
ing correlated with antitumor efficacy. After an initial brief stage of
systemic distribution, TCR-redirected and genetically labeled T cells
demonstrated an early pattern of specific distribution to antigen-
matched tumors and locoregional lymph nodes, followed by a more
promiscuous distribution 1 wk later with additional accumulation in
antigen-mismatched tumors. This approach of TCR engineering and
molecular imaging reporter gene labeling is directly translatable to
this mode of therapy.
adoptive cell transfer therapy|molecular imaging|tumor immunotherapy
tigen-specific T cells generated by ex vivo expansion of cytotoxic
T lymphocytes (CTLs) from peripheral blood mononuclear cells
(PBMC) (1) or by expanding tumor antigen-reactive tumor-in-
filtrating lymphocytes (TIL) (2). These approaches have resulted
in significant antitumor activity in patients with metastatic mela-
noma, but they are primarily limited by the need for lengthy ex
vivo cell expansion time (several weeks) followed by the selection
of antigen-specific cells for ACT. T cell receptor (TCR) engi-
neering represents an alternative approach that attempts to
shorten this process because the transfer of the alpha and beta
TCR genes is necessary and sufficient to endow recipient T cells
with the specificity of donor T cells (3, 4). The pioneering work by
investigators at the Surgery Branch, National Cancer Institute,
provided proof of principle that the ACT of TCR-engineered
lymphocytes in humans is feasible and leads to objective tumor
responses in patients with metastatic melanoma (5, 6).
However, early clinical studies with ACT of TCR-engineered
cells suggest that their antitumor activity lags behind the response
rates achieved with ACT of TILs (2). Using two different TCRs,
the response rate of ACT of TCR transgenic cells to patients with
metastatic melanoma was in the range of 25%, whereas the same
full ACT protocol but using TILs generated response rates in the
50–70% range in patients with metastatic melanoma (6, 7). There
are several possible explanations for this discrepancy, one of them
being a differential trafficking of peripheral blood lymphocytes
doptive cell transfer (ACT) of antigen-specific T cells
involves the administration of large pools of autologous an-
genetically modified to express transgenic TCRs compared with
the ability of TILs to traffic back to peripherally located tumors.
The study of the in vivo dynamics of the infused cells and how they
specifically target tumors would provide information about po-
tential problems, such as lack of specific tumor homing following
ex vivo expansion, inappropriate sequestration in nonantigen
positive sites, or rapid cell death and inability for the adoptively
transferred TCR transgenic cells to persist in vivo. The study of
these possibilities can be achieved with modern molecular imaging
techniques with reporter gene labeling of cells to allow non-
invasive detection of adoptively transferred TCR transgenic cell
populations in recipients (8). In the current work we have taken
the molecular imaging gene marking approach of antigen-specific
T cells one step closer to the clinic by simultaneously redirecting
the TCR specificity of T cells and providing genetic labeling for
molecular imaging demonstrating robust antitumor activity cor-
related with specific tumor targeting.
Efficient TCR and Reporter Transgene Expression and Function with
2A-Linked Viral Constructs. We used a TCR obtained from a TIL
clone (TIL 1383I) that specifically recognizes the MHC class
I-restricted tyrosinase368–376peptide presented by HLA-A2.1 (9,
10). A chimeric murine/human modification of this tyrosinase-
specific TCR, with proximal constant TCR subunits being murine
and the distal variable subunits being human and restricted to
HLA-A2.1 (Fig. 1A), allowed its testing in a fully immunocom-
petent mouse model, recognizing tyrosinase antigen-expressing
tumors that had been engineered to express a corresponding chi-
meric murine/human MHC molecule derived from HLA-A2.1/Kb
mice (11). This modification turned the tumors syngeneic to the
HLA-A2.1/Kbmice, which express the HLA-A2.1 α1 and α2
domains that allow their cells to present the same epitopes as
HLA-A2.1 subjects and maintain the murine α3 domain, permit-
ting murine CD8 coreceptor engagement (11). We generated
lentiviral and retroviral vectors coexpressing the alpha and beta
TCR chains of this TCR and molecular imaging reporter genes
linked by picornavirus-derived “self-cleaving” 2A-like sequences
(Fig. 1B). The 2A sequences allow the stoichiometric expression of
Author contributions: R.C.K., C.G.R., M.I.N., O.N.W., and A.R. designed research; R.C.K., S.M.,
B.C.-A., and T.C. performed research; R.C.K., B.C.-A., C.G.R., M.I.N., O.N.W., and A.R. con-
tributed new reagents/analytic tools; R.C.K., C.G.R., M.I.N., O.N.W., and A.R. analyzed
data; and R.C.K., O.N.W., and A.R. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
See Commentary on page 13977.
1To whom correspondence may be addressed. E-mail: email@example.com, aribas@
mednet.ucla.edu or firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 10, 2010
| vol. 107
| no. 32www.pnas.org/cgi/doi/10.1073/pnas.1008300107
several transgenes under a single promoter in a reasonably sized
gene transfer vector (12, 13), which are key features for the success
of this approach. The reporter genes were luciferase for bio-
luminescence imaging (BLI) and an optimized herpes simplex
virus 1 thymidine kinase (HSV1-tk) termed sr39tk for microPET
imaging (14, 15). Transduction of cells with viral supernatants
efficiently induced the expression of both alpha and beta TCR
chains with more than 90% cleavage efficiency mediated by the
2A sequences as assessed by immunoblotting (Fig. 1C). A high
level of cell surface expression of the TCRs was confirmed
by flow cytometry with a clonotypic antibody to the beta chain
(Fig. 1D) and by a specific HLA-A2.1 tetramer loaded with ty-
rosinase368–376 peptide (Fig. 1E). The vector constructs also
allowed expression of high levels of the fluorescent marker GFP
(Fig. 1 D and E) concomitant with the PET reporter gene sr39tk.
The functionality of sr39tk in transduced cells was analyzed by
uptake of radiolabeled penciclovir (Fig. 1F) and by its use as
a suicide gene when treated with ganciclovir (Fig. S1).
Transduced Primary T Cells Specifically Recognize Antigen-Matched
Cell Targets. We then generated tyrosinase TCR retargeted pri-
mary murine T cells from HLA-A2/Kbtransgenic mice by ret-
roviral transduction with a limited expansion protocol of a total of
4 d (including the 2 d of viral vector transduction), in an attempt
to limit the alteration of the functional phenotype of T cells used
for ACT. These cells displayed specific functional activity dem-
onstrated by high tyrosinase-restricted polyfunctional cytokine
production (Fig. 2 and Fig. S2).
Potent Melanoma Tumor Eradication in Vivo with TCR-Transduced
Syngeneic Primary T Cells. Fully immunocompetent HLA-A2/Kb
transgenic mice with flank B16-A2/Kbmurine melanoma tumors
of 4 mm average diameters underwent whole body myelodepleting
irradiation followed by i.v. ACT of tyrosinase TCR-transduced
syngeneic splenocytes, tyrosinase368–376peptide-pulsed DC vacci-
nation, and high-dose IL-2 (Fig. 3A). The melanoma tumors grew
rapidly in the control group where mice received the complete
therapy including myelodepletion, DC vaccination, and IL-2 but
the splenocytes were transduced with a control retrovirus. Mice
receiving adoptive transfer of tyrosinase TCR T cells had robust
antitumor activity (Fig. 3B) and improved survival (P = 0.0006).
In Vivo T Cell Tracking with Bioluminescence Imaging Shows Discrete
Patterns of TCR Transgenic Distribution and Specific Tumor Targeting.
ACT of TCR transgenic cells need to repopulate a lymphopenic
host, expand in vivo, target antigen-matched tumors, and then
exert their specific cytotoxic activity. This process can be se-
quentially studied using noninvasive molecular imaging. HLA-
A2/Kbtransgenic mice had isogenic tumors implanted that stably
expressed tyrosinase (EL4-A2/Kb-Tyr) or did not express this
tumor antigen (EL4-A2/Kb) in contralateral lower abdominal
flanks. When tumors reached average diameters of 6 mm, mice
were conditioned with whole body irradiation and then re-
ceived i.v. ACT of tyrosinase TCR/firefly luciferase retroviral
vector-transduced syngeneic T cells, tyrosinase368–376 peptide
Schematic of the chimeric murine/human interaction between the trans-
genic TCR and MHC molecules in the A2.1/Kbmouse model. In gray are the
proximal murine sequences, and in white the distal human sequences
allowing the presentation and recognition of peptide antigen with human
restriction. (B) Tyrosinase (Tyr)-TCR/sr39TK-GFP and firefly luciferase vector
schematic representation. (C) Immunoblotting of 293T cells transduced with
control lentiviral vector expressing GFP (LV-GFP) or the tyrosinase TCR and
sr39tk (LV-TCR/sr39TK-GFP) vectors, incubation with rabbit anti-2A primary
antibody. Arrows indicate cleaved products with sizes corresponding to
the TCR α and β chains. (D) Flow-cytometric analysis of 293T cells expressing
CD3 transduced with LV-TCR/sr39TK-GFP vector, stained with specific clono-
typic anti-Vβ12 antibody. (E) Tyrosinase368–376specific and negative control
peptide HLA-A2.1 tetramer assay of Jurkat cells transduced with LV-TCR/
sr39TK-GFP vector. (F) Penciclovir uptake assay of Jurkat cells transduced with
negative control LV-GFP, positive control LV-L/GFP/TK, or LV-TCR/sr39TK-GFP
Model system, vector schematic, and transgene expression. (A)
pg/ ml 106 cells/24h
tyrosinase TCR retroviral supernatants. (A) ELISPOT assay for cellular IFN-γ
secretion of control T cells and tyrosinase TCR transduced T cells incubated
with control scrambled or tyrosinase368–376peptides. (B) ELISA for total IFN-γ
secretion of 24 h-collection supernatants of EL4-A2/Kbpulsed with control or
tyrosinase peptides coincubated with control T cells or tyrosinase TCR
transduced T cells.
In vitro functional analysis of murine primary T cells transduced with
Koya et al.PNAS
| August 10, 2010
| vol. 107
| no. 32
pulsed DCs, and high-dose IL-2 with serial BLI of T cell locali-
zation and persistence. After an initial brief period of systemic
distribution, a strong BLI signal was observed in the tyrosinase-
expressing tumors with peak intensity on day 5 after ACT (Fig. 4A
and Fig. S3). The decrease in BLI intensity paralleled the cor-
responding marked tumor shrinkage in response to tyrosinase
TCR ACT therapy (Fig. 4B). The BLI signal on tyrosinase nega-
tive tumors was relatively low initially, but gradually increased as
the tumors grew in size (Fig. 4A). We confirmed the identity of
adoptively transferred tyrosinase TCR/firefly luciferase T cell
accumulation in the tumors and lymph nodes by CD4, CD8,
and luciferase immunofluorescence staining of histological sec-
tions (Fig. 4C).
Micro-PET/CT Imaging of TCR-Transduced T Cell Localization with
Higher Spatial Refinement and Quantification Confirming the Phases
of T Cell Distribution in Vivo. There is signal attenuation from deep
tissues with BLI analysis, which may limit the study of tyrosinase
TCR transgenic cell distribution in vivo and its clinical applica-
bility. Therefore, to confirm the findings with an approach with
higher spatial/anatomical localization of ACT with engineered
tyrosinase TCR T cells, micro-PET/CT imaging was used. Murine
T cells were transduced with a retroviral vector coexpressing
tyrosinase TCR and sr39tk-GFP and adoptively transferred i.v.
to syngeneic mice with EL4-A2/Kbtumors expressing tyrosinase
and negative control EL4-A2/Kbtumors (Fig. S4) or B16-A2/Kb
murine melanoma tumors (Fig. 5) and followed over different
time periods. The phases of tyrosinase TCR transgenic cell dis-
tribution upon ACT defined by BLI were confirmed with PET
imaging. As observed in the BLI studies, tyrosinase TCR trans-
genic T cells equally distributed to both tyrosinase positive and
negative tumors on day 1. Scans on subsequent days showed
higher accumulation of tyrosinase TCR T cells in tyrosinase-
expressing tumors (Fig. S4) followed by a final phase of systemic
distribution. With the higher spatial resolution provided by mi-
cro-PET/CT imaging, a punctuated pattern demonstrating
a heterogeneous accumulation of T cells within the tumor con-
finements could be observed (Fig. 5A and Fig. S4). Another
advantage of micro-PET/CT imaging is the possibility of tri-
dimensional imaging reconstruction (Fig. 5B and Movie S1).
Because [18F]FHBG tracer scans in mice (but not in humans)
(16) have high nonspecific background signal in organs involved
in its clearance such as bladder, kidneys, ureters, liver, gall
bladder, and intestines, the signals from these organs were sub-
tracted (Fig. S4). The utilization of Fenestra VC contrast agent
for CT imaging allowed us to analyze and quantify the signal
localized in the spleen (Fig. 5B and Fig. S4B). In addition, micro-
PET/CT scanning could distinguish discrete signal corresponding
to axillary lymph nodes from the closely located tumor (Fig. 5B).
This work demonstrates a close relationship between antitumor
activity and early specific antigen homing of TCR-engineered
ACT therapy. This was achieved using lymphocytes that were
simultaneously genetically redirected and labeled to allow the
generation of large numbers of uniformly antigen-specific cells
that could be visualized as they accumulated in tumors express-
ing their cognate antigen, leading to profound antitumor activity
using this system. We demonstrate that the i.v. adoptive transfer
of tyrosinase TCR-engineered T cells to lymphopenic hosts goes
through an orderly systemic distribution and antigen-specific
There is a need to optimize the ACT of TCR transgenic cells
to achieve the antitumor activity levels of ACT with TIL (2).
Improvements in viral vector design, high-efficiency gene modi-
fication of lymphocytes, and selection of TCR chains with
dominant pairing or with molecular alterations to avoid mis-
pairing with endogenous TCR chains (6, 17) allow rapid gener-
ation of large pools of antigen-specific lymphocytes for ACT with
limited ex vivo manipulation. The limitation derived from the
need for ex vivo expansion of lymphocytes to transduce T cells is
important because this process is likely to modify the phenotype
and function of these cells (18), resulting in altered in vivo dis-
tribution to secondary lymphoid organs and peripheral tissues.
Potential scenarios include the inability of T cells to maintain
a naïve phenotype with this manipulation, the loss of specific
memory T cell phenotypes, and the acquisition of late effector or
exhausted T cell phenotypes with prolonged ex vivo expansion
(19). Therefore, minimizing ex vivo manipulation and analyzing
the in vivo kinetics of adoptively transferred cells would allow
optimization of this therapeutic approach.
Our fully immunocompetent and syngeneic animal model
should be useful for the testing of the distribution of TCR
transgenic cells with relevance to the clinic. It provides a signifi-
cant advancement over xenogeneic ACT studies in immunode-
ficient animals (20), because those studies lack fully developed
lymphoid organs and therefore cannot provide deeper insight
into the engraftment and biodistribution of adoptively trans-
ferred TCR-engineered T cells. In a clinical scenario, the
patient’s own peripheral blood T cells would be engineered to
express TCR specific to tumor antigens, which is similar to our
use of murine splenocytes with limited ex vivo manipulation to
insert TCR genes and molecular imaging genes. Using this
model we demonstrated that the retargeted T cells are very ef-
ficient in controlling specific tumor growth in vivo and they lo-
calized in different organs in an exquisite temporal pattern. The
model could be expanded to preclinically screen the in vivo
function of newly cloned TCRs, because HLA-A2.1 is the most
prevalent HLA allele in the general population, accounting for
roughly 45% of patients with metastatic melanoma. Conse-
quently, HLA-A2/Kbtransgenic mice provide a powerful model
to study human MHC class I-restricted antigen epitopes recog-
nized by human T cells, which potentially provide a direct ap-
plication of the same TCRs studied in these transgenic mice for
future clinical use.
TCR retrovirus. (A) HLA-A2/Kbtransgenic mice with s.c. B16-A2/Kbtumors
received the full protocol of myelodepletion with hematopoietic stem cells
(HSC)/ bone marrow (BM) transplantation, adoptive cell transfer (ACT) of
control T cells or tyrosinase TCR-transduced T cells followed by tyrosinase368–
376peptide-pulsed dendritic cell (DC) vaccination and high-dose IL-2. (B) Mice
were followed for tumor growth measurements (product of two diameters
as mm2), P < 0.01.
In vivo function of murine primary T cells transduced with tyrosinase
| www.pnas.org/cgi/doi/10.1073/pnas.1008300107Koya et al.
The definition of the pattern of distribution of adoptively
transferred cells eliciting robust antitumor activity can be used to
later analyze individual components of this combinatorial ap-
proach before going into clinical trials. In addition to screening
different TCRs with human MHC class I restriction elements,
their skewing while being activated ex vivo for TCR transduction
affecting their in vivo distribution upon ACT, the maximization of
conditioning regimen, the use of high-dose IL-2, and antigen-
specific vaccines can be systematically analyzed. Therefore, mo-
lecular imaging with reporter genes enables monitoring of adop-
tively transferred TCR-engineered retargeted cells to study their
biodistribution, expansion/contraction, and persistence, allowing
pattern-based prediction of T cell-based immunotherapeutic ef-
ficiency and clinical outcome.
Materials and Methods
Subcloning and Vector Construction. The αandβ chains ofthe TIL 1383I TCR(9,
counterparts bystandardPCRtechniques togenerateahybridhuman/murine
TCR construct. The 2A self-cleaving sequences were inserted between the
a self-inactivating third generation lentiviral vector (21) or a retroviral vector
derived from a murine stem cell virus (pMSCV) backbone (22) containing a 5′
long terminal repeat-driven truncated version of the sr39tk (15) fused with
obtain the HLA-A2/Kbtransgene to engineer syngeneic tumor targets, total
mRNA was obtained from hepatocytes of HLA-A2/Kbtransgenic mice (from
Linda Sherman, The Scripps Research Institute, La Jolla, California) (11). This
HLA-A2/Kbtransgene was inserted into a lentiviral vector with a MND-driven
B16-A2/Kband EL4-A2/Kb, respectively. Similarly, the full-length human ty-
rosinase cDNA was obtained by PCR cloning and used to generate a lentiviral
vector with MND promoter for the transduction of EL4-A2/Kbcells to express
the tyrosinase gene (EL4-A2/Kb-Tyr).
Analysis of Transgene Expression by TCR Engineering Viral Vectors. Lentivirus
vectors were produced using a transient transfection protocol (21). Testing of
transgene expression was performed by transducing 293T-CD3 cells (from
David Baltimore, California Institute of Technology, Pasadena, California).
Cells were analyzed by Western blot using an anti-2A antibody (from Dario
Vignali, St. Jude Children’s Research Hospital, Memphis, Tennessee) as pre-
viously described (13). TCR expression upon transduction of 293T-CD3, the
and control EL4-A2/Kb(Right) tumors received the full protocol of adoptive cell transfer (ACT) with tyrosinase TCR/fLuciferase-transduced T cells. Mice
were followed from day 1 to 10 post-ACT and bioluminescence signal of ventral views were recorded and quantified on region of interest (ROI) drawn on
tumor sites. Representative animals are shown. Pink, EL4-A2/Kb-tyrosinase+; yellow, control EL4-A2/Kb. (B) These mice were followed for tumor growth
measurements (product of two diameters as mm2). (C) EL4-A2/Kb-tyrosinase+ tumors were costained with DAPI (nucleus localization), anti-CD8, and anti-
Bioluminescence imaging (BLI) of T cell trafficking in vivo. (A) HLA-A2/Kbtransgenic mice with inguinal s.c. EL4-A2/Kb-expressing tyrosinase (Left)
Koya et al.PNAS
| August 10, 2010
| vol. 107
| no. 32
human T cell lymphoma line Jurkat or primary murine splenocytes was an-
alyzed by flow cytometry using a clonotypic TCR β12 antibody (BD Bio-
sciences) and tyrosinase368–376MHC tetramers (Beckman Coulter). An in vitro
ganciclovir lysis assay was performed by adding titrated amounts of ganci-
clovir to transduced 293T-CD3 cells and viable cells analyzed by an MTS as-
transduced cells incubated for 120 min with [3H]-Penciclovir at 3.7 kBq/mL
(1.48 TBq/mmol) (Moravek Biochemicals) were assayed for radioactivity con-
centrations with a TriCarb 1600 β-spectrometer (Canberra Packard).
3H-Penciclovir accumulation assays, transduced and control un-
In Vitro Activation of Murine T Cells and Viral Vector Transduction. High-titer
helper-free lentivirus or retrovirus stocks were prepared by transient
cotransfection of 293T cells (21). RBC lysed murine splenocytes from HLA-A2/
Kbtransgenic mice were cultured in X-Vivo 15 (Biowhittaker) supplemented
with 10% heat inactivated FBS (HyClone), 50 μM β-mercapto-ethanol and 50
IU/mL of rhIL-2 (Novartis) with anti-CD3 and anti-CD28-coated plates (BD
Biosciences). At 48 h postactivation, cells underwent two rounds of spin-
fection with retrovirus supernatants (10 multiplicity of infection) in retro-
nectin (Takara Bio) coated plates at 1,000 g, 120 min, 32 °C.
Adoptive Transfer, Vaccination, and Tumor Treatment. HLA-A2/Kbtransgenic
mice had s.c. B16-A2/Kbtumor implanted, or EL4-A2/Kbtumors expressing or
not expressing tyrosinase protein. When tumors reached 4–6 mm in di-
ameter, lymphopenia was induced by sublethal irradiation (500 cGy) or
myeloablation with 900 cGy irradiation (followed by bone marrow trans-
plant as described in ref. 24). On the next day (day 0), groups of mice were
randomized into control marker vector or tyrosinase TCR vector transduced
cells for i.v. (tail vein) injection. DCs were differentiated from bone marrow
progenitor cells obtained from HLA-A2/Kbmice by in vitro culture in murine
granulocyte macrophage colony-stimulating factor (GM-CSF, 50 ng/mL) and
murine IL-4 (50 ng/mL; R&D Systems) as described (25) and pulsed with ty-
rosinase368–376peptide at 10 μM in serum-free media for 90 min at room
temperature. Each mouse received 105pulsed DCs s.c. on day 0. Recombi-
nant human IL-2 (250,000 IU) was injected intraperitoneally on days 0, 1, and
2. Murine studies were performed under the University of California Los
Angeles Animal Research Committee (ARC) approval number 2004–159.
Functional Antigen Recognition Assays. Transduced cells were tested for ty-
rosinase-specific reactivity by coculturing responder cells with stimulator cells
in a 1:1 ratio in 96-well U-bottomed plates. Stimulator cells included tyros-
inase368–376peptide-pulsed or unpulsed K562-A2.1, EL4-A2/Kb, EL4-A2/Kb-
Tyr, or B16-A2/Kbcells. The amount of IFN-γ released was measured by ELISA
(R&D Systems) and ELISPOT assays as described (26). For the multiplex cy-
tokine-release assay, supernatants were obtained at 24, 48, and 72 h of
coincubation and analyzed following the manufacturer’s instructions using
a Bio-Plex Mouse Cytokine Panel (Bio-Rad Laboratories).
Bioluminescence Imaging (BLI). BLI was performed with a Xenogen IVIS 200
Imaging System (Xenogen/Caliper Life Sciences) as previously described (14).
Micro-PET/Computed Tomography Imaging. Mice were anesthetized with
2% isoflurane. PET was performed 1 h after i.v. administration of 7.4 MBq
(200 μCi) of [18F]FHBG and mice were scanned using a FOCUS 220 micro-PET
scanner (Siemens) (energy window of 350–750 keV and timing window of 6
ns) as described previously (15). Additional details on the performance of
PET scans are included in SI Materials and Methods.
Histological Analysis. Freshly isolated tissues were frozen in optimum cutting
temperature (OCT) compound (Sakura Finetek). The immunohistochemical
reaction was carried out with the following antibodies: rat anti-mCD4 or anti-
mCD8 (BD Biosciences), and rabbit anti-fLuciferase (Abcam) and then with
secondary donkey anti-rat antibodies conjugated to DyLight 488 and anti-
rabbitDyLight 549(Jackson ImmunoresearchLaboratories), respectively,with
4,6-diamidino-2-phenylindole for nuclei visualization. Immunofluorescence
was assessed with a fluorescence microscope (Carl Zeiss).
Statistical Analysis. Data were analyzed with GraphPad Prism (version 5)
software (GraphPad Software). A Mann–Whitney test or ANOVA with Bon-
ferroni posttest was used. Survival analysis was performed with the Kaplan–
Meier method, and curves were compared in a log-rank test.
ACKNOWLEDGMENTS. This work was funded by the National Institutes of
Health Award P50 CA086306, the California Institute for Regenerative Medicine
New Faculty Award RN2-00902-1, the California Institute of Technology–Univer-
sity of California Los Angeles Joint Center for Translational Medicine (to A.R.),
and the California Institute for Regenerative Medicine Tools and Technology
Award RT1-01126 (to C.G.R.). R.C.K. was supported by the V Foundation-Gil
Nickel Family Endowed Fellowship in Melanoma Research. O.N.W. is an investi-
gator of the Howard Hughes Medical Institute.
post-ACT. (A) HLA-A2/Kbtransgenic mice with thoracic dorsal s.c. B16-A2/Kb(Right) and control EL4-A2/Kb(Left) tumors were adoptively transferred with
tyrosinase TCR/sr39TK/GFP transduced T cells. Representative animals are shown. Specific signal quantification ratio above background: Left ROI = 1.05 ±
0.59%ID/g; Right ROI = 3.00 ± 0.61%ID/g. (B) Schematic representation of tumor location and reconstructed tridimensional PET CT scan image with the
nonspecific signal from abdominal excretion of [18F]FHBG subtracted from the final image. Yellow arrows, signals on axillary lymph nodes (specific signal
quantification ratio above background: Right LND = 2.21 ± 0.52%ID/g; Left LND = 2.05 ± 0.63%ID/g). Blue arrow, signal in B16-A2/Kbtumor. R, right; L, left; D,
dorsal; V, ventral sides.
PET CT imaging of T cell trafficking in vivo in mice with control EL4-A2/Kbtumors or with contralateral tyrosinase-positive B16-A2/Kbtumors at day 5
| www.pnas.org/cgi/doi/10.1073/pnas.1008300107Koya et al.
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| August 10, 2010
| vol. 107
| no. 32