Protective immunosurveillance and therapeutic antitumor activity of gammadelta T cells demonstrated in a mouse model of prostate cancer.
ABSTRACT In contrast to Ag-specific alphabeta T cells, gammadelta T cells can kill malignantly transformed cells in a manner that does not require the recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that gammadelta T cells provide protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a rationale for developing novel clinical approaches to exploit the innate antitumor properties of gammadelta T cells for the treatment of cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish that gammadelta T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP mice, which predictably develop prostate adenocarcinoma, were backcrossed with gammadelta T cell-deficient mice (TCRdelta(-/-) mice) yielding TRAMP x TCRdelta(-/-) mice, a proportion of which developed more extensive disease compared with control TRAMP mice. By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with supraphysiological numbers of syngeneic gammadelta T cells (C57BL/6-derived) developed measurably less disease compared with untreated mice. Disease-bearing mice treated i.v. with gammadelta T cells also displayed superior survival compared with untreated mice. These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous gammadelta T cells for the treatment of prostate cancer.
- SourceAvailable from: fhcrc.org[show abstract] [hide abstract]
ABSTRACT: T cells with variable region Vdelta1 gammadelta T cell receptors (TCRs) are distributed throughout the human intestinal epithelium and may function as sentinels that respond to self antigens. The expression of a major histocompatibility complex (MHC) class I-related molecule, MICA, matches this localization. MICA and the closely related MICB were recognized by intestinal epithelial T cells expressing diverse Vdelta1 gammadelta TCRs. These interactions involved the alpha1alpha2 domains of MICA and MICB but were independent of antigen processing. With intestinal epithelial cell lines, the expression and recognition of MICA and MICB could be stress-induced. Thus, these molecules may broadly regulate protective responses by the Vdelta1 gammadelta T cells in the epithelium of the intestinal tract.Science 04/1998; 279(5357):1737-40. · 31.03 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Here we describe a family of GPI-anchored cell surface proteins that function as ligands for the mouse activating NKG2D receptor. These molecules are encoded by the retinoic acid early inducible (RAE-1) and H60 minor histocompatibility antigen genes on mouse chromosome 10 and show weak homology with MHC class I. Expression of the NKG2D ligands is low or absent on normal, adult tissues; however, they are constitutively expressed on some tumors and upregulated by retinoic acid. Ectopic expression of RAE-1 and H60 confers target susceptibility to NK cell attack. These studies identify a family of ligands for the activating NKG2D receptor on NK and T cells, which may play an important role in innate and adaptive immunity.Immunity 07/2000; 12(6):721-7. · 19.80 Impact Factor
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ABSTRACT: Physiologic immune responses are an integration of the activities of several lymphoid cell types that include qualitatively distinct T cell subsets. The contributions that specific T cell subsets make during infection, inflammation, and carcinogenesis is becoming increasingly clear from a variety of mouse models and, importantly, their backcrossing onto different genetic backgrounds. This review considers what we have learned in the mouse about the crucial roles played by gammadelta T cells. We consider how the cells' associations with specific tissues have revealed that T cell responses are regulated locally as well as systemically, and we discuss the implications of this for understanding and enhancing immune surveillance in the clinical setting.Chemical immunology and allergy 02/2005; 86:136-50.
Protective Immunosurveillance and Therapeutic Antitumor
Activity of ?? T Cells Demonstrated in a Mouse Model of
Zhiyong Liu,* Isam-Eldin A. Eltoum,‡Ben Guo,* Benjamin H. Beck,§Gretchen A. Cloud,*
and Richard D. Lopez2*†
In contrast to Ag-specific ?? T cells, ?? T cells can kill malignantly transformed cells in a manner that does not require the
recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that ?? T cells provide
protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a
rationale for developing novel clinical approaches to exploit the innate antitumor properties of ?? T cells for the treatment of
cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish
that ?? T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP
mice, which predictably develop prostate adenocarcinoma, were backcrossed with ?? T cell-deficient mice (TCR??/?mice)
yielding TRAMP ? TCR??/?mice, a proportion of which developed more extensive disease compared with control TRAMP mice.
By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate
cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in
otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with
supraphysiological numbers of syngeneic ?? T cells (C57BL/6-derived) developed measurably less disease compared with un-
treated mice. Disease-bearing mice treated i.v. with ?? T cells also displayed superior survival compared with untreated mice.
These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous ?? T
cells for the treatment of prostate cancer. The Journal of Immunology, 2008, 180: 6044–6053.
focused primarily upon adaptive cellular immune responses medi-
ated primarily by tumor Ag-specific ?? CTL. However, despite
major advances in the field, clinical approaches that rely upon the
generation of highly specific adaptive cellular immune responses
directed against tumor-associated or tumor-specific Ags have only
been modestly successful. With this result in mind, particularly in
the context of developing and testing new cell-based cancer ther-
apies, it becomes important to consider and explore tumor Ag-
independent (innate) cellular immune responses mediated by such
cells as NK cells and ?? T cells.
Unlike ?? T cells, which recognize specific peptide Ags pre-
sented by MHC molecules, ?? T cells in contrast can recognize
generic Ags, which are physiologically expressed by stressed cells,
he view that cellular immune responses might be ex-
ploited for the treatment of cancer is not new. Interest-
ingly, to date, the majority of studies in this regard have
including cells that have undergone malignant transformation. In-
deed, cancerous cells are now known to display a number of stress-
induced Ags (e.g., MICA/MICB in humans; Rae-1 in mice) that
although neither tumor-specific nor tumor-derived per se, can
nonetheless serve as recognition determinants for human and
mouse ?? T cells (1–6). Although a functional homology between
mouse and human ?? T cells has yet to be firmly established in this
specific regard, the complementary study of both mouse and hu-
man ?? T cells has yielded important insight into how ?? T cells
recognize and kill malignantly transformed cells in vitro and in
vivo. For example, as expertly reviewed by Girardi (5) and Kabe-
litz et al. (6), it is now evident that both mouse and human ?? T
cells use various pairings of specific TCR?? chains, often in com-
bination with key coreceptors such as NKG2D, to interact with
determinants commonly expressed on tumor cells that are suscep-
tible to ?? T cell-mediated killing. Indeed, it is this ability of both
mouse and human ?? T cells to recognize and kill cancer cells of
various histological subtypes in a tumor Ag-independent manner
that has contributed to the emerging view that ?? T cells provide
protective immunosurveillance against cancer. This view is sup-
ported by the findings that mice lacking ?? T cells are more sus-
ceptible to the development of chemically induced cutaneous tu-
mors and are likewise less able to resist challenges with
tumorigenic melanoma or squamous cell carcinoma cell lines
In this report, we begin with proof-of-concept studies that both
confirm and extend these important initial findings of others (7–9).
In this study, we demonstrate for the first time that ?? T cells are
indeed capable of providing protective immunosurveillance
against a spontaneously arising noncutaneous solid tumor of epi-
thelial origin, in this instance, mouse prostate cancer. For these
*Division of Hematology and Oncology and†Bone Marrow Transplantation and Cell
Therapy Program, Department of Medicine,‡Anatomic Pathology, and§Program in
Molecular and Cellular Pathology, Department of Pathology, The University of
Alabama at Birmingham, Birmingham, AL 35294
Received for publication February 24, 2008. Accepted for publication February
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work is supported in part by a Congressionally Directed Medical Research
Programs Award DAMD17-03-1-0265 from the U.S. Department of Defense.
2Address correspondence and reprint requests to Dr. Richard D. Lopez, BMI
Program, 541 Tinsley Harrison Tower, The University of Alabama at Birming-
ham, 1900 University Boulevard, Birmingham, AL 35294. E-mail address:
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
studies, transgenic mice that spontaneously develop prostate can-
cer were bred with ?? T cell-deficient mice, which allowed for
observation of tumor development occurring in the absence of ??
T cells. Findings from these initial proof-of-concept studies were
then used as the rationale for performing subsequent studies to
conversely examine how the adoptive transfer of supraphysiologi-
cal numbers of syngeneic ?? T cells might be used to treat tumors
established in otherwise healthy mice.
The model of transgenic adenocarcinoma of the mouse prostate
(TRAMP) was chosen for these studies because it is a model of
spontaneously arising mouse prostate cancer that mimics human
disease (10–13). As disease that develops in TRAMP mice histo-
logically resembles human disease with pathology ranging from
noncancerous prostatic intraepithelial neoplasia to aggressive ad-
enocarcinoma of the prostate (10–13), the TRAMP model has
been widely adopted for use in a variety of studies designed to
assess novel therapies directed against prostate cancer (14–17).
Accordingly, we have adopted this model to first assess the extent
to which the absence of ?? T cells might be permissive for the
development or progression of spontaneously arising mouse pros-
tate cancer. Extending from these studies, immunotherapy studies
were then performed by first establishing disease in healthy wild-
type C57BL/6 mice using TRAMP-C2 cells, a syngeneic mouse
prostate cancer cell line originally derived from TRAMP mice
(C57BL/6 genetic background) (18). Disease-bearing mice were
then treated with supraphysiological numbers of ex vivo expanded
syngeneic (C57BL/6-derived) ?? T cells, thus approximating a
clinical situation in which a tumor-bearing patient might be treated
with supraphysiological numbers of ex vivo expanded autologous
?? T cells.
Given the recognized capacity of ?? T cells to innately kill
malignant cells, efforts are now actively underway to develop the
means to exploit the antitumor properties of ?? T cells for clinical
purposes (6, 19). Although it remains to be determined specifically
how this exploitation might best be accomplished, two general
approaches are currently being taken in this regard. One approach
includes strategies primarily designed to activate or expand en-
dogenous ?? T cells in vivo, for example through the clinical ad-
ministration of pharmacological agents such as the aminobisphos-
phonates pamidronate (Aredia) or zoledronate (Zometa). Though
commonly used to prevent skeletal fractures in cancer patients,
these drugs have recently been found to cause the in vivo activa-
tion or expansion of human ?? T cells, particularly when admin-
istered in conjunction with IL-2 (20, 21). Importantly, recently
published results from a phase I clinical trial strongly support the
view that such activated ?? T cells found in zoledronate-treated
patients contribute either directly or indirectly to the clinical re-
sponses observed in patients with hormone-refractory prostate can-
Alternatively, the innate antitumor properties of human ?? T
cells might also be exploited through the adoptive transfer of ?? T
cells first expanded ex vivo, then subsequently reinfused into tu-
mor-bearing patients. Though such approaches are far less well
developed given the difficulty in generating a sufficient number of
?? T cells, recent advances by our group and other researchers
have now made possible the efficient large-scale ex vivo expansion
of human ?? T cells that importantly retain potent innate antitumor
activity against a variety of human cancer cell lines in vitro (23–
27). Thus, irrespective of the methods used for the ex vivo expan-
sion or activation of ?? T cells, it is now feasible to propose clin-
ical trials designed explicitly to assess the therapeutic effectiveness
of administering a large number of ?? T cells to patients with
cancer. With this reasoning in mind, our specific findings derived
using the TRAMP model of mouse prostate cancer are presented in
this study to be considered in the broader context of developing
new strategies for treating human prostate cancer, or other com-
mon human cancers of epithelial origin, through the adoptive
transfer of ex vivo expanded autologous ?? T cells.
Materials and Methods
C57BL/6 wild-type mice; C57BL/6 TRAMP mice (C57BL/6-Tg(TRAMP)
8247Ng/J); C57BL/6 TCR?-deficient (TCR??/?) mice (B6.129P2-
Tcrdtm1Mom/J); C57BL/6 TCR?-deficient (TCR??/?) mice (B6.129P2-
GFP)30Scha/J) were purchased from The Jackson Laboratory. All mice
were maintained in pathogen-free facilities in accordance with guidelines
of the Animal Care and Use Committee at The University of Alabama at
Birmingham (Birmingham, AL).
Breeding and selection of TRAMP mice lacking ?? T cells
To obtain TRAMP mice on a TCR??/?background, TRAMP mice and
TCR??/?mice were first interbred. Tail DNA isolated by standard proce-
dures was used for genotyping of mice by PCR using the following specific
primers: TCR? 5?-CAA ATg TTg CTT gTC Tgg Tg-3? and 5?-gTC AgT
CgA gTg CAC AgT TT-3?; and TRAMP 5?-CAg AgC AgA ATT gTg gAg
Tgg-3? and 5?-ggA CAA ACC ACA ACT AgA ATg CAg Tg-3?. Offspring
carrying the TRAMP transgene that were found to be heterozygous for
TCR? (TCR??/?) were then backcrossed with homozygous TCR??/?
mice. Male offspring expressing the TRAMP transgene but lacking ??
T cells were then identified for further study. Absence of ?? T cells in
male TRAMP ? TCR??/?was further confirmed by FACS of periph-
Assessment of spontaneously arising tumors of the genitourinary
Upon sacrifice, GU tracts were removed en bloc, weighed, then fixed in
formalin. Digital photographs of gross specimens were taken using a Nikon
CoolPix 5700 digital camera. Formalin-fixed specimens were subsequently
embedded in paraffin and stained with H&E as previously described (16).
Prostate lesions were then scored blindly by an experienced pathologist
using a 1–6 scale established for TRAMP mice (16).
TRAMP-C2 cells were provided by Dr. N. Greenberg (Fred Hutchinson
Cancer Research Center, Seattle, WA) and were also purchased from
American Type Culture Collection. Cells were maintained as described
Preparation of ex vivo expanded mouse ?? T cells
?? T cells used for adoptive transfer studies were obtained from spleen
cells derived from C57BL/6 mice lacking ?? T cells (TCR??/?mice) as
described (28). Spleen mononuclear cells were isolated by density gradient
centrifugation (800 ? g, 15 min) using Ficoll-Paque Plus (Amersham Bio-
sciences). Cells were cultured in a manner similar to methods developed
for expanding human ?? T cells (23), but using mouse cytokines and re-
agents as indicated. Spleen cell cultures were initiated at a density of 5 ?
106cells/ml in RPMI 1640 with 10% FBS, 2 mmol/L L-glutamine, 100
U/ml penicillin, 100 U/ml streptomycin, and 50 ?M 2-ME. On the day of
culture initiation (day 0), cells were transferred to tissue culture wells first
coated with rat anti-mouse CD2 mAb clone RM2-5 (BD Biosciences).
Recombinant mouse IFN-? (1000 U/ml; R&D Systems) and recombinant
mouse IL-12 (10 U/ml; R&D Systems) were then added. After 24 h (day
1), three volumes of fresh culture medium was added. Cultures were then
stimulated with 10 ng/ml anti-CD3 mAb clone 145-2C11 (BD Biosciences)
and 300 U/ml mouse recombinant IL-2 (R&D Systems). Fresh medium
with 10 U/ml human IL-2 (Roche Diagnostics) was added every 3 days. At
day 8, cells were harvested. Purity of ?? T cells was assessed using a
FACSCalibur flow cytometer (BD Biosciences) employing directly conju-
gated hamster anti-mouse Abs CD3-allophycocyanin, 145-2C11, TCR??-
FITC, or GL3 (all BD Biosciences). Cell viability was determined by
FACS or by fluorescent microscopy as described (23).
3Abbreviation used in this paper: GU, genitourinary.
6045The Journal of Immunology
In vitro cytotoxicity assay
?? T cells derived from C57BL/6 mice lacking ?? T cells (TCR??/?mice)
as well as control ?? T cells (derived from TCR??/?mice) were prepared
identically, as described. These cells were then used as effector cells
against51Cr-labeled mouse TRAMP-C2 target cells in standard 4-h in vitro
51Cr release cytotoxicity assays, as described (23). As previously described
(24), the calcium dependency of ?? T cell-mediated cytotoxicity was as-
sessed by adding EGTA and MgCl2to cell cocultures at 1 and 1.5 mM,
respectively. To restore killing, 3 mM CaCl2was added in culture
Preparation of ex vivo expanded GFP??? T cells
Mice lacking ?? T cells (TCR??/?mice) and mice transgenic for GFP
were first interbred. After backcrossing, offspring expressing GFP but lack-
ing ?? T cells were identified by FACS analysis of peripheral blood.
Spleen mononuclear cell preparations derived from these mice were then
cultured as described, yielding GFP??? T cells that were used for sub-
sequent adoptive transfer studies. FACS analysis and fluorescence micros-
copy were used to confirm that resulting cultured cells were GFP??? T
Treatment and assessment of tumor-bearing mice
TRAMP-C2 cells were used to establish tumors in healthy syngeneic
C57BL/6 male mice. On experimental day 0, tumor cells (3 ? 106cells
suspended in 200 ?l of DMEM) were injected s.c. into a flank of each
mouse. On experimental days 14, 19, 21, 26, and 28, ex vivo expanded
syngeneic (C57BL/6) ?? T cells (20 ? 106cells suspended in 400 ?l of
RPMI 1640) were administered i.v. by tail vein injection into tumor-bear-
ing animals. Control animals received only RPMI 1640. As described (29),
tumor measurements were converted to a calculated tumor weight (in mil-
ligrams) using the formula (width (mm2) ? length (mm))/2.
Imaging of adoptively transferred GFP??? T cells in tissues
TRAMP-C2 cells were used to establish tumors in wild-type male
C57BL/6 mice. Five weeks after tumor establishment, syngeneic GFP???
T cells (100 ? 106cells suspended in 400 ?l of RPMI 1640) were intro-
duced by tail vein injection. After 8 days, mice were sacrificed. Upon
sacrifice, tumors were excised then rinsed with PBS. Normal muscle tissue
was excised from the opposite hind leg of the same treated animal. Whole
fresh tumor specimens or whole muscle sections were examined directly
using a Nikon Eclipse TE 2000-U inverted fluorescence microscope fit
with a CoolSNAPESdigital camera (Photometrics). Digital photos shown
are at a magnification of ?100 and are presented as pseudocolor images
derived using IPLab image processing software (version 3.9.2/Mac OS X;
Detection and enumeration by FACS of GFP??? T cells in
tumors and blood
Tumors were removed at necropsy from mice that had previously received
i.v. GFP??? T cells. Whole fresh tumors were mechanically disaggregated
using a HandiChopper Plus food chopper (Black & Decker). Dead cells and
debris were removed by density centrifugation (800 ? g, 15 min) using
Ficoll-Paque Plus. Interface cells were resuspended in Hanks’ buffer (con-
taining 3% FBS) then incubated with Abs to mouse CD3 and TCR??.
Using multicolor FACS, GFP?cells were electronically gated then ana-
lyzed with respect to CD3 and TCR?? surface staining. Peripheral blood
samples taken from animals before sacrifice were treated with ACK lysis
buffer before staining and analysis.
Establishment, treatment, and assessment of disseminated
Disseminated pulmonary metastatic disease was established in mice as has
been described (30). On experimental day 0, TRAMP-C2 cells (6 ? 105
cells suspended in 400 ?l of DMEM) were introduced by tail vein injection
into healthy male C57BL/6 mice. Treated mice received 20 ? 106?? T
cells (suspended in RPMI 1640) by tail vein injection on experimental days
4, 8, and 11, whereas control mice received RPMI 1640 only. Assessment
of pulmonary metastases in treated and untreated mice was performed, as
described (30) with the following modifications. At necropsy, lungs from
each animal were removed en bloc, weighed then fixed in formalin for
subsequent analysis. Photographs of gross specimens were taken using a
Nikon Coolpix 5700 digital camera. Formalin-fixed lungs were sectioned
sagittally (superior to inferior) then embedded in paraffin for H&E staining.
Digital images of lung sections were produced using a Nikon Coolscan V
ED scanner using Nikon Scan 4.0 software. Morphometric analysis of lung
section photomicrographs was performed using ImageJ software (version
1.37v) from W. Rasband (National Institutes of Health, Bethesda, MD)
(http://rsb.info.nih.gov/ij/). Tumor involvement of lung sections was de-
termined by expressing the area of lung parenchyma involved with tumor
(dark blue) as a percentage of total lung parenchyma.
TRAMP mice, and TRAMP mice lacking ?? T cells (TRAMP ? TCR??/?
mice). A, GU tracts from male mice were removed at necropsy and photo-
graphed. A representative initial experiment shows the en bloc resected GU
kidney and ureter) removed from a 7-mo-old wild-type mouse (BL/6 wild-
type) (left), a 7-mo-old TRAMP mouse (center), and a 5-mo-old TRAMP ?
TCR??/?mouse (TRAMP ??/?) (right). B, As a measure of tumor burden,
GU tract weight was determined at necropsy and expressed as a percentage of
total body weight for each mouse. Data are shown for control wild-type mice
(BL/6 wild type) (n ? 12), control TCR??/?mice on the C57BL/6 back-
ground (BL/6 ??/?) (n ? 11), TRAMP mice (n ? 20), and TRAMP ?
TCR??/?mice (TRAMP ??/?) (n ? 22) with the mean ? SD and median
(with range) for each genotype shown. Animals in this representative experi-
ment were pooled from cohorts of mice sacrificed at 6.5 or 7 mo of age. In
accordance with our Institutional Animal Care and Use Committee guidelines,
animals were sacrificed when grossly massive tumors began to become evi-
dent. Thus, to allow for meaningful comparison between experimental and
control animals within a cohort, all mice within a given cohort were sacrificed
when it became necessary to sacrifice any mouse within the same cohort.
a cohort were invariably TRAMP ? TCR??/?mice and never TRAMP mice.
Gross comparison of the GU tracts of wild-type mice,
6046ADOPTIVE IMMUNOTHERAPY OF CANCER WITH ?? T CELLS
The median GU tract to body weight ratio of TRAMP mice and TRAMP ?
TCR??/?mice was compared using the Kruskal-Wallis test, whereas mean
ratio was compared by t test. Pathological scores of prostate lesions oc-
curring in TRAMP mice and TRAMP ? TCR??/?mice were compared
using Fisher’s one-sided exact test. The relationship between GU tract to
body weight ratio and pathological score in TRAMP mice was examined
using a one-way ANOVA. The analysis of GU tract to body weight ratio
by pathological score in TRAMP ? TCR??/?mice was examined using
ANOVA with Duncan’s Multiple Range Test (tested at 5% level) used to
control for pairwise comparisons. In immunotherapy studies, mean tumor
size in treated and untreated mice was compared using the Student’s t test.
Median lung weight of treated and untreated mice and tumor involvement
of lung parenchyma in treated and untreated mice were compared using the
Kruskal-Wallis test. Kaplan-Meier survival curves of treated and untreated
mice were compared according to the Mantel-Haenszel log-rank test.
Evidence shows that ?? T cells provide protective
immunosurveillance against spontaneously arising mouse
As reported, male TRAMP mice spontaneously develop prostate
adenocarcinomas in a predictable manner with disease developing
in 100% of male mice with a relatively short latency period (10–
13). Accordingly, these mice were used in initial proof-of-concept
studies designed to establish the extent to which mouse ?? T cells
are involved in providing protective immunosurveillance against
spontaneously arising mouse prostate cancer. To accomplish this
study, TRAMP mice were backcrossed with mice lacking ?? T
cells. Images of the GU tracts obtained from mice sacrificed during
performance of one of our earliest pilot studies are shown in Fig.
1A. The GU tracts of a male wild-type mouse, a TRAMP mouse,
and a TRAMP ? TCR??/?mouse are grossly compared. Given
that these initial pilot studies were performed using only a small
number of mice, subsequent more extensive studies were then per-
formed to follow up and confirm these highly suggestive findings.
Results from these larger studies are shown in Fig. 1B. In this
experiment, in accordance with convention, assessment of tumor
burden in each mouse is presented graphically as the GU tract
weight normalized to body weight (i.e., GU tract weight to body
weight ratio). In comparing tumor burdens of TRAMP mice vs
TRAMP ? TCR??/?mice, the mean tumor burden (6.8 vs 10.5,
respectively; p ? 0.06, strong trend) as well as median tumor
burden (7.0 vs 7.6, respectively; p ? 0.11) were found to differ,
though these differences did not reach statistical significance. Nev-
ertheless, descriptive comparison of these data importantly reveals
that only TRAMP ? TCR??/?mice developed very large tumor
burdens, arbitrarily defined as a tumor burden at least double the
TRAMP mice. A, After weighing, GU tracts were fixed in formalin then embedded in paraffin. Sections of prostate glands were cut from paraffin blocks
and stained with H&E for examination by microscopy. Prostate lesions were scored blindly by an experienced pathologist using a 1–6 scale that has been
established for TRAMP mice. Noncancerous lesions were graded as 1 (normal tissue); 2 (low prostatic intraepithelial neoplasia); or 3 (high prostatic
intraepithelial neoplasia). Cancerous lesions (adenocarcinomas) were graded as 4 (well-differentiated); 5 (moderately differentiated); or 6 (poorly differ-
entiated). Representative photomicrographs are shown at the same magnification. No animal in either group was scored grade 2. The number (and
percentage) of TRAMP mice and TRAMP ? TCR??/?mice with the corresponding grade is shown. †, p ? 0.057 (suggesting a trend) by Fisher’s one-sided
exact test comparing the number of TRAMP and TRAMP ? TCR??/?mice with grade 6 scores. B, GU tract weight to body weight ratio plotted against
the corresponding pathological score for each TRAMP mouse and each TRAMP ? TCR??/?mouse. In TRAMP mice (left), the GU tract weight to body
weight ratio shows no relationship to pathological score by one-way ANOVA (p ? 0.15). In TRAMP ? TCR??/?mice (right), the GU tract weight to
body weight ratio in animals with a grade 6 pathological score is significantly greater than the GU tract weight to body weight ratio of mice with less than
grade 6 pathological scores (p ? 0.0006 by ANOVA with Duncan’s Multiple Range Test comparing all pairwise differences, at 5% level).
Tumors developing in TRAMP ? TCR??/?mice are more likely to be high-grade and larger when compared with tumors developing in
6047The Journal of Immunology
median tumor burden of the group. Thus, whereas four TRAMP ?
TCR??/?mice developed large tumor burdens (2-, 3.4-, 3.7-, and
5-fold greater than the median tumor burden of all TRAMP ?
TCR??/?mice), in contrast no TRAMP mice developed corre-
spondingly large tumor burdens. Indeed, the largest tumor burden
observed in any TRAMP mouse measured only 1.5-fold greater
than the median tumor burden of the TRAMP mice group. In ad-
dition to the normalized GU tract weight to body weight ratio
shown in Fig. 1B, tumor burdens are also presented in this exper-
iment as actual GU tract weights (mean ? SD) of all groups of
mice: BL/6 wild-type mice (1.1 ? 0.3 g); BL/6 TCR??/?mice
(0.9 ? 0.2 g); TRAMP mice (2.3 ? 0.7 g); and TRAMP ?
TCR??/?mice (3.9 ? 3.7 g). In comparing GU tract weights of
TRAMP mice and TRAMP ? TCR??/?mice, the value of p ?
0.06 is indicating a strong trend. Importantly, at the time of sac-
rifice, no significant differences were observed in whole body
weights when comparing control TRAMP mice (mean body
weight, 36 ? 5 g) and experimental TRAMP ? TCR??/?mice
(mean body weight, 36 ? 4 g). This latter point indicates that
differences in the normalized GU tract weight to body weight ratio
shown in Fig. 1B are reflections of differences in tumor burden
(GU tract weight) and not simply reflections of variations in whole
body weight between mice.
Histological sections of the GU tracts resected from TRAMP
mice and TRAMP ? TCR??/?mice were analyzed and scored
using an established grading system (16). As shown in Fig. 2A,
whereas 5 of 22 TRAMP ? TCR??/?mice (23%) developed
high-grade disease (adenocarcinoma, grade 6), in comparison, only
1 of 20 TRAMP mice (5%) developed high-grade disease. Reveal-
ingly, by plotting tumor size (GU tract weight to body weight
ratio) against the corresponding tumor grade (pathological score)
for each individual mouse, TRAMP ? TCR??/?mice (Fig. 2B,
right) appear not only more likely to develop high-grade (grade 6)
tumors, but that when high-grade tumors do develop, tumor
growth is less well contained (larger tumor burden). In contrast,
TRAMP mice (Fig. 2B, left) appear not only less likely to develop
high-grade tumors, but that when high-grade tumors do develop,
tumor growth is relatively contained (smaller tumor burden). For
these studies, a score of 6 was used as the cutoff for statistical
analysis based upon accepted conventions in the TRAMP model
literature in which this score is the highest grade lesion developing
in TRAMP mice. In numerous chemoprevention studies, the abil-
ity of compounds to inhibit prostate cancer progression is assessed
by the capacity of such compounds to retard the development le-
sions of score 6 in TRAMP mice. Accordingly, for these current
studies, we reasoned that if an experimental condition (in this case,
cells demonstrated against TRAMP-C2 cells. A, ?? T cells used for adop-
tive transfer studies were obtained by in vitro expansion of spleen cells
derived from C57BL/6 mice lacking ?? T cells (TCR??/?mice) using a
modified version of a previously published method (28). FACS analysis
performed on cultures after 1 wk shows that ?95% of viable cells in these
cultures are ?? T cells. B, ?? T cells used for adoptive transfer studies were
routinely found to be cytolytic in vitro against TRAMP-C2 cells, whereas
control ?? T cells expanded in an identical manner were not. TRAMP-C2
cells were labeled with51Cr then cocultured in vitro with effector ?? T cells
(F) or control ?? T cells (E) at the indicated E:T ratios. The percentage of
specific target cell lysis as mean ? SD of triplicate determinations is shown
in a representative in vitro cytotoxicity assay. C, The in vitro lysis of
TRAMP-C2 cells by highly purified ex vivo expanded ?? T cells is cal-
cium-dependent, indicating that the perforin-granzyme pathway and not the
Fas-Fas ligand pathway is primarily involved.51Cr-labeled TRAMP-C2
cells were cocultured with effector ?? T cells in the presence (?) or ab-
sence (?) of EGTA. Specific lysis of TRAMP-C2 cells occurring when
cocultured with effector ?? T cells under standard control conditions (a);
when cocultured with effector ?? T cells under conditions in which Ca2?
is depleted by the addition of EGTA (b); and when cocultured with effector
?? T cells in the presence of EGTA but after the addition (replacement) of
Ca2?(c). Addition of EGTA has no effect on target cell51Cr release (d).
The percentage of specific target cell lysis is expressed as a mean ? SD of
triplicate determinations. All E:T ratios are 10:1.
In vitro cytolytic activity of expanded spleen-derived ?? T
growth of established mouse prostate cancer cells in vivo. A, Serial mea-
surement of tumor size in treated and untreated mice. TRAMP-C2 cells
previously derived from tumors arising in TRAMP mice (C57BL/6 origin)
were used to establish s.c. tumors in healthy syngeneic wild-type C57BL/6
mice. Fourteen days after tumorigenic cells were implanted, animals were
randomly assigned to either the control group (untreated, left) or to the
treatment group (treated, right). Animals in the treated group received syn-
geneic C57BL/6 ?? T cells (20 ? 106cells per treatment) administered i.v.
by tail vein injection on experimental days 14, 19, 21, 26, and 28. Tumor
size was serially determined and is shown for each individual mouse in
both groups. B, Mean tumor size ? SD (in milligrams) is shown on cor-
responding experimental days 14, 21, 26, 33, 40, and 47 for both the treated
group (n ? 7) (f) and the untreated group (n ? 7) (?). Values for p were
calculated using Student’s t test comparing the mean tumor size of the
treated and untreated groups on the indicated days. NS, Not significant.
Adoptively transferred syngeneic ?? T cells moderate the
6048ADOPTIVE IMMUNOTHERAPY OF CANCER WITH ?? T CELLS
the absence of ?? T cells) is statistically shown to enhance the
development of large, high-grade (score 6) tumors, then we would
be justified in concluding that ?? T cells play a role in inhibiting
either the development or progression of prostate cancer in
TRAMP mice. Collectively, we thus interpret the data from Figs.
1 and 2 to indicate that ?? T cells do indeed play a role in pro-
viding protective immunosurveillance against spontaneously aris-
ing mouse prostate cancer.
Adoptive transfer of syngeneic mouse ?? T cells can moderate
the growth of established localized disease
With the described proof-of-concept findings in mind, we con-
versely designed the following series of experiments to determine
whether the adoptive transfer of supraphysiological numbers of ??
T cells might result in enhanced innate antitumor immune re-
sponses directed against established disease in tumor-bearing mice
(adoptive cellular immunotherapy model). For these studies, dis-
ease was first established by the s.c. injection of TRAMP-C2 cells
into the flanks of otherwise healthy wild-type male C57BL/6 mice,
as previously described (18). Tumor-bearing mice were then
treated i.v. with highly purified (Fig. 3A) syngeneic (C57BL/6 or-
igin) ?? T cells that were shown to be cytolytic in vitro against
TRAMP-C2 cells (Fig. 3B). In addition, Fig. 3C shows that the
lysis of TRAMP-C2 cells by purified ex vivo expanded ?? T cells
is calcium-dependent, indicating that the perforin-granzyme path-
way and not the Fas-Fas ligand pathway is primarily involved.
A representative immunotherapy study is shown in Fig. 4A in
which tumor size measured serially in each individual treated and
control mouse is shown. Fig. 4B compares the mean tumor size of
both groups. These results indicate that the growth of tumors es-
tablished using mouse prostate cancer cells can be moderated by
the i.v. introduction of supraphysiological numbers of ex vivo ex-
panded syngeneic ?? T cells. Importantly, during the course of
these adoptive immunotherapy studies, no untoward side effects
were observed in mice receiving repeated i.v. infusions of ex vivo
expanded syngeneic ?? T cells.
Adoptively transferred ?? T cells localize to sites of disease
To demonstrate that adoptively transferred ?? T cells can subse-
quently be detected within the tumors of host mice, the following
studies were performed. As discussed, TRAMP-C2 cells were used
to establish s.c. tumors in healthy male C57BL/6 mice. However in
these adoptive transfer studies, syngeneic ?? T cells expressing
GFP were introduced i.v. into tumor-bearing mice. Subsequently,
samples of tumor, normal tissue, and blood were obtained and
analyzed by fluorescence microscopy or FACS. As shown in Fig.
5A, GFP??? T cells are readily detectable within tumor tissues
removed from treated animals, but are not readily detectable in
lished s.c. tumors as demonstrated by direct imaging and by FACS. A,
TRAMP-C2 s.c. tumors were first established in wild-type C57BL/6 mice.
After 35 days, 100 ? 106GFP??? T cells were administered i.v. by tail
vein injection into tumor-bearing mice that were subsequently sacrificed 8
days later. Immediately upon sacrifice, tumors were excised then rinsed
with PBS. Freshly excised tumors were then imaged using an inverted
fluorescence microscope using the appropriate filters for the visualization
of GFP. Representative digital images show a tumor resected from an
animal treated i.v. with GFP??? T cells (left), normal muscle resected
from the same animal (center), and a tumor resected from a mouse that
received no GFP??? T cells (right), included as a control for nonspecific
autofluorescence. Images are shown at ?100 magnification. In addition
(data not shown), in normal healthy mice (i.e., mice without tumors) re-
ceiving GFP? ?? T cells, although occasional GFP?events could be di-
rectly observed, we failed to detect by microscopy the gross accumulation
of GFP??? T cells within various normal tissues including muscle, skin,
brain, GU tract, intestine, and lungs. Although some GFP??? T cells were
also evident in spleen and lymph nodes, no gross accumulations were noted
in these tissues as well. B, TRAMP-C2 tumors were removed at necropsy
from mice that had received GFP??? T cells i.v. 8 days before. After
mechanical disaggregation followed by density centrifugation to remove
debris, cell suspensions were stained with Abs specific for mouse CD3 and
TCR??, then analyzed by FACS. GFP?cells were first identified (left) then
analyzed with respect to CD3 and TCR?? surface staining (right) where the
percentage of GFP?cells that are ?? T cells is shown (upper right quadrant).
C, TRAMP-C2 tumors (top) were removed at necropsy from mice that had
received GFP??? T cells i.v. 8 days before. After disaggregation and
The i.v. administered GFP??? T cells localize to estab-
centrifugation, cell suspensions were stained with Abs allowing for the
identification of ?? T cells (left). These cells were then analyzed for the
expression of GFP (right) where the percentage of tumor-infiltrating ?? T
cells of donor origin (GFP?) is shown. Peripheral blood (bottom) taken
from the same animal at necropsy was similarly analyzed for the percent-
age of peripheral blood ?? T cells of donor origin (GFP?). As shown in the
FACS analysis strategy used in both the blood and the tumor cell prepa-
rations, acquired data were first plotted as TCR?? PE-positive events by
side scatter (left). This analysis was performed to allow the identical elec-
tronic amorphous gate to be used to identify ?? T cells in both tumor
tissues (top) and blood (bottom), thus assuring consistency in subsequent
comparative analysis of ?? T cells. When represented as forward scatter by
side scatter plots, blood cell preparations demonstrated the characteristic
forward scatter by side scatter profile of ACK-lysed blood (data not
6049The Journal of Immunology
display superior survival compared with untreated animals. Widespread systemic disease was first established in healthy male C57BL/6 mice by the i.v.
injection of 6 ? 105TRAMP-C2 cells on experimental day 0. Mice were then randomly assigned to a treatment group or a control group. On days 4, 8,
and 11, treated mice received ?? T cells by tail vein injection, whereas control mice received medium alone. Mice were monitored for systemic signs of
progressive disease and were all sacrificed on day 66. At necropsy, lungs from each animal were removed en bloc, weighed, and fixed in formalin for
subsequent analysis. A, Representative photographic images comparing gross lung size and visible tumor nodules of formalin-fixed lungs removed from
treated mice (left) and untreated mice (right). Scale size is shown in centimeters. B, For this experiment, the weight of freshly resected lungs was used as
a quantitative measure of tumor burden. In this representative cohort, four mice in the untreated group died before the termination of the study and could
not be included for pathologic or histologic analysis. Median lung weight (in milligrams) and range are shown for treated mice (n ? 13) and untreated mice
(n ? 11). p ? 0.0005 using the Kruskal-Wallis test. C, Formalin-fixed lungs were sectioned sagittally (superior to inferior) then embedded in paraffin for
routine H&E staining. Representative low-power photomicrographic images of lung sections from treated mice (left) and untreated mice (right) are shown.
D, Fixed and stained lung sections were analyzed using ImageJ morphometric software. Tumor involvement of lung sections was determined by expressing
the area of lung parenchyma involved with tumor (dark blue) as a percentage of total lung parenchyma. Median tumor involvement (percentage) and range
are shown for treated mice (n ? 13) and untreated mice (n ? 11). p ? 0.0004 using the Kruskal-Wallis test. E, Comparison of survival of treated and
untreated animals used in the experimental model of metastatic disease. Life tables (left) and Kaplan-Meier survival curves (right) of the pooled cohorts
of mice collectively comparing treated mice (n ? 26) to untreated control mice (n ? 29). Data are censored at day 66. p ? 0.002 according to the
Mantel-Haenszel log-rank test comparing treated to untreated mice.
Mice treated with adoptively transferred syngeneic ?? T cells develop less extensive disseminated pulmonary disease and also appear to
6050 ADOPTIVE IMMUNOTHERAPY OF CANCER WITH ?? T CELLS
nontumor tissues from the same animal. These findings suggest
that adoptively transferred ?? T cells are preferentially localizing
to tumors. FACS analysis (Fig. 5B) confirms that the majority of
GFP?cells found in tumor tissue resected from treated mice are
indeed ?? T cells.
In addition, the proportion of ?? T cells of donor origin (GFP?)
was determined in both tumor tissue and blood obtained simulta-
neously from treated tumor-bearing mice. In this representative
study (Fig. 5C), over 70% of ?? T cells within tumor tissue were
found to be of donor origin (GFP?), compared with only 17% in
peripheral blood. These data suggest that the GFP??? T cells
detected within tumor tissues are not simply contaminating periph-
eral blood-derived cells, but rather that these GFP?cells are adop-
tively transferred ?? T cells, which have localized to tumor tissues.
Although these studies (Fig. 5) establish neither the mechanisms
involved in homing nor the mechanisms by which adoptively
transferred ?? T cells actually moderate tumor growth, these find-
ings nevertheless demonstrate that adoptively transferred ?? T
cells can infiltrate established tumors and that this infiltration may
be important for these cells to subsequently exert their therapeutic
antitumor effects in vivo.
Adoptive transfer of syngeneic mouse ?? T cells can moderate
the growth of established widespread disease
The immunotherapeutic potential of ?? T cells was additionally
demonstrated using a model of metastatic disease. In this experi-
ment, the ability of adoptively transferred ?? T cells to moderate
the growth or progression of widespread disease was assessed by
first introducing TRAMP-C2 cells i.v. into healthy male C57BL/6
mice, as has been previously described (30). Mice were then
treated i.v. with syngeneic ?? T cells. Upon sacrifice, both by gross
appearance (Fig. 6A) and by assessment of lung weight (Fig. 6B),
treated mice were found to have lower tumor burdens compared
with control, untreated mice. Histological analysis of formalin-
fixed lung sections (Fig. 6C) as well as morphometric analysis of
these sections (Fig. 6D) confirm that lungs removed from untreated
mice were more heavily infiltrated with tumor cells compared with
treated mice. Finally, although these particular studies were not
designed as survival studies, Kaplan-Meier analysis of pooled co-
horts of treated and untreated mice reveals that mice receiving ??
T cells displayed superior survival compared with untreated con-
trols (Fig. 6E).
The studies undertaken in this report were performed with two
distinct yet logically related objectives in mind. Our first objective
was to directly test the hypothesis that ?? T cells play a role in
limiting the development or progression of spontaneously arising
mouse prostate cancer (proof-of-concept studies) (Figs. 1 and 2).
Our second objective was to test a specific prediction of this hy-
pothesis, a prediction that asserts that if ?? T cells do indeed pro-
vide protective immunosurveillance against mouse prostate cancer,
then introducing supraphysiological numbers of syngeneic ?? T
cells into tumor-bearing mice would result in measurable control
of established disease (adoptive immunotherapy studies) (Figs.
By breeding TRAMP mice with mice lacking ?? T cells, the
opportunity is afforded to observe the development or progression
of prostate cancer as it occurs in the specific absence of ?? T cells.
Although potentially an important approach to directly assess
whether ?? T cells play a role in limiting tumor development or
progression, we nonetheless remained mindful of some of the lim-
itations of this study or any study designed to assess immune re-
sponses directed against tumors arising in transgenic animals.
First, in a general sense, it should be noted that even in fully
immunocompetent TRAMP mice, disease development and pro-
gression can vary substantially between individual animals (10–
13). Second, in a more specific sense, it should be noted that in the
TRAMP model, spontaneously arising tumors that develop in male
TRAMP mice are known to express simian virus SV40 oncopro-
teins, which in this model are authentic tumor Ags that in theory
could elicit strong protective adaptive antitumor responses by Ag-
specific ?? T cells (10–13). Importantly, however, it is now rec-
ognized that strong tolerance to SV40 Ags commonly develops in
otherwise immunocompetent TRAMP mice. This development ap-
pears to occur through a variety of mechanisms, including possibly
the clonal deletion of SV40-specific ?? T cells (31, 32). Accord-
ingly, we believe that it is the fortuitous absence of a strong, pro-
tective adaptive SV40-specific immune response that ultimately
permits us to detect the subtly enhanced tumor development oc-
curring in some TRAMP ? TCR??/?mice, as evidenced by our
ability to observe a strong statistical trend (p ? 0.06) for larger
tumor burdens in TRAMP ? TCR??/?mice (Fig. 1B) as well as
the strong statistical trend (p ? 0.057) for greater numbers of
TRAMP ? TCR??/?mice developing high-grade disease as com-
pared with immunocompetent TRAMP mice (Fig. 2A).
The observation that large, high-grade tumors develop in only
some but not all TRAMP ? TCR??/?mice strongly suggests that
?? T cells are providing protective immunosurveillance in a par-
tially redundant, or possibly cooperative manner with other dis-
crete elements of the innate or adaptive immune response. We
surmise that ?? T cells may be cooperating with NK cells or with
?? T cells, including possibly SV40-specific ?? T cells that have
remained functional. In the final analysis though, protective anti-
tumor immunosurveillance that ?? T cells might provide in
TRAMP mice is not absolute, given that eventually 100% of male
TRAMP mice develop prostate cancer (10–13). For this reason, as
well as the limitations discussed, we feel that our proof-of-concept
studies though important, ultimately serve best as logical support
for the subsequent adoptive immunotherapy research presented in
the balance of this study.
Although current standard treatments for prostate cancer are
usually effective at achieving initial disease control, prostate can-
cer often recurs. Moreover, salvage therapy for recurrent disease,
or therapy for prostate cancer presenting initially as widespread
metastatic disease, is often only modestly effective. Clearly, en-
tirely new forms of therapy for recurrent or metastatic prostate
cancer are needed. Although cell-based immunotherapies have been
developed in this regard, the vast majority of studies have relied
upon the generation of tumor Ag-specific adaptive immune re-
sponses (33–36). These approaches, however, have been largely
unsuccessful owing primarily to 1) the difficulties encountered in
identifying tumor Ags that are truly prostate cancer-specific, and 2)
the well-documented ability of prostate cancer cells to specifically
evade adaptive immune responses through a variety of mecha-
nisms, including the down-regulation of MHC class I molecules
(33, 37–39). The ability of innately functioning ?? T cells to pro-
vide an alternative or complementary means to recognize and kill
tumor cells, particularly those that have escaped Ag-specific,
MHC-restricted adaptive immune responses, makes the develop-
ment of ?? T cell-based cellular therapies particularly attractive in
this regard (5, 40).
By injecting otherwise healthy mice with a predetermined num-
ber of tumorigenic TRAMP-C2 prostate cancer cells, we have de-
veloped a model to quantitatively determine the extent to which
the i.v. introduction of syngeneic ?? T cells can subsequently mod-
erate the growth of disease (Figs. 4 and 6). The use of TRAMP-C2
6051The Journal of Immunology
cells in this model is important for several reasons. First, by spe-
cifically using the syngeneic TRAMP-C2 prostate cancer cell line
to establish disease in otherwise healthy wild-type C57BL/6 mice,
this process has allowed us by extension to approximate a clinical
situation whereby a patient with prostate cancer is treated with a
supraphysiological number of autologous ?? T cells. With this
particular point in mind, it is important to note that in all studies,
disease-bearing animals that received ex vivo expanded syngeneic
?? T cells tolerated repeated injections very well without any ob-
served untoward side effects. Second, unlike primary tumors cells
that develop in TRAMP mice, TRAMP-C2 cells do not express
SV40 oncoproteins either in vitro, or subsequently in vivo upon
transfer into otherwise healthy mice (18). Consequently, we be-
lieve that the confounding effects of adaptive immune responses
directed against TRAMP-C2-derived tumors are potentially mini-
mized, which is supported in part by our finding that in vitro,
TRAMP-C2 cells are readily killed by ?? T cells but not by control
?? T cells (Fig. 3B).
As shown in Fig. 4, i.v. administered syngeneic ?? T cells are
capable of moderating the growth of palpable, established s.c.
TRAMP-C2-derived tumors. That antitumor efficacy is so clearly
evident against grossly palpable disease would strongly suggest
that adoptively transferred ?? T cells would also be effective
against minimal residual disease, such as microscopic disease that
might persist after surgical resection or radiation therapy. Related
to this point is the observation that adoptively transferred synge-
neic ?? T cells are also effective against nonbulky but neverthe-
less, widespread disseminated disease (Fig. 6). Thus, we would
argue that the adjuvant delivery of ?? T cells would be a logical
strategy to develop as a means to prevent local treatment failure or
to prevent the development of widespread metastatic disease, par-
ticularly if given at a time when metastatic disease is only micro-
Biologically, it is likely that s.c. implanted tumors derived using
TRAMP-C2 cells may not necessarily be comparable to tumors
that develop spontaneously in TRAMP mice. Indeed, we originally
wished to examine the ability of adoptively transferred ?? T cells
to prevent the development of spontaneous disease in TRAMP
mice. However, this approach was rejected on account that in
TRAMP mice, both the latency period for disease development
and the natural history of disease progression once disease devel-
ops can be quite variable from mouse to mouse (10–13), thus
making the interpretation of any immunotherapy studies extremely
difficult. In contrast, by using the s.c. disease model, we were able
to establish predetermined tumor burdens in otherwise healthy
mice and then initiate therapy after a predetermined interval, al-
lowing for more accurate measure of the immunotherapeutic ef-
fects of adoptively transferred ?? T cells against tumor cells (Fig.
4). Although choosing to use the s.c. model represents somewhat
of a compromise, importantly the robust immunotherapy data gen-
erated using the metastatic disease model (Fig. 6) strongly corrob-
orate the findings generated using the s.c. disease model. Interest-
ingly in some respects, the metastatic model can be thought of as
mimicking the advanced stages of disease found in TRAMP mice
in which the development of widespread metastatic disease is not
uncommon. Ultimately, we take these findings (Figs. 4 and 6) to
support the view that adoptively transferred ?? T cells can mediate
potent antitumor activity against mouse prostate cancer cells, ir-
respective of where or how disease might be established.
Despite the strong theoretical promise and growing interest in
?? T cell-based immunotherapies, it still remains to be determined
how best to exploit ?? T cells for the treatment of prostate cancer
or any human malignant disease. Two distinct approaches are cur-
rently being developed in this regard (6, 19–23, 27, 41). One ap-
proach is based upon the in vivo activation or expansion of en-
dogenous ?? T cells present within patients through the clinical
administration of pharmacologic agents capable of stimulating ??
T cells. The second approach is based upon the adoptive transfer
of ?? T cells first expanded ex vivo, then infused into tumor-
bearing patients, as approximated in our current studies undertaken
using the TRAMP mouse model. Clearly, although both ap-
proaches are rational and will likely lead to important advances in
cancer treatment, from a conjectural point of view, we believe that
the latter approach has several advantages that merit discussion.
First, in adopting the latter approach it becomes possible to pro-
spectively identify and consequently, selectively administer, spe-
cific ?? T cell subsets deemed more likely to be clinically effective
against tumor cells. For example, using state-of-the-art cell sepa-
ration technologies, ?? T cell subsets within ex vivo expanded
cultures could be selectively enriched on the basis of specific phe-
notypic features found, or postulated, to be associated with more
potent antitumor activity. Specific ?? T cell subsets could in theory
be selected on the basis of defined criteria, including activation
profile (determined by surface markers such as CD45 and CD27),
unique patterns of cytokine production, expression of specific
homing receptors, and other criteria. Studies in mice specifically
addressing this point are ongoing in our laboratory.
Second, in adopting the latter approach it becomes possible to
experimentally track ?? T cells after administration, an important
correlative tool for use in future clinical trials. Thus, in a manner
analogous to our animal studies using GFP-marked ?? T cells for
tracking (Fig. 5), in human clinical trials it will be possible to first
radiolabel ex vivo expanded ?? T cells with isotopes such as111In
or99mTc, then track these ?? T cells to sites of disease using
various clinical imaging techniques.
Third, the latter approach makes possible the ex vivo expansion
of ?? T cells derived from healthy donors and not just patients.
Though beyond the scope of this discussion, the introduction of
tumor-reactive allogeneic (donor-derived) ?? T cells might be-
come feasible in the setting of newer, nonmyeloablative allogeneic
hematopoietic stem cell transplants (42) in which donor-derived
?? T cells could be delivered as a posttransplant donor lymphocyte
infusion as is commonly done after immunological tolerance is
established. This option may become especially important given
our observation (manuscripts in preparation) and the observation
of others (43) that numeric and functional deficits appear to exist
within the ?? T cell compartment of patients newly diagnosed with
even the earliest-stage malignancies.
In conclusion, it is important to acknowledge that our adoptive
immunotherapy results presented in this study were not designed
to establish the mechanisms involved in the homing of adoptively
transferred ?? T cells to sites of disease. Indeed, as these studies
were performed using unfractionated, ex vivo expanded spleen-
derived ?? T cells, they do not address the possibility that distinct
subsets of ?? T cells might be relatively more (or less) efficient at
homing to specific tissues where disease is established. In addition,
these studies were not designed to directly establish the mecha-
nisms by which adoptively transferred ?? T cells recognize and
kill tumor cells in vivo, though it is unlikely that in this model,
adoptively transferred ?? T cells are recognizing and killing tumor
cells through mechanisms other than those already known (1–7).
Nevertheless, despite these shortcomings, these studies are impor-
tant demonstrations that the adoptive transfer of unfractionated, ex
vivo expanded syngeneic ?? T cells is an effective and well-tol-
erated form of treatment for murine prostate cancer. Accordingly,
these findings provide a strong biological rationale for the design
of the corresponding studies in human disease, particularly now
6052 ADOPTIVE IMMUNOTHERAPY OF CANCER WITH ?? T CELLS
that the means exist to generate the larger numbers of ?? T cells
required for such studies (23, 27).
We thank Dr. Larry Lamb for thoughtful review of this manuscript.
The authors have no financial conflict of interest.
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6053The Journal of Immunology