synergistic effect of adoptive t-cell therapy and intratumoral interferon γ-inducible protein-10...

Synergistic effect of adoptive T-cell therapy and intratumoralinterferon c-inducible protein-10 transgene expression

in treatment of established tumors

Hui Huang, YongQing Liu, and Jim Xiang*

Research Unit, Saskatchewan Cancer Agency, Departments of Microbiology, Oncology and Pathology, University of Saskatchewan,

Saskatoon, Saskatchewan, Canada S7N 4H4

Received 20 March 2002; accepted 21 June 2002

Abstract

The lack of efficient T-cell infiltration of tumors is a major obstacle to successful adoptive T-cell therapy. We have previously

shown that transplanted SP2/0 myeloma tumors engineered to express lymphotactin invariably induced tumor regress mediated by

SP2/0 tumor-specific T cells. Herein, we further systemically characterize these activated T cells and investigate their therapeutic

efficacy, either alone or with the chemokine interferon c (IFN-c)-inducible protein-10 (IP-10) gene therapy. Following stimulationwith SP2/0 cells, these activated T cells were CD25þFasLþ L-selectinlow, expressed CXCR3 receptor and were chemoattracted by

IP-10 in vitro. They comprised 64% CD4þ Th1 and 36% CD8þ Tc1 cells, both of which expressed IFN-c, perforin, and TNF-a, butnot IL-4. The activated T cells were strongly cytotoxic for SP2/0 tumor cells (79% specific killing; E:T ratio, 50), mainly via perforin-

mediated pathway. Cell tracking using labeled T cells confirmed that these T cells infiltrated better into the IP-10-expressing tumors

than non-IP-10-expressing ones. In vivo, combined intratumoral IP-10 gene transfer and adoptive T-cell immunotherapy for well-

established SP2/0 tumors eradicated the tumors in 7 of the 8 mice. Control or IP-10 adenoviral treatments by themselves neither

alter the lethal outcome for tumor-bearing mice nor did T-cell therapy by itself, although the latter two treatments did slow its time-

frame. Taken together, our data provide solid evidence of a potent synergy between adoptive T-cell therapy and IP-10 gene transfer

into tumor tissues, which culminated in the eradication of well-established tumor masses.

� 2002 Elsevier Science (USA). All rights reserved.

Keywords: Adenoviral vector; IP-10 gene therapy; Adoptive T-cell therapy; Tumor immunity

1. Introduction

Cytotoxic T lymphocytes (CTLs) play a crucial role

in the host immune response to cancer. Effective adop-

tive cancer immunotherapy with tumor-sensitized CTLs

has been well-documented in animal models [1–4],

where transfer of such tumor-specific cells into micebearing established tumors has resulted in tumor erad-

ication. However, even in such model systems this

therapeutic approach is limited to early stage tumors

(e.g., 3-day lung metastases) or established s.c. tumors

that have been previously irradiated (to facilitate T-cell

infiltration; [3–5]). In clinical settings only limited

numbers of patients have responded to CTL therapy

[6–8], with an objective response rate of �30% [9]. In

general, it is assumed that the anti-tumor efficacy of the

transferred T cells is, to a large extent, determined by

their ability to leave the vasculature and infiltrate the

tumor [5,10], but the overall fraction of transferred T

cells that accumulate in tumors is rather small [11–13].The resistance of tumors to T-cell infiltration may be

due in part to a limited expression on the neovascula-

ture within these growths of the adhesion molecules that

are essential for T-cell adherence and transendothelial

migration [e.g., vascular cell adhesion molecule-1

(VCAM-1) and intercellular adhesion molecule-1

(ICAM-1)] [14].

The trafficking of lymphocytes from the systemiccirculation into tissues is a dynamic, multistep process.

Cellular Immunology 217 (2002) 12–22

www.academicpress.com

*Corresponding author. Fax: +1-306-655-2635.

E-mail address: [email protected] (J. Xiang).

0008-8749/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

PII: S0008 -8749 (02 )00508-7

mail to: [email protected]

It requires selectin-mediated rolling and tethering, lym-phokine-induced activation of integrins, firm adhesion

of the lymphocytes to endothelial cells and their diape-

desis through the endothelium, as well as migration

within the connective tissues along established chemo-

attractant gradients [15]. While adhesion to endothelial

cells and various extracellular matrix proteins is crucial

for successful T-cell recruitment into inflammatory sites,

the signals that regulate these processes have not beenfully elucidated. It is believed that essential steps are

mediated by chemokines produced at the sites of in-

flammation [16].

Chemokines are a superfamily of cytokines that at-

tract and activate leukocytes [17]. They are produced by

multiple cell types (e.g., leukocytes, endothelial cells,

fibroblasts, and tumor cells) in response to viruses,

bacteria, lipopolysaccharide (LPS), and pro-inflamma-tory cytokines [e.g., interleukin (IL)-1, tumor necrosis

factor (TNF)-a; [18]]. The superfamily�s four major

branches are defined by the spacing of the first two

cysteines in a conserved four-cysteine motif. The two

cysteines of the a-subfamily (C–X–C) are separated byanother residue, those of the b-subfamily (C–C) are

adjacent, while the c-subfamily (C) has only one cysteineat its N-terminus [19], and the newly identified C–X3–Csubfamily has two cysteines separated by three other

residues [20]. Interferon (IFN)-c inducible protein-10

(IP-10) [21] belongs to the CXC chemokine family

known to stimulate the IP-10 receptor CXCR3 [22].

IP-10 binds to a seven-trans-membrane G protein-cou-

pled receptor, CXCR3, expressed on activated T cells,

leading to chemotaxis [22]. In addition, it can also in-

hibit tumoral angiogenesis [23,24]. Therefore, IP-10 hasbeen involved in the anti-tumor immune responses by

recruitment of T cells to the malignancies and the non-

immune responses by its anti-angiogenesis effect.

Recently, we genetically modified a mouse myeloma

cell line SP2/0 into an engineered tumor cell line

SP2/0-Lptn expressing the C chemokine lymphotactin

(Lptn) [25]. We found that (i) the engineered SP2/0-Lptn

tumor cells invariably regressed upon implantation intosyngeneic BALB/c mice and (ii) the vaccination of SP2/

0-Lptn cells induced SP2/0-specific T-cell responses.

More recently, we conducted a double gene therapy with

adenovirus-mediated IP-10 and IL-18 gene transfer in

treatment of established tumors. We found that trans-

gene expression of IP-10 in tumors induced significant

T-cell infiltration, leading to tumor apoptosis and ne-

crosis [26]. Since IP-10 can induce T-cell migration invitro and in vivo [22], we reasoned that IP-10 expression

within tumors by adenovirus-mediated IP-10 gene

transfer should also enhance infiltration of adoptively

transferred tumor-specific T cells and thus the thera-

peutic efficiency of adoptive T-cell therapy of cancer. In

the present study, we tested this hypothesis. We

first phenotypically characterized the SP2/0-specific T

lymphocytes from the SP2/0-Lptn tumor immunizedmice, and then studied the efficacy of adoptive T-cell

therapy using these activated T cells and intratumoral

adenovirus-mediated IP-10 gene transfer.

2. Materials and methods

2.1. Antibodies, tumor cell lines, recombinant adenoviral

vectors, and animals

Mouse anti-mouse major histocompatibility complex

(MHC) class I (H-2Kd) and class II (Iad) antibodies and

rat anti-mouse CD3, CD4, CD8, CD25, L-selectin, Fas,

and FasL antibodies as well as phycoerythrin (PE)-

conjugated anti-mouse IL-4, IFN-c, perforin, and TNF-a antibodies were all obtained from Pharmingen(Mississauga, Ont., Canada). Fluorescein isothiocyanate

(FITC)-conjugated goat anti-rat IgG antibody was ob-

tained from Bio/Can Scientific (Mississauga, Ont.,

Canada). Recombinant mouse IP-10 and IL-2 were

purchased from R&D Systems (Minneapolis, MN). The

myeloma cell line SP2/0, B cell lymphoma cell line A20

(of BALB/c mouse origin), and the mouse lymphoma

cell line Yac-1 were obtained from the American TypeCulture Collection (ATCC, Rockville, MD) and main-

tained in Dulbecco�s modified Eagle�s medium (DMEM)

(Gibco, Gaithersburg, MD) containing 10% fetal calf

serum (FCS) and gentamycin (50 lg/ml). Engineeredtumor cell line SP2/0-Lptn secreting the C chemokine

lymphotactin (Lptn) was generated in our lab [25] and

maintained in DMEM containing 10% FCS and G418

(0.5mg/ml). The adenoviruses (AdVs) AdVIP-10 andAdVLacZ expressing IP-10 and the Escherichia coli

b-galactosidase, respectively, and the control adenovirusAdVpLpA (i.e., with no gene insert) were previously

constructed in our laboratory [27]. Female BALB/c mice

(4–6 weeks old) were obtained from the Animal Re-

source Center, University of Saskatchewan. Female

athymic nude mice (4–6 weeks old) were obtained from

Charles River Laboratories (St. Constant, PQ, Canada).All mice were maintained in our animal facility at the

Saskatoon Cancer Center.

2.2. Preparation of SP2/0 tumor-specific activated T

lymphocytes

Spleens were removed from immunized mice that had

previously rejected SP2/0-Lptn tumors [34] and singlecell suspensions were prepared by pressing the spleens

through fine nylon mesh. The red blood cells were lysed

using 0.84% ammonium chloride. The splenic lympho-

cytes (5� 106) were cocultured in 24-well plates (Costar,

Cambridge, MA) with 1� 105 c-irradiated (6000 rad)

SP2/0 cells in 2ml of DMEM supplemented with 10%

FCS and IL-2 (10U/ml) (DMEM/FCS/IL-2 medium).

H. Huang et al. / Cellular Immunology 217 (2002) 12–22 13

After 4 days, the lymphocytes were purified from thecultures using Ficoll–Paque density gradient centrifu-

gation and, for some experiments, the CD4þ and CD8þ

T cells were fractionated by negative selection using

anti-mouse CD8 (Ly2) and CD4 (L3T4) paramagnetic

beads, respectively (DYNAL, Lake Success, NY), ac-

cording to manufacturer�s protocols. Hereafter, thesepopulations of cells are referred to as CD4þ and CD8þ

T cells, respectively. These T cells were then subjected to(i) the phenotypic analysis by flow cytometry and cy-

tokine enzyme-linked immunosorbent assay (ELISA),

(ii) the functional analysis by chemotaxis and cytotox-

icity assays, and (iii) the animal studies.

2.3. Phenotypic characterization of activated T lympho-

cytes and SP2/0 tumor cells

In the phenotypic analyses, splenic T lymphocytes

from na€ııve BALB/c mice were used as negative controlcells. Red cells were lysed using 0.84% ammonium

chloride and the T cells purified using nylon wool col-

umns, as described [28]. na€ııve or activated T lympho-

cytes were incubated for 1 h on ice with rat anti-mouse

antibodies against CD3, CD4, CD8, CD25, L-selectin or

FasL (each, 2 lg/ml), washed with phosphate-bufferedsaline (PBS), then incubated for another 1 h on ice with

FITC-conjugated anti-rat IgG antibody (5 lg/ml). SP2/0tumor cells were incubated for 1 h with mouse anti-H-

2Kd and Iad antibodies, and rat anti-Fas antibody (each,

2 lg/ml), washed with PBS, then incubated for another1 h on ice with FITC-conjugated anti-rat or mouse IgG

antibody (5 lg/ml). After another three washes with

PBS, the cells were analyzed by flow cytometry. Isotype-matched irrelevant specificity monoclonal antibodies

were used as controls. To examine the intracellular ex-

pression of cytokines by the purified CD4þ and CD8þ Tcells, the cells were processed using a commercial kit

(Cytofix/CytoPerm Plus with GolgiPlug; Pharmingen),

and stained with PE-conjugated anti-IL-4, -IFN-c,-TNF-a, or -perforin antibodies, according to manu-

facturer�s protocols.

2.4. Chemotaxis assay

The chemotactic response of SP2/0-specific activated

T lymphocytes to IP-10 [23] was examined using modi-

fied Boyden microchemotaxis chambers (Neuroprobe,

Gaithersburg, MD) and polyvinyl pyrrolidone-free

5-lm pore-size polycarbonate membranes, essentially asdescribed [29]. Recombinant IP-10 (R&D Systems,

Minneapolis, MN), diluted in DMEM/0.1% bovine se-

rum albumin (BSA) to 0.1–1000 ng/ml, was added to

triplicate lower chambers of the wells, and 105 activated

T lymphocytes in DMEM/BSA were added to the upper

chambers. After incubation for 2 h at 37 �C, the cellsthat had not migrated into the membranes were wiped

from the upper surfaces of the membranes, which werethen fixed in 70% methanol and stained using a Diff-

Quik Kit (American Scientific Products, McGraw-Hill,

IL). The lymphocytes that were associated with the

membranes were enumerated by direct counting in a

blinded fashion of at least nine 40� objective fields per

well. The results are expressed as the mean number of

cells/40� field (�SEM).

2.5. T-cell cytotoxicity assay

In the T-cell cytotoxicity assays, the freshly activated

T lymphocytes were used as effector cells, while SP2/0 or

irrelevant control A20 tumor cells were used as target

cells. The effector T cells were also preincubated with

different concentrations of concanamycin A (CMA) (0–

1000 nM) and emetin (0–5 lM) (Sigma, St. Louis, MO)for 2 h to prevent perforin (CMA)- or Fas/FasL inter-

action (emetin)-mediated cytotoxicity [30,31]. The target

cells were radiolabeled by culture for 1 h in the presence

of 50 ll of sodium ½51Cr�chromate (36mCi/ml, Amer-sham), then washed twice with DMEM. Approximately

1� 105 labeled target cells per triplicate well were mixed

with effector cells at various effector/target cell ratios,

and were then incubated for 6 h. The percent specificlysis was calculated as: 100� ½ðexperimental CPM�spontaneous CPMÞ=ðmaximal CPM�spontaneous CPMÞ�[28]. Spontaneous counts per minute (CPM) release in

the absence of effector cells was less than 10% of specific

lysis. The maximal CPM release was determined by lysis

of the target cells with 1% Triton X-100.

2.6. Adenoviral infection of SP2/0 tumors in vitro and in

vivo

To test the susceptibility of SP2/0 tumor cells to

adenoviral infection, serial dilutions of AdVLacZ stock

[2� 1011 plaque-forming units (PFU)/ml] were added

to SP2/0 cells seeded in triplicate in 96-well plates

(1� 105 cells/well) to form different �multiplicities-of-infection� (MOI). The cells were incubated with theadenovirus in 293 serum-free medium (Gibco,

Gaithersburg, MD) for 2 h at 37 �C, then the mediumwas replaced with DMEM/10% FCS and the cells

incubated for an additional 24 h at 37 �C. To assess

b-galactosidase expression [32] the cells were fixed in

formaldehyde/glutaraldehyde, then stained and counter-

stained with X-gal and nuclear fast red, respectively.

The proportions of positive (i.e., blue-staining) cellswere determined from triplicate wells and taken as the

percentage of transduction. Control SP2/0 cells trans-

fected with AdVpLpA did not exhibit any intrinsic

b-galactosidase activity or false-positive staining. We

observed a dose-dependent response to the infecting

dose of adenovirus, with maximal staining (70%) at a

MOI of P 200. Therefore, a MOI of 200 was selected

14 H. Huang et al. / Cellular Immunology 217 (2002) 12–22

for transfection of SP2/0 cells with AdVIP-10 in thisstudy. For transfection of SP2/0 cells with AdVIP-10 at

200 MOI, following viral adsorption for 1 h at 37 �C in

one well of a 24-well culture plate, the culture medium

was replaced with 1ml DMEM/10% FCS and the cells

(1� 106 cells/well) incubated for another 24 h at 37 �C.SP2/0 tumor cells transfected with AdVIP-10 (termed

SP2/0IP-10) or AdVpLpA (termed SP2/0pLpA) and their

culture supernatants were then harvested for examina-tion of IP-10 expression using the reverse transcription

polymerase chain reaction (RT-PCR) and functional

analysis of secreted IP-10, respectively.

The ability of AdVLacZ to infect SP2/0 tumors in

vivo was also investigated. Briefly, 0:5� 106 SP2/0 cells

were subcutaneously (s.c.) injected into each athymic

nude mouse. Twelve days later, when the SP2/0 tumors

were 8mm diameter, bolus 50 ll aliquots of AdVLacZvirus (2� 109 PFU or 5-fold dilutions thereof) were in-

jected into the SP2/0 tumors. One day after viral injec-

tion, each SP2/0 tumor was removed and cut into three

approximately equal sections. These tumor tissues were

mounted in tissue-freezing medium (Triangle Biomedi-

cal Sciences, Durham, NC) and snap-frozen by immer-

sion in 2-methylbutane (J.T. Baker, Phillipsburg, NJ)

that had been chilled over liquid nitrogen. Frozen 6-lmsections were obtained for the analysis of b-gal expres-sion [27]. To accomplish this, transfected SP2/0 tumor

tissue sections were fixed in PBS containing 37% form-

aldehyde and 25% glutaraldehyde, stained with X-gal,

and then counterstained with nuclear fast red (Poly

Scientific, Bay Shore, NY). The mean proportion of

blue-staining SP2/0 cells from triplicate wells or from

three tumor sections were taken as the percentage oftransfection. We observed a dose-dependent response to

the adenovirus, with maximum staining (45% of cells)

at 2� 109 PFU AdVLacZ. Thereafter, 2� 109 PFU was

selected as the optimal dose for delivery of AdVIP-10 to

tumors in vivo.

2.7. Reverse transcription polymerase chain reaction

analysis

Total RNA was obtained from the na€ııve and SP2/0-specific activated T cells, and the SP2/0 and AdVIP-10-

transfected SP2/0 tumor tissues [32]. The first-strand

cDNA synthesis for the RT-PCR was performed with

5 lg of RNA using a commercial kit (Stratagene, La

Jolla, CA), following manufacturer�s instructions. ThePCR primers were specific for IP-10 (the sense primer, 50

atgaa cccaa gtgct gccgt c 30; the anti-sense primer, 50

ttaag gagcc ctttt agacc ttt 30), chemokine (IP-10) recep-tor CXCR3 (the sense primer, 50 cctac gatta tggg aaacgag 30; the anti-sense primer, 50 tgatt ctctc cgtga agatgacg 30, [22]), and glyceraldehyde phosphate dehydroge-nase (GAPDH: sense primer, 50 caggt tgtct cctgc gactt 30;anti-sense primer, 50 cttgc tcagt gtcct tgctg 30). The PCR

conditions comprised 1 cycle at 94 �C (5min), 54 �C(1min), and 72 �C (1min) and 25 cycles at 94 �C (1min),55 �C (1min), and 72 �C (1min). All PCR products were

resolved on 1% agarose gels with ethidium bromide

staining.

2.8. Assessment of T-cell infiltration of the SP2/0 tumors

in vivo

To track T-cell migration into the tumors, on exper-

imental day 0 we injected 2� 109 PFU of AdVIP-10 and

AdVpLpA into well-established (i.e., 10-day) SP2/0 tu-

mors in the right and left thighs, respectively, of the mice.

The next day, 5� 106 51Cr-labeled activated T lympho-

cytes were injected into the tail vein of each mouse, as

previously described [32], and 2 or 24 h later the mice

were sacrificed. Each tumor and a panel of organs ortissues were removed and weighed, then its associated

radioactivity was determined using a c-counter. Thebiodistribution of labeled T cells was expressed as the

localization index (CPM per gram of tissue).

2.9. Histology

Tumor nodules were removed for histological analy-sis 24 h subsequent to the injection of AdVIP-10 or

AdVpLpA. They were then fixed in 10% formaldehyde

and embedded in paraffin. Sections of 6–7 lm thickness

were stained with hematoxylin–eosin according to the

standard procedures.

2.10. Effects of adoptive immunotherapy and intratumoral

IP-10 transgene expression on animal mortality and

tumor growth

Mice (8 per group) were injected s.c. with 0:5� 106

SP2/0 tumor cells into each thigh. At 10–12 days post-

inoculation, when the tumors were 5mm in diameter,

each was injected with 2� 109 PFU of AdVIP-10 or

AdVpLpA. One and two days after virus injection, each

mouse was injected i.v. with 1� 107 activated T lym-phocytes. Animal mortality and tumor growth were

monitored daily for up to 60 days; for humanitarian

reasons, all mice with tumors that achieved a size of

1.5 cm in diameter were sacrificed.

3. Results

3.1. Activated T lymphocytes generated from SP2/0-Lptn-

immunized mice comprise CD4þ Th1 and CD8þ Tc1 cells

Activated T lymphocytes were prepared by cocul-

turing splenocytes of SP2/0-Lptn-immunized mice with

irradiated SP2/0 tumor cells for 4 days and then sub-

jected to phenotypic characterization by flow cytometry.


As shown in Fig. 1 (upper panel), these cells were es-

sentially all CD3þ, with around 64% CD4þ and 36%

CD8þ T cells. They uniformly displayed high level ex-

pression of CD25 (IL-2R), indicating that they wereactivated, and low- and high-level L-selectin and FasL,

respectively, all relative to T cells from na€ııve mice.The CD4þ and CD8þ cells from this activated T-cell

pool were fractionated by negative selection using the

anti-CD8 and -CD4 DYNAL paramagnetic beads, re-

spectively. Their expression of IFN-c and IL-4 was thenassessed by flow cytometry (intracellular cytokines).

Following in vitro SP2/0 cell challenge, both populationsstained positively for intracellular IFN-c (Fig. 1, lowerpanel). However, neither population stained strongly for

intracellular IL-4. This pattern of cytokine expression is

consistent with the type 1 immune responses usually as-

sociated with anti-tumor immunity [28], and indicates

that the CD4þ and CD8þ populations comprised Th1

and Tc1 phenotype cells, respectively. High proportions

of both populations also expressed significant amountsof intracellular TNF-a and perforin.

3.2. Activated T lymphocytes display tumor-specific

cytotoxicity in vitro

Since the phenotypic markers associated with these

activated T cells were consistent with cytotoxic T

lymphocytes, we next tested their cytotoxic activities

against 51Cr-labeled SP2/0 target cells. The phenotypes

of target SP2/0 tumor cells were analyzed by flow cy-

tometry. As shown in Fig. 2, the wild-type SP2/0 tumorcells expressed cell surface Fas molecule and H-2Kd, but

not Iad. These in vitro -activated T cells from the SP2/0-

Lptn-immunized mice did induce significant cytotoxicity

for SP2/0 cells (79% specific killing at an E:T cell ratio of

50), but not for the irrelevant A20 cells or for natural

killer (NK) cell-sensitive Yac-1 cells (Fig. 3), indicating

that the killing activity of these activated T cell is SP2/0

tumor specific. The T lymphocytes from na€ııve mice didnot display discernable SP2/0 cell killing (data not

shown). To study the killing mechanism, concanamycin

Fig. 1. SP2/0-stimulated T cells from SP2/0-Lptn-immune mice comprise activated CD4þ Th1 and CD8þ Tc1 cells. Upper panel: Splenic lymphocytesfrom SP2/0-Lptn-immune mice were stimulated in vitro for 4 days with irradiated SP2/0 cells (activated T cells), then immunostained for assessment

of multiple markers by flow cytometry using specific antibodies. Unstimulated cells from na€ııve mice were used as negative control cells (na€ııve T cells).

Cell surface CD3, CD4, CD8, CD25, L-selectin, and FasL were detected using marker-specific primary antibodies (solid lines) or isotype-matched

irrelevant control antibodies (dotted lines) and FITC-conjugated secondary antibodies. Lower panel: The CD4þ and CD8þ cells from the activated T

cell pool were purified, and then either stained as above (CD4 and CD8), or permeabilized and stained for intracellular IL-4, IFN-c, TNF-a or

perforin using a commercial kit and PE-conjugated antibodies (solid lines). Unpermeabilized CD4þ and CD8þ T cells were used as negative controlcells (dotted lines). It is readily apparent that the SP2/0-immune cells comprise CD4þ or CD8þ;CD25þFasLþ L-selectinlow cells that express IFN-c,TNF-a, and perforin, but not IL-4.

Fig. 2. Phenotypic analysis of SP2/0 tumor cells by flow cytometry.

SP2/0 tumor cells were stained with the anti-mouse H-2Kd, Iad, and

Fas antibodies (solid lines). Isotype-matched antibodies were used as

controls (dotted lines).


A (CMA) and emetin were used to inhibit the perforin-

and Fas/FasL interaction-mediated cytotoxicity, re-

spectively. Our data showed that both CMA and emetin

demonstrated dose-dependent inhibition of T-cell cyto-

toxicity (data not shown). The treatment of CMA at1 lM induced nearly a complete inhibition (98%) of

T-cell cytotoxicity, while emetin treatment at 5 lM re-

sulted in only a maximum of 8% inhibition, indicating

that the perforin-mediated pathway plays a major role

in CTL cytotoxicity.

3.3. Functional IP-10 secreted by transfected SP2/0IP -10tumor cells

To examine IP-10 expression of transfected SP2/0IP-10cells, RNA extracted from SP2/0IP-10 cells was subjected

to RT-PCR analysis. As shown in Fig. 4A, IP-10 ex-

pression was found in AdVIP-10-transfected SP2/0IP-10,

but not in the control adenovirus-transfected SP2/0pLpAcells. To evaluate whether the secreted IP-10 is func-

tional, the culture supernatant of SP2/0IP-10, whichcontained IP-10 was subjected to chemotaxis analysis.

To validate the IP-10-mediated T-cell chemotaxis re-

sponse, we first demonstrated using RT-PCR that highly

purified mouse activated T cells expressed substantial

levels of the IP-10 receptor CXCR3 whereas SP2/0 tu-

mor cells did not (Fig. 4B). We then assessed the acti-

vated T-cell chemotactic properties of recombinant

IP-10, as well that of the culture supernatants of SP2/

0IP-10 tumor cells. Our data demonstrated that both therecombinant IP-10 and the SP2/0IP-10 supernatant (but

not the control SP2/0pLpA one) were able to chemoat-

tract the activated T cells in a dose-dependent fashion

(Fig. 5), indicating that IP-10 secreted by transfected

SP2/0IP-10 cells is functional. The amount of IP-10 se-

creted by SP2/0IP-10 cells (1� 106 cells/24 h) in superna-

Fig. 4. Expression of chemokine IP-10 and chemokine receptor

CXCR3. (A) RNA was obtained from the AdVIP-10-transfected SP2/

0IP-10 and the control AdVpLpA-transfected SP2/0pLpA tumor cells for

examination of IP-10 expression. (B) RNA was obtained from the

activated T and SP2/0 tumor cells for examination of CXCR3 ex-

pression. The first strand cDNA was synthesized from RNA using the

reverse transcriptase, and the PCRs were conducted using sets of

primers for CXCR3 and the control �house-keeping� gene GAPDH,respectively.

Fig. 3. The activated T cells from SP2/0-Lptn-immune mice affect the

anti-tumor cytotoxic activities against SP2/0 tumor cells. Activated T

cells were generated as in Fig. 1, then used as effector cells against 51Cr-

labeled SP2/0 cells or irrelevant A20 tumor cells or natural killer (NK)

cell-sensitive YAC-1 target cells, as detailed in the Materials and

methods. The data are presented as the percent specific lysis of the

target in a 6 h 51Cr-release assay. Each point represents the mean of

triplicate cultures. One representative experiment of two is depicted.

Fig. 5. T cells expressing the chemokine receptor CXCR3 chemotac-

tically respond to the recombinant IP-10 and the IP-10 in SP2/0IP-10supernatant. Activated SP2/0-specific T cells were generated as in Fig.

1. Their responses to IP-10 (0–1000 ng/ml) were assessed using 2 h

microchemotaxis assays. The chemotaxis assay results are expressed as

the mean number of cells/40� field (�SEM). The chemotactic activityof SP2/0IP-10 supernatant is much stronger than that of the control

SP2/0pLpA supernatant (Student�s t test, p < 0:05). The data comprise

one representative experiment of two.


tant was estimated to be 2 ng/ml in the chemotaxis assay.In addition, the chemotatic activity of SP2/0IP-10 super-

natant is much stronger than that of the control SP2/

0pLpA supernatant (p < 0:05).

3.4. AdVIP-10 transfection in tumors induce enhanced

T-cell infiltration and anti-angiogenesis

Since IP-10 has T-cell chemotactic effects in vitro, wenext assessed the potential in vivo relevance of this ob-

servation. We assessed the infiltration of T cells into

SP2/0 tumors that had been transfected with the ex-

perimental or control adenovirus. One day after injec-

tion of the virus into tumors on the right (AdVIP-10)

and left (AdVpLpA) thighs of each mouse, the animals

were injected i.v. with 51Cr-labeled activated T cells

from SP2/0-immune mice, and at 2 or 24 h post-treat-ment, the distribution of the radiolabeled T cells within

the organ systems of the mice was determined. At 2 h the

radiolabel had largely accumulated in the capillary-rich

organs, such as the spleen, lungs, livers, and kidneys

(Table 1). The levels of radioactivity detected in the

AdVIP-10- and AdVpLpA-treated tumors were much

lower at this time (Table 1), and there were no signifi-

cant differences between the experimental and controltumors (p > 0:05). As expected, the amount of radio-activity in the lungs decreased substantially over 24 h,

when most of the residual activity was in the spleens and

livers. Of note is the fact that at this time the levels of

radioactivity in the AdVIP-10-treated tumors was morethan three times than that of the AdVpLpA-treated

tumors (p < 0:05; Table 1), clearly suggesting that the51Cr-labeled T cells were preferentially localizing to the

tumors that expressed IP-10.

Since IP-10 can inhibit tumoral angiogenesis [23,24],

we also investigated the anti-angiogenesis effect of ade-

novirus-mediated transgene IP-10 expression in tumors.

Tumor nodules were removed for histological analysis

Table 1

Distribution of adoptively transferred tumor-specific T cells in tumor-

bearing mice

Organ Localization index (�104 CPM=g)a

2 h post-injection 24 h post-injection

Blood 2:60� 0:07 1:89� 0:05

Lung 36:89� 2:37 8:63� 0:13

Liver 22:34� 2:90 62:87� 3:47

Spleen 102:06� 4:49 74:39� 5:54

Kidney 26:49� 1:33 52:79� 3:71

Stomach 0:37� 0:06 0:39� 0:08

Muscle 0:68� 0:08 0:56� 0:04

AdVIP-10-treated

tumors

1:63� 0:07 3:18� 0:08b

AdVpLpA-treated

tumors

1:51� 0:01 0:98� 0:05b

aMice were s.c. inoculated with 0:5� 106 SP2/0 tumor cells in their

right and left thighs. When the tumors were approximately 5mm in

diameter (10 days), AdVIP-10 or AdVpLpA (2� 109 PFU) were

injected into the tumors. One day later, 51Cr-labeled activated T cells

from SP2/0-immune mice (5� 106 cells/mouse) were injected i.v. into

the mice (n ¼ 5), and 2 or 24 h later various organs/tissues were as-

sessed for their 51Cr content by c-counting. The distribution of theradiolabeled T cells in these tissues is expressed as the localization

index (CPM per gram of tissue). One representative experiment of two

is shown.bThe localization index in AdVIP-10-treated tumors is significantly

greater (p < 0:05) than that of the AdVpLpA-treated tumors (Stu-

dent�s t test).

Fig. 6. Histologic photographs of SP2/0 tumors. (A) Section from a

tumor 10 days after injection of 0:5� 106 SP2/0 tumor cells showing

brisk mitotic activity (arrows). (B) Sections from tumors 1 day after

intratumoral injection of AdVIP-10 (2� 109 PFU), in which throm-

bosed vessels (arrows) and significant necrosis (arrow heads) can be

seen. (C) Sections from tumors after intratumoral injection of the

control AdVpLpA (2� 109 PFU), in which some small focal necrosis

was seen (arrows). Magnifications for A, B, and C: 100�.


24 h subsequent to the injection of AdVIP-10 or Ad-VpLpA. As shown in Fig. 6A, the wild-type SP2/0 tu-

mors grew aggressively with many mitosis. Twelve hours

subsequent to the treatment of AdVIP-10, there were

vessel injuries including the complete occlusion by

thrombi and significant necrotic tumor tissues (Fig. 6B),

as previously described by Sgadari et al. [33]. In con-

trary, only small focal necrosis was found in tumor

nodules injected with AdVpLpA (Fig. 6C). These datasuggest that transgene IP-10 expression in tumors can

induce anti-angiogenesis effect.

3.5. Synergistic enhancement of anti-tumor immunity with

adoptively transferred tumor-specific T cells and intratu-

moral IP-10 transgene expression

In clinical practice, most candidates for cancer ther-apy are patients with existing tumor burdens. While we

and others have demonstrated efficacious prophylactic

treatment of experimental mice [28,34], if we are to ac-

curately model the clinical case we must direct our

questions of therapeutic efficacy to the elimination of

established tumors in animals. To this end, we next

tested the effects of combining an induced intratumoral

expression of IP-10 with the adoptive transfer of tumor-specific T cells, using the model system outlined above

(i.e., 10-day established tumors). At this time, these tu-

mors were 5mm in diameter and had a well-developedvasculature [32]. As above, we directly injected into the

tumors with 2� 109 PFU of AdVIP-10 or AdVpLpA,

then at the 24 and 48 h timepoints each mouse was in-

jected i.v. with 1� 107 activated T lymphocytes, for a

total of 2� 107 cells/mouse. Control mice were given i.t.

injections of AdVIP-10 or PBS (day 0), but not the T

cells. Tumor growth within the mice and their mortality

rates were then monitored daily. As expected, the tumorswithin the PBS-treated mice grew very aggressively (Fig.

7) and all mice in this group died within 21 days sub-

sequent to the tumor inoculation (Fig. 8). On the con-

trary, mice with tumors that had been treated with the

AdVIP-10 virus alone, or with the control AdVpLpA

virus in combination with an adoptive T-cell transfer,

experienced somewhat slower tumor growth than the

PBS-treated animals (Fig. 7), although both groups ofmice died within 26–38 days of tumor inoculation (Fig.

8). Of great significance here was the observation that 7

of the 8 (88%) mice that had been treated with the Ad-

VIP-10 virus and had received the adoptive T-cell

transfers were entirely tumor-free at 60 days post-inoc-

ulation of tumor cells. These results compellingly dem-

onstrate that a combined adoptive T-cell therapy and

adenovirus-mediated IP-10 gene transfer can synergisti-cally enhance anti-tumor immunity in mice with preex-

isting SP2/0 tumors of substantial mass.

Fig. 7. Impact of combined IP-10 gene therapy and adoptive T cell immunotherapy on the growth of well-established SP2/0 tumors in mice. Mice

bearing well-established (i.e., 5mm) SP2/0 tumors were given intratumoral injections of 2� 109 PFU AdVIP-10, AdVpLpA or PBS (no therapy)

alone or in conjunction with activated T cells obtained from SP2/0-Lptn-immune mice. The activated T cells (1� 107 cells/day) were administered i.v.

into each mouse 1 and 2 days subsequent to the gene therapy. Tumor growth was monitored and the tumor size (diameter) measured daily using an

engineering caliper. The evolution of the tumors in individual mice is depicted, as are the fractions of mice in each treatment group that were tumor-

free at 60 days post-inoculation of tumor cells. One representative experiment of two is shown.


4. Discussion

Previously, we demonstrated that inoculation of mice

with SP2/0 myeloma tumor cells engineered to express

the chemokine Lptn resulted in a failure of otherwise

inevitable tumor establishment, as a consequence of a

strongly induced SP2/0 tumor-specific T-cell response

[25]. In this study, we further characterized the SP2/0

tumor-specific T cells from mice immunized with SP2/0-Lptn tumor cells. Our data show that, following cocul-

ture with irradiated SP2/0 cells, the activated T cells

from these immunized mice expressed CD25high, L-

selectinlow, and FasLhigh on their cell surfaces and

comprised 64% CD4þ Th1 and CD8þ Tc1 cells. Their invitro cytotoxicity was specific for SP2/0 tumor cells, but

not for the irrelevant A20 tumor cell line.

CTLs including CD4þ and CD8þ cytotoxic T cells,NK cells and lymphokine-activated killer (LAK) cells

can kill appropriate allogeneic targets, virus-infected

cells and tumor cells by using one or more of pathways.

These include the secretion of cytotoxic cytokines such

as TNF-a and IFN-c in the region of their target and thecontact-dependent mechanisms of CTL-induced cell

death through the T-cell receptor (TCR) and members

of integrin family [35]. The latter include the calcium-dependent pathway which relies on the secretion of cy-

totoxic granules such as perforin and granzyme [36]

onto the target cells and the calcium-independent

pathway via the interaction of FasL on the CTLs and

the Fas receptor on the target cells [37]. CD8þ cytotoxicT cells, NK and LAK cells depend primarily on the

perforin/granzyme system to kill the targets, while

CD4þ cytotoxic T cells utilize Fas or other mechanismsto induce target cell death [34]. In our study, we showed

that SP2/0 tumor cells expressed Fas on their cell sur-

faces and the SP2/0 tumor-specific activated T cells ex-pressing both FasL and perforin displayed SP2/0 tumor-

specific cytotoxicity. To study the killing mechanisms,

CMA and emetin were used to inhibit the perforin- and

Fas/FasL interaction-mediated cytotoxicity, respec-

tively. Our data showed that CMA treatment almost

completely inhibits CTL cytotoxicity while emetin

treatment only reduces 8% of CTL cytotoxicity, indi-

cating CMA-mediated pathway plays a major role inCTL cytotoxicity, which is also consistent with a recent

report by others [38].

L-selectin is an adhesion molecule that is highly ex-

pressed on na€ııve T cells [39]. It has been recently re-

ported that tumor-specific L-selectinlow T cells play a

major role in tumor cell killing in vitro, as well as in the

eradication of lung metastasis or s.c. tumors in vivo

after adoptive T-cell transfers [4,5,40]. However, inthose studies the target tumors were either very small

(i.e., 3- or 10-day metastases) [4,5] or were well-estab-

lished ones for which prior tumor irradiation was a

prerequisite of the desired immunotherapeutic outcome

[5,40]. In our hands, the SP2/0 tumor-specific T cells

from SP2/0-Lptn tumor immunized mice were all

L-selectinlow. Nevertheless, these cells alone were insuf-

ficient to achieve curative effects against well-establisheds.c. tumors, very likely because the tumors were so well-

established in our system. This observation is consistent

with those of Narvaiza et al. [41] also with otherwise

untreated well-established tumors.

The importance to successful T-cell immunotherapy

of T-cell infiltration into the target tumor tissues is in-

creasingly being recognized [42,43], as is the direct rel-

evance to this process of chemokines expressed in thetissues and the receptors for these molecules on the T

cells (e.g. [44]). In the present study, our working hy-

pothesis was that transgene-derived intratumoral IP-10

expression would enhance the infiltration of adoptively

transferred CXCR3þ T cells into the tumors and thus

improve the therapeutic efficiency of the T-cell therapy.

Narvaiza et al. [45] have recently reported the treatment

of established tumor with intratumoral coinjection oftwo adenoviral vectors expressing IL-12 and IP-10. In

their report, they also showed that the adoptive transfer

of CD4þ T cells in combination with coinjection of thesetwo adenoviral vectors did not cure any established tu-

mors, and suggested the therapeutic effect may be de-

rived from the combined function of both CD4þ and

CD8þ T cells. In this study, we did investigate the

therapeutic effect of adoptive transfer of both CD4þ andCD8þ T cells with intratumoral injection of AdVIP-10.

Consistent with a previous report [46], we found that

adenovirus-mediated IP-10 gene transfer alone did not

cure any of the mice that were bearing well-established

tumors. Similarly, a combination of the adoptive T-cell

transfers and the control adenovirus AdVpLpA treat-

ments did not effect tumor elimination, although for

Fig. 8. Impact on mortality rates of combined IP-10 gene therapy and

adoptive T-cell immunotherapy for well-established SP2/0 tumors in

mice. The tumor inductions and treatments were as in Fig. 7, but the

readout in this figure represents the long-term mortality among the

animals, as determined by daily assessments across 60 days post-in-

oculation of tumor cells. The data closely mirror that in Fig. 7. One

representative experiment of two is shown.


both of these groups (i.e., AdVIP-10 and T cell +Ad-VpLpA) tumor growth was significantly dampened. In

contrast, combinational immunotherapy with adeno-

virus-mediated IP-10 gene transfer and adoptive T-cell

therapy not only substantially inhibited tumor growth

but also eradicated 10-day SP2/0 tumors in 7 of the 8

mice. These results clearly highlight the synergistic

therapeutic effects of a local expression of IP-10 within

tumors in the context of adoptive T (both CD4þ andCD8þ) cell therapy.Nevertheless, whatever the mechanism(s), our data

provide clear evidence of a potent synergy between

adoptive T-cell immunotherapy and adenovirus-medi-

ated IP-10 gene transfer into tumor tissues. This com-

binational immunotherapeutic strategy resulted in the

cure of well-established tumors and thus becomes a tool

of considerable conceptual interest in the implementa-tion of future clinical objectives.

Acknowledgments

This study was supported by a research grant (ROP-

15151) of the Canadian Institutes of Health Research.

We thank Connie Percy and Xuguang Bi for their flowcytometric analysis and technical support in this study.

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