行政院國家科學委員會專題研究計畫期中進度報告:NK細胞受不表現MHC且生產TGF-b之CTVT腫瘤中之細胞素的影響,並開發治療不表現MHC且生產TGF-b腫瘤的基因(2/3)

行政院國家科學委員會專題研究計畫 期中進度報告

NK 細胞受不表現 MHC 且生產 TGF-b 之 CTVT 腫瘤中之細胞素

的影響,並開發治療不表現 MHC 且生產 TGF-b 腫瘤的基因

(2/3)

中 華 民 國 93 年 6 月 25 日

Joint Immunogene therapy with IL-6 and IL-15 enhances antitumor activity by augmenting the cytotoxicity of NK cells

inhibited by tumor-derived TGF-β

Ching-Yi Lin, Kuang-Wen Liao, Tien-Fu Chuang, Ya-Wen Hsiao, Rea-Min Chu Department of Veterinary Medicine, National Taiwan University, Taipei,

Taiwan, ROC *Corresponding Author: RM Chu, Prefessor 142 Chou-San Rd Taipei 106 Taiwan, ROC Tel: 886 2 2368 6570 Fax: 886 2 2365 5147 Email: [email protected]

Kuang-Wen Liao’s current address: Neuromedical Science Center, Buddhist Tzu Chi General Hospital. Tzu Chi College of Technology, Taiwan, ROC

Running title: anti-tumor NK cytotoxicity restored by IL-6 and IL-15

Summary

Lowering the expression of major histocompatibility complex (MHC) molecules

is one way tumors evade host immune surveillance. Natural killer (NK) cells are

activated by low MHC expression. However, in many tumors, NK cell cytotoxicity is

severely suppressed. Immunogene therapy with plasmid IL-6 and plasmid IL-15

(pIL6/pIL15) stimulated NK cells. The plasmids were delivered by muscle

electroporation. IL-6 antagonized the inhibitory effect of TGF-β on NK cell

cytotoxicity and IL-15 promoted NK cell cytotoxicity. In cell cultures, treatment with

IL-6/IL-15 effectively relieved the inhibitory effect of TGF-β and activated NK cell

cytotoxicity. IL-6 or IL-15 alone did not enhance or only moderately promoted NK cell

cytolytic activity. In BALB/c mice, electroporation with pIL-6/pIL-15 increased the

ratio of NK cells in the spleen and promoted NK cell cytotoxicity. pIL-6 or pIL-15

alone did not have a significant effect. In C.B-17 SCID mice, gene therapy with

pIL-6/pIL-15 strongly inhibited the establishment, and the growth of established canine

transmissible venereal tumor (CTVT), a tumor with low MHC expression and high

TGF-β secretion. Treatment with anti-asialo GM-1 antibody depleted NK cells and

restored tumorigenicity. We demonstrated that the anti-TGF-β effect of IL-6 was

essential, but that stimulation of NK cell cytotoxicity by IL-15 also was necessary to

therapeutic regimenagainst tumors with low MHC expression and high TGF-β

Introduction

Major histocompatibility complex (MHC) class I antigens are 44-kDa

glycoproteins expressed on the cell plasma membrane associated with β2 -

microglobulin. Cytotoxic T cells can recognize tumor cells that display peptides

developed from tumor-associated antigens on their surface and in association with

MHC class I Ag. However, total loss or down-regulation of MHC class I antigens has

been documented in many tumors, including head and neck squamous cell carcinomas1,

lung cancer2, glioma3, melanoma4, laryngeal carcinomas 5, breast6, ovarian and

peritoneal carcinomas7, and canine transmissible venereal tumor (CTVT) 8-10. Tumor

cells that do not express MHC class I escape CTL recognition, but switch on natural

killer (NK) cells.However, many tumors secrete transforming growth factor-beta

(TGF-β) to inhibit NK cell cytotoxicity11.

TGF-β is a 25 kDa homodimeric protein with very potent pleiotropic regulatory

effects on the mammalian immune system. Addition of exogenous TGF-β to

lymphocyte culture decreases proliferation of B cells, mature T cells, thymocytes, NK

cells and lymphokine-activated killer (LAK) cells 12-16. TGF-β1, at physiologic

concentrations in the nanomolar range, inhibited NK cell proliferation and NK

cell-dependent cytolytic activity, and reduced production of the cytokines IFN-β,

association between the yield of TGF-β from malignant tumor cells, suppression of NK

cell cytotoxicity and tumorigenicity 18. Because NK cells are able to recognize and lyse

tumor cells that lack or express few MHC molecules on their surface, restoring the

cytotoxicity of NK cells inhibited by tumor-derived TGF-β is an important area in

cancer therapy research.

Up-regulation of IL-6 within ocular tissues during endotoxin-induced uveitis (EIU)

antagonizes TGF-β, restoring T cell proliferation19. IL-6 inhibits TGF-β-induced

apoptosis through the PI-3 kinase/Akt and the STAT3 pathways in hepatocytes20. In a

recent study, we demonstrated, for the first time, that tumor infiltrating lymphocytes

(TIL) isolated from regressing CTVT secreted high concentrations of IL-6, restoring

TGF-β 1-inhibited NK cell cytotoxicity10. IL-15 is a critical cytokine for the

development, survival, and activation of NK cells 21. IL-15 shares many biological

activities with IL-2, including inducing the proliferation of

phytohemagglutinin-stimulated normal peripheral blood mononuclear cells (PBMC),

NK cells, B cells, and the generation of CTL and LAK cells in vitro22,23. In addition,

IL-15 enhances NK cell cytotoxic activity and induces cytokine production of activated

NK cells23.

Based on IL-6 antagonism of TGF-β and the activation of NK cell cytotoxicity by

cytokines against tumors that down-regulate MHC class I antigens and produce TGF-β.

The use of IL-6 and IL-15 together inhibited TGF-β activity and promoted NK cell

cytotoxicity against CTVT cells. This is an effective strategy for enhancing NK

cell-mediated anti-tumor immunity against a low MHC tumor that secretes high levels

Results

Effects of TGF-β on IL-2 and IL-15-induced NK cell cytotoxicity

TGF-β strongly inhibited the cytotoxicity of IL-2-induced NK cells, from the

spleens of BALB/c mice, against YAC-1 cells (Fig. 1). The cytotoxicity of spleen NK

cells cultivated for 6 days with TGF-β and IL-2 or IL-15 was inhibited significantly

(P<0.01). However, while TGF-β completely canceled the effect of IL-2, that of IL-15

was only partially inhibited (Fig. 2).

TGF-β inhibition of NK cell cytotoxicity overcome by treatment with IL-6 and IL-15

Treatment using IL-6 and IL-15 significantly promoted TGF-β-inhibited NK cell

cytotoxicity of freshly isolated splenocytes. IL-15, alone, only moderately enhanced

NK cell cytotoxicity. Treatment with only IL-6 failed to promote spleen NK cell

cytotoxicity (Fig. 3). The direct effect of IL-6 on NK cell activity was investigated by

generating a dose response curve (Fig. 4a). Fresh BALB/c spleen cells were cultivated

for 6 days with IL-6 at concentrations ranging from 100 U/ml to 2000 U/ml. IL-6

activation of cytotoxic was only slightly concentration-dependent from 100 U/ml to

800 U/ml. High concentrations of IL-6 (1400 U/ml to 2000 U/ml) actually inhibited NK

cell cytotoxicity. We then tested whether IL-6 antagonized the inhibitory effect of

spleen LAK cells, we found IL-6 (400U/ml) completely countered the

immunosuppressive effect of TGF-β (Fig. 4b).

Expression of bioactive IL-6 and IL-15

The bioactivity of IL-6 and IL-15 in the supernatants from transfectants was

evaluated with TF-1 and HT-2 proliferation bioassays. Supernatant from BALB/3T3

cells transfected with IL-6 plasmids stimulated proliferation of IL-6-dependent TF-1

cells; human-anti-IL-6 antibody blocked proliferation (Fig. 5a). A similar experiment,

using IL-15-dependent HT-2 cells, demonstrated that BALB/3T3 cells transfected with

a chimeric IL-2SP/IL-15MP gene expressed bioreactive IL-15 protein (Fig. 5b).

Anti-IL-15 antibody also blocked HT-2 cell proliferation.

IL-6 and IL-15 gene expression in serum

Blood samples were collected from mice in each treatment (mock, pIL-6, pIL-15,

pIL-6/pIL-15) 0, 3, 5, 8, 12, and 15 days after muscle electroporation. In the pIL-6

treatment, serum IL-6 peaked on day 8 . In the pIL-6/pIL-15 treatment, serum IL-6

peaked on day 5 (Fig. 6a). In the pIL-15 only, and pIL-6/pIL-15 treatments, serum

IL-15 peaked on day 5 (Fig. 6b). In addition, low concentrations of IL-6 occurred in the

serum of mice in the pIL-15 and mock treatments. No IL-15 was detectable, within the

sensitivity limit of the ELISA assay (< 11 pg/ml), in the sera of mice that received pIL-6

Changes in cell populations following the transfer of pIL-6 and pIL-15 by muscle electroporation

To investigate the mechanism by which the pIL-6/pIL-15 treatment affected host

cells, we analyzed (with flow cytometry) changes in T, B, and NK cell populations, and

NK cell cytotoxicity, in the spleens of mice that received different plasmids. The

percentage of T or B cells was not changed significantly by any of the treatments (Fig.

7a, b). However, the percentage of NK cells was elevated significantly by treatment

with pIL-6 and pIL-15 together (P<0.01) (Fig. 7c). NK cells from BALB/c mice that

received both IL-6 and IL-15 plasmids exhibited much stronger cytotoxic activity

against YAC-1 cells than NK cells from mice in other treatments. pIL-15, alone,

augmented NK cell cytotoxicity somewhat, but pIL-6 failed to elevate NK cell

cytotoxicity (Fig. 8).

Effect of pIL-6 and pIL-15 transfer by muscle electroporation on tumor establishment

To determine whether intramuscular delivery of pIL-6 and pIL-15 inhibited CTVT

establishment, we electroporated 100 µg of mock plasmid, pIL-6, pIL-15, and

pIL-6/pIL-15 into C.B-17 SCID mice 7 days after the mice were inoculated with CTVT

cells. On day 7, CTVT tumors were still not detectable in the subcutaneous tissue by

development and growth (P<0.01) (Fig. 9a). In the pIL-6/pIL-15 treatment, 2 of 5 mice

were tumor-free for the duration of the observation period (56 days) and the long-term

survival rate of mice was significantly higher than that of mice in other treatments

(P<0.01) (Fig. 9b). Intraperitoneal injection of anti-asialo GM-1 antibodies blocked

pIL-6/pIL-15 activity, resulting in tumors that grew faster and larger than in any other

treatment. Administration of pIL-6 or pIL-15 alone did not decrease tumor incidence or

retard CTVT growth (Fig. 9a).

Effect ofpIL-6 and pIL-15 transfer by muscle electroporation on the growth of established tumors

The effect of pIL-6 and pIL-15 on the growth of established CTVT in C.B-17 SCID

mice was evaluated in a separate experiment. Fourteen days after inoculation with

tumor cells, when the tumor was approximately 5 mm in diameter, plasmids (mock,

pIL-6, pIL-15, or pIL-6/pIL-15) were delivered to the mice via muscle electroporation.

pIL-6/pIL-15 significantly inhibited CTVT growth (P<0.01), but pIL-6 or pIL-15 alone

did not (Fig. 10a). Again, intraperitoneal injection of anti-asialo GM-1 antibodies

blocked the effect of pIL-6/pIL-15 allowing unrestricted CTVT growth (P<0.05) (Fig.

Discussion

Immuno-regulatory inhibition of NK cells by TGF-β has been documented in

several studies including: inhibition of peripheral blood lymphocyte (PBL) NK activity

15_{, inhibition of NK cell-mediated cytolysis in the aqueous humor of the anterior}

chamber of the eye24, and diminished TIL cytotoxicity against CTVT in dogs10.

Decreased cytotoxicity can be blocked by adding anti-TGF-β antibody. Similarly, the

TGF-β of human breast cancer cell line suppresses NK cell activity in athymic mice and

treatment of anti-TGF-β antibody suppresses the tumor growth and lung metstasis18. In

our study, TGF-β inhibited IL-2- and IL-15 stimulated NK cell cytotoxic activity. In

addition, we found that the immuno-regulatory effect of TGF-β is so potent, that

whenever TGF-β is present, NK cell cytotoxicity could not be enhanced significantly.

Therefore, only pIL-6 and pIL-15 delivered together significantly suppressed tumor

establishment and the growth of an established tumor. Only when TGF-β is antagonized

by IL-6 can IL-15 be fully effective. This explains why treatment with pIL-6 or pIL-15

alone did not inhibit the growth of CTVT in C.B-17 SCID mice. Furthermore, the

survival rate of CTVT-laden C.B-17 SCID mice treated with pIL-6/pIL-15 was greatly

higher than the survival rates of mice receiving other treatments. Eliminating the effect

of TGF-β is essential for effective anti-tumor treatment and should be included in the

In this study, we demonstrated that IL-6 effectively blocked the function of TGF-β

and restored the cytotoxicity of IL-2-activated mice LAK cells. This activity was first

described in our previous report on CTVT10. IL-6 did not significantly increase the NK

cell cytotoxicity of freshly isolated splenocytes; high IL-6 concentrations actually

suppressed the cytotoxicity. IL-6 did not enhance the cytotoxicity of fresh splenocytes

in the presence of TGF-β, but restored the TGF-β inhibited cytotoxicity of

IL-2-dependent LAK from mice splenocytes. Therefore, IL-6 did not act on NK cells

directly to enhance the cytotoxicity, but functioned mainly to antagonize the TGF-β

effect. In BALB/c mice, treatment with pIL-6 and pIL-15 increased NK cell

cytotoxicity significantly. They acted synergistically and were more effective than

IL-15 alone. IL-6 enhances the cytotoxic activity of thymocyte-derived NK cells with

the help of IL-2 produced by IL-6-activated T cells25. Thus, IL-6 might stimulate IL-2

production to work in concert with IL-15 on NK cell activation. This IL-2 associated

activity may be more important in an immune-intact animal than the C.B.-17 SCID

mice used in this study.Thus, IL-6 had two distinct roles contributing to the

suppression of the tumor. The dominant role was to antagonize TGF-β, the minor one

was to activate NK cells. However, IL-6 did not enhance the NK cell cytotoxicity of

mice splenocytes that were not activated. Thus, muscular electroporation of pIL-6 in

number of NK cells in the spleen. Different reports on the effect of IL-6 on NK cell

functions present some results that are contradictory. For example, it has been reported

that IL-6 directly activates the proliferation and surface Ag expression of

IL-2-independent, human CD3-CD56+ NK cells26. However, IL-6 did not induce

significantly the LAK lytic activity of PBMC-derived large granular lymphocytes,

mice spleen cells, or thymocyte-derived CD56+ cells15,27,28. Others studies even

reported that IL-6 secreted from ductal breast carcinoma and high doses of recombinant

IL-6 suppressed NK and LAK activity in vitro29,30.

IL-15 plays a pivotal role in the differentiation, survival, and function of NK

cells31. IL-2 and IL-15 receptors share two receptor chains, IL-2Rβ and γc, but each also

has a unique third chain, IL-2Rα and IL-15Rα, respectively32. The α chains appear to

aid the formation of a high-affinity complex. In the absence of the β and γc chains, IL-2

has low binding affinity to IL-2Rα (10-8 M) but the binding affinity of IL-15 to IL-15Rα

is still relatively high (10-11 M)33,34. IL-15 moderately increased the cytotoxicity of

mice spleen NK cells. Delivery of pIL-15 increased serum concentrations of IL-15 and

enhanced NK cell cytotoxicity. Thus, S.D.-17 SCID mice that received only pIL-15

suppressed tumor establishment. However, mice in this treatment, without the

anti-TGF-β activity of IL-6, could not inhibit the growth of an established tumor.

no changes between species in the phenotype and genotype of CTVT, including the

lack of MHC expression35,36. Therefore, CTVT grows normally in C.B-17 SCID mice,

a strain of mice that lacks functional T and B cells, and Thy-1+ dendritic cells, but

exhibits normal NK cell differentiation and function37-40. These properties nicely fit one

of the objectives of this study: to determine whether NK cell cytotoxicity is important

against tumors that express low levels of MHC molecules and secrete TGF-β. Injection

of anti-asialo GM-1 antibodies abolished the inhibitory effects of pIL-6/pIL-15 on

tumor establishment and the growth of an established tumor. In S.D-17 SCID mice in

the pIL-15 treatment, NK cell number and cytotoxicity increased, and tumor

establishment was inhibited. These results demonstrate the important anti-cancer

activity of NK cells. Furthermore, because two-thirds of CTVT cells do not express

MHC molecules during regression, NK cell cytotoxicity must play a major role in the

process of regression. CTL, with IL-6 and/or other cytokines, should also help to

eliminate tumor cells expressing surface MHC.

In conclusion, we have presented a new strategy for cancer gene therapy that

reverses the inhibitory effect of TGF-β on NK cells and promotes NK cell cytotoxicity.

The anti-tumor effects against CTVT on C.B-17 SCID mice were achieved by

delivering pIL-6/pIL-15 by intramuscular electroporation. Our strategy was to use IL-6

cytotoxicity. Treatment with only IL-6 or IL-15 did not significantly delay or inhibit

tumor cell growth. It is important to integrate the anti-TGF-β concept into the treatment

of cancers that produce TGF-β. This novel approach provides a promising rationale for

using immunotherapy against tumors with low MHC expression and high TGF-β

production, and it may yield a new therapeutic tactic for cancer treatment.

Finally, tumor cells evade host immune surveillance by lowering MHC expression,

which leads to NK cell activation. However, tumor cells secrete high concentrations of

TGF-β to inhibit NK cell cytotoxicity. In turn, host TIL, activated through an unknown

mechanism, produce large amounts of IL-6 to antagonize TGF-β, and to cooperate with

other immunocytes and immuno-stimulating cytokines to suppress tumor growth. This

information is in the literature about tumors of animals and humans, but the linkages

Materials and Methods Mice

Female BALB/c and C.B-17 SCID mice (6-8 weeks of age) were obtained from

the Laboratory Animal Center at the National Taiwan University College of Medicine

(NTULAC) (Taipei, Taiwan). BALB/c mice were used for plasmid delivery via muscle

electroporation and their splenocytes were used for NK cell cytotoxicity assays.

Xenotransplantation of CTVT and cytokine gene therapy were manipulated in the

C.B-17 SCID mice. All animal experiments were performed in compliance with

NTULAC standard operating procedures.

Target cell labeling

YAC-1 were used as NK target cells. They were grown in a complete medium

RPMI-1640 (Gibco-BRL, UK) supplemented with 10% fetal bovine serum (FBS;

Gibco-BRL, UK), 2mM L-glutamine adjusted to final concentration of 1.5 g/L, sodium

bicarbonate (Merck, South St. Paul, MN), 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM

sodium pyruvate (Sigma, St. Louis, MO). A lipophilic carbocyanine membrane dye,

3,3’-dioctadecyloxacarbocyanine (DIO C18) (Sigma), was used for FITC-target cell

labeling41. YAC-1 cells (5×105) were incubated with DIO C18 (10 µl, 3 mM in

dimethylsulfoxide) for 16 hrs at 37℃. Cells were rinsed 3 times with RPMI-1640

Effector cells preparation

BALB/c mice were euthanized by intraperitoneal injection of 1 ml Pentothal®

(Abbott, Kurnell, Australia). Each dead mouse was given an aseptic splenoctomy.

Splenocyte suspensions were prepared in RPMI-1640 containing 10% FBS. ACK lysis

buffer was used to remove the erythrocytes. Freshly isolated splenocytes were directly

used for the inhibitory effect of TGF-β. Splenocytes were also cultured for 6 days in

complete medium with 50 µM 2-mercaptoethanol and 500 U recombinant human (rh)

IL-2 (kindly provided by Dr. Mi-Hua Tao) per 1 × 106 mice splenocytes to generate

LAK cells. On day 3, the cells were fed again with 500 U rhIL-2. To study IL-6 and

IL-15 antagonism of TGF-β (Peprotech, London, UK ), the rhIL-2 was replaced with

rhIL-6 or rhIL-15.

Cytotoxicity assay

Triplicate 0.1 ml aliquots of each concentration of effector cells were placed in the

round-bottom wells of a microtiter plate. To each well, 100 µl of DIO C18-labeled

YAC-1 (6×105cells / ml) were added to make mixtures with different E/T ratios. The

mixtures were centrifuged at 300xg for 5 mins to enhance cell contact, and then

incubated in a complete RPMI medium at 37℃ in 5% CO2. After 6h incubation, the cell

mixtures were harvested and propidium iodide (PI, 2500μg/ml) (Sigma, St. Louis, MO)

flow cytometry (FACscan, Becton Dickinson, Mountain View, CA, USA).

Plasmid construction and preparation

The human IL-6 coding sequence was obtained from a PCR product amplified

from a IL-6 plasmid (kindly provided by Dr. Min-Liang Kuo, National Taiwan

University College of Medicine, Taipei, Taiwan) which was created by inserting a

human IL-6 gene into pcDNA3. The forward primer sequence was

5’-atgaactccttctccacaag-3’ and the reverse primer sequence was

5’-catttgccgaagagccctca-3’. The human IL-15 gene was designed as described by

Suzuki21. It contained hIL-2 SP and hIL-15 MP coding regions and proved to be a

highly secreting form of IL-15 gene. The chimeric IL-15 gene was artificially

synthesized with 27 primers, which covered the entire length of the gene with PCR

reactions. PCR products of both IL-6 and IL-15 were TA-cloned into

pcDNA3.1-V5-His-TOPO vectors (Invitrogen, San Diego, CA). The sequences were

confirmed. Plasmid DNA was purified from transformed Escherichia coli (One shot®

TOP10 competent E.coli) (Invitrogen, San Diego, CA) using a Nucleobond AX

plasmid purification kit (Macherey-Nagel, Duren, Germany) according to the

manufacturer’s instructions, and stored at –20℃. For experiments, plasmid DNA was

DNA transfection and cytokine biofunctional assay

BALB/3T3 cells were maintained in growth medium (Dulbecco’s modified

Eagle’s medium, with 4mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose,

and 10% FBS). TF-1 is a cell line of human lymphoblasts that responds to a variety of

cytokines such as IL-1, IL-4, IL-6, IL-9, IL-11, and IL-13. To culture TF-1 cells, 2

ng/ml rhGM-CSF were added to the complete medium. The HT-2 cell line is highly

responsive to IL-2. Because IL-15 shares the β and γc chains of the IL-2 receptor32, we

used the HT-2 cell line to evaluate the biofunction of the pIL-15 transfectant. HT-2

cells were maintained in complete medium supplemented with 50 µM

2-mercaptoethanol and 50 U/ml of rhIL-2. The day before transfection, BALB/3T3

cells were seeded in 6-well plates and grown for 24-h to 75% confluence. Then 3 µg of

pIL-6, pIL-15, pIL-6/pIL-15 (3 µg each), or pcDNA3.1-V5-his-TOPO (the mock) were

transfected using LipofectamineTM_{2000(Invitrogen, CA, USA) following the}

manufacturer’s protocol. The cells were incubated in growth medium for 48 h and the

supernatants of transfected cells were collected for IL-6 and IL-15 biofunctional assays.

Supernatants from pIL-6, pIL-15, and the mock were used for cell proliferation assays,

the MTS（3-(4,5-dimethylthiazol-2-yl)

-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium）(CellTiter 96

measure IL-6 biofunction, 2 × 104 TF-1 cells were washed three times with RPMI-1640

to remove residual rhGM-CSF. The cells were plated in microtiter-plate wells, in

triplicate, in 100 µl complete medium. Then, 100 µl of rhIL-6, supernatants of the mock

or pIL-6 were added to the wells. In another treatment, pIL-6 supernatant was added to

the wells in the presence of 5 µg rabbit-anti-human polyclonal IL-6 antibody (Endogen,

MA., USA). After 72h incubation, 20 µl MTS were added to each microtiter-plate well

and incubated for 4h at 37℃ in a humidified, 5% CO2 atmosphere. The absorbance was

read with a MRX Microplate Reader (Dynex Technologies, Chantilly, VA, USA) at 490

nm. For each treatment, the results were expressed as the mean optical density (OD) of

the 3-well set. To measure IL-15 bioactivity, 6 × 103 HT-2 were used and the

procedures described above for IL-6 were followed. For one treatment, 100 µl pIL-15

supernatant was added to the HT-2 cells in the presence of 3.75 µg rabbit-anti-human

polyclonal IL-15 antibody (Endogen, MA., USA).

Intramuscular plasmid injection and electroporation

BALB/c mice and CTVT-bearing C.B-17 SCID mice were anesthetized with 30-50

µl acepromazine maleate (Fermenta Animal Health Co., Kansas, Mo., USA). A

disposable insulin syringe with a 27-gauge needle was used to inject plasmid DNA into

bilateral quadriceps muscles. A 100 µg each of plasmid DNA, pIL-6, pIL-15, pIL-6/

mouse. After 5 minutes incubation, a pair of electrodes was inserted 5 mm into the

muscle in the area of the plasmid injections. A total of 10 electric pulses (50 ms each,

100V) were delivered segmentally with an electric pulse generator (Electro Square

Porator ECM 830; BTX, San Diego, Calif., USA). The electrodes were made of a pair

of gold-plated stainless steel needles, 5 mm in length and 0.8 mm in diameter, with a 5

mm, immobile distance between them.

Determination of IL-6 and IL-15

hIL-6 and hIL-15 levels in serum samples were measured with hIL-6 and hIL-15

ELISA kits fromEndogen (MA, USA) and Biosource (CA., USA) with detection levels

of 15.6 and 11 pg/ml, respectively. Mice were bled from the tail vein beforeand at

different times after electroporation. The blood was centrifuged at 6000 rpm for 5

minutes and serum was collected and stored at –20℃ until analyzed. ELISA kits were

used according to manufacturers’ instructions. Color change was read using a MRX

Microplate Reader (Dynex Technologies, Chantilly, VA, USA).

Flow cytometric analysis

Spleens taken from sacrificed BALB/c mice 14 days after muscle electroporation

were homogenized gently with 230 µm stainless steel mesh. The erythrocytes were

lysed with ACK lysis buffer. Rat-anti-mouse fluorescein isothiocyanate (FITC)

anti-NK 1.1 antibody were obtained from Serotec (Oxford, UK) and BD PharMingen

(San Diego, CA, USA) for T, B, and NK cells staining. Splenocyte samples were

stained separately with anti-CD3, anti-CD-19, or anti-NK antibodies for 45 minutes in

ice, washed three times with PBS, and analyzed on a FACscan (Becton Dickinson,

Mountain View, CA, USA).

Tumor challenge, therapy, and growth monitoring

Experimentally inoculated CTVT on beagles was used for xenotransplantation to

C.B-17 SCID mice. Aseptic tumor samples were surgically excised from beagles and

homogenized in HBSS (Gibco-BRL, UK) following the procedures described

previously9 to obtain single cell suspensions. Mice were subcutaneously inoculated

with 1×108 viable CTVT cells on both dorsal flanks. For the tumor establishment

inhibition experiment, intramuscular electroporation of target plasmids was performed

on day 7 post CTVT inoculation. There were 5 groups and 5 mice in each group. The

mice in 3 groups received the mock, pIL-6 or pIL-15, the other 2 groups received both

pIL-6 and pIL-15 plasmids. Mice in two combined pIL-6 and pIL-15 replicates were

injected intraperitoneally with 30 µl /mice of anti-asialo GM-1 antibodies or 30 µl

/mice of normal rabbit serum intraperitoneally (Wako Pure chemical, Osaka, Japan) on

the day before tumor inoculation and two times per week thereafter. For the growth

reached a measurable size (about 5 mm in diameter), mice were randomly divided into

groups of 5-6 mice each. Plasmids were delivered by muscle electroporation of the

mock plasmid, pIL-6, pIL-15, or pIL-6/pIL-15. Mice treated with anti-asialo GM-1

antibodies (30 µl /mice) also received 100 µg each of pIL-6 and pIL-15. Tumor size was

measured twice a week with calipers. Tumor volume was calculated using the formula8:

length (mm) ×width (mm) ×height (mm) ×π/4.

Statistics

Our results were analyzed with two-tailed Student’s t-tests. Differences were significant when P < 0.05.

Acknowledgments

This project was supported by National Science Council of Taiwan

(

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Titles and legends to figures

Fig. 1 Modulation of IL-2-activated NK activity by TGF-β. Fresh splenocytes

(2x106 cells) of BALB/c mice were cultured with IL-2 (1000 U/ml), TGF-β (200 U/ml),

or both for 6 days.The cytokines were fed again on day 3. NK cell cytotoxicity was

assessed by adding 100μl of DIO C18-labeled YAC-1 (6×105cells/ml) into each well

at an E/T ratio of 6.25/1. Lysis was measured with flow cytometry. (N=3). NC: No

cytokine control.

Fig. 2 Effect of TGF-β on IL-2- and IL-15-induced NK cell cytotoxicity. Freshly

isolated splenocytes (2x106 cells) of BALB/c mice were cultured for 6 days with

growth medium only, IL-2 (2000 U/ml), IL-2 (2000 U/ml) plus TGF-β (200 U/ml),

IL-15 (400 U/ ml), or IL-15 (400 U/ ml) plus TGF-β (200 U/ml). More IL-2 was added

on day 3. NK cell cytotoxicity was assessed by adding 100μl of DIO C18-labeled

YAC-1 (6×105cells/ml) into each well at an E/T ratio of 6.25/1. Lysis was measured

with flow cytometry (N=3).

Fig. 3 Treatment with IL-6 and IL-15 reversed TGF-β inhibition of NK

cytotoxicity. Fresh splenocytes (2x106 cells) of BALB/c mice were cultured for 6 days in growth medium only, TGF-β (200 U/ml), TGF-β (200 U/ml) plus IL-6 (400 U/ ml),

IL-15 (400 U/ ml), or IL-6 (400 U/ ml) plus IL-15 (400 U/ ml). The same cytokines

were added again on day 3. NK cell cytotoxicity was assessed by adding 100μl of DIO

C18-labeled YAC-1 (6×105cells/ml) into each well at an E/T ratio of 6.25/1. Lysis was

measured with flow cytometry (N=3).

Fig. 4 IL-6 antagonized TGF-β inhibition of NK cytotoxicity. To assess the ability

of IL-6 to induce NK activity, fresh BALB/c splenocytes (2x106 cells) were incubated

for 6 days with different concentrations (100 U/ml to 2000 U/ml) of IL-6 (a).

IL-2-stimulated LAK cells from mice spleen were incubated for 3 days with complete

medium (CM), IL-6 (400 U/ml),TGF-β (100 U/ml), or IL-6 (400 U/ml) and TGF-β

(100 U/ml) to evaluate the anti-TGF-β effect of IL-6 (b). Values are means with the

standard error (SE) of triplicate samples. NK cell cytotoxicity was assessed by adding

100μl of DIO C18-labeled YAC-1 (6×105cells/ml) into each well at an E/T ratio of

6.25/1. The lysis was measured with flow cytometry (N=3).

Fig. 5 Biofunctional assay for pIL-6 and pIL-15. IL-6-dependent TF-1 cells (2 × 104) were incubated with 10 U/ml rhIL-6 or transfectant supernatants for 72 h, followed by

4h MST pulse. Rabbit-anti-human polyclonal IL-6 Ab (5 µg) was added to pIL-6

IL-2-dependent HT-2 cells (6× 103) were cultured with 10 U/ml rhIL-15 or transfectant

supernatants followed by 4h MTS pulse. Rabbit-anti-human polyclonal IL-15 Ab (3.75

µg) was added to pIL-15 transfectant to verify that HT-2 cell proliferation was due to

IL-15 secretion. (b). The absorbance was read with a MRX Microplate Reader (Dynex

Technologies, Chantilly, VA, USA) at 490 nm. For each treatment, the results are the

mean optical density (OD) of the 3-well set. Vector: mock; PC: recombinant human

IL-15; NC: lipofactamine only

Fig. 6 Serum levels of IL-6 and IL-15 in BALB/c mice following pIL-6 and pIL-15 delivery by muscle electroporation. Six mice were used for each treatment and

control. The bilateral quadriceps muscles of each BALB/c mouse were injected with

100μg (50μg in each muscle) of pIL-6, pIL-15, or pcDNA3.1 (mock) plasmid DNA.

Then the muscles were electroporated (10 pulses, 50 milliseconds each, at 100V).

Serum samples were collected before, and 3, 5, 8, 12 and 15 days after, electroporation.

The levels of IL-6 (a) and IL-15 (b) were determined with ELISA. Results are the mean

Fig. 7 Effect of IL-6 or IL-15 genes delivered by muscle electroporation on

BALB/c mice splenocyte immune cell populations. The bilateral quadriceps muscles

of each BALB/c mouse were injected with 100μg (50μg in each muscle) of pIL-6,

pIL-15, or pcDNA3.1 (mock) plasmid DNA. Then the muscles were electroporated (10

pulses, 50 milliseconds each, at 100V). On day 14 after electroporation, the CD3+ (a),

CD19+ (b) and NK1.1+ (c) populations in the spleen (N=4) were analyzed by flow

cytometry. Results are the mean ± the standard error (SE). There were six mice in each

group.

Fig. 8 Effect of pIL-6 or pIL-15 delivered by muscle electroporation on the NK killing activity of BALB/c mice splenocytes. The bilateral quadriceps muscles of each

BALB/c mouse were injected with 100μg (50μg in each muscle) of pIL-6, pIL-15, or

pcDNA3.1 (mock) plasmid DNA. Then the muscles were electroporated (10 pulses, 50

millisecondseach, at 100V). On day 14 after muscle electroporation, spleen NK cell

cytotoxicity was measured by adding 100μl of DIO C18-labeled YAC-1 (6×105cells /

ml) into each well at the E/T ratio indicated. Lysis was measured with flow cytometry

(N=4). Results are the mean ± the standard error (SE; error bars). There were four mice

Fig. 9 pIL-6 and pIL-15 delivered by muscle electroporation inhibit tumor establishment. There were two control groups: a mock plasmid control, and the

intraperitoneal injection of normal rabbit serum in mice treated with pIL6/pIL15 as a

control for the injection of anti-asialo GM-1 antibodies in the mice with the same

treatment. The bilateral quadriceps muscles of each S.D.-17 SCID mouse were injected

with 100μg (50μg in each muscle) of pIL-6, pIL-15, pIL-6 and pIL-15 (100μg of

each), or pcDNA3.1 (mock) plasmid DNA. Then the muscles were electroporated (10

pulses, 50 milliseconds each, at 100V). Tumor size was measured twice a week with

calipers to determine the effect of the pIL6 and pIL15 on the inhibition of tumor

establishment (a). Tumor volume was calculated with the formula: length (mm) ×width

(mm) ×height (mm) ×π/4. Bars show the mean ± standard error (SE, error bar). There

were significant differences in tumor volume between groups by the end of each

experiment (P<0.01). The arrowhead shows the date of muscle electroporation. The

percentage of survivors in each treatment (b) was determined.There were five mice in

Fig. 10 pIL-6 and pIL-15 delivered by muscle electroporation suppress growth of established CTVT. Mice in each treatment received 100μg of plasmid DNA followed

by muscle electroporation on day 14, when the diameter of the tumor was about 5 mm.

The bilateral quadriceps muscles of each S.D.-17 SCID mouse were injected with 100

μg (50μg in each muscle) of pIL-6, pIL-15, pIL-6 and pIL-15 (100μg of each), or

pcDNA3.1 (mock) plasmid DNA. Then the muscles were electroporated (10 pulses, 50

milliseconds each, at 100V). Mice treated with anti-asialo GM-1 antibodies also

received pIL-6/ pIL-15 by muscle electroporation. Tumor size was measured twice a

week with calipers. Tumor volume was calculated using the formula: length (mm) ×

width (mm) ×height (mm) ×π/4. The mean tumor volume (a) and the effect of

anti-asialo GM-1 antibodies (b) were determined. The bars in each graph represented

the mean±standard error (SE, error bar). There were significant differences in tumor

volume between groups by the end of each experiment (P<0.01). The arrowhead shows

the date of muscle electroporation. Five to six mice were used for each treatment and