Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells

    Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells Guojun Zhang, Mingkai Xu, Huiwen Zhang, Yubo Song, Jian Wang, Chenggang Zhang PII: DOI: Reference:

S0041-008X(16)30310-6 doi:10.1016/j.taap.2016.10.009 YTAAP 13784

To appear in:

Toxicology and Applied Pharmacology

Received date: Revised date: Accepted date:

7 July 2016 24 September 2016 10 October 2016

Please cite this article as: Zhang, Guojun, Xu, Mingkai, Zhang, Huiwen, Song, Yubo, Wang, Jian, Zhang, Chenggang, Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells, Toxicology and Applied Pharmacology (2016), doi:10.1016/j.taap.2016.10.009

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ACCEPTED MANUSCRIPT Title: Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1-6 cells · Mingkai Xu1 * · Huiwen Zhang1 · Yubo

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The affiliation, address of authors:

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Song1, 2 · Jian Wang1 · Chenggang Zhang1

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Authors: Guojun Zhang1,

1 Institute of Applied Ecology, Chinese Academy of Sciences, No.72

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Wenhua Road Shenhe Dis., Shenyang, Liaoning, China

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2 University of Chinese Academy of Sciences, Beijing, China Correspondence author: Mingkai Xu

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Address: Institute of Applied Ecology, Chinese Academy of Sciences,

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110016 Shenyang, People’s Republic of China.

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Phone: 86-24-83970380

Fax number: 86-24-83970381

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T

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lymphocytes. With this property, SEC2 has already been used in clinic as

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a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by

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site-directed mutagenesis, and the molecular mechanism of the enhanced

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antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with

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SEC2, and exhibited significantly enhanced immunocyte stimulation and

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antitumor activity in vitro. The synthetic peptide sequencing the residues

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of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4

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up-regulated granzyme B and perforin at both mRNA and protein level. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulate in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced 2

ACCEPTED MANUSCRIPT tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising

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Staphylococcal

enterotoxins

Granzyme

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B · Perforin · Apoptosis · Cytotoxicity

·

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Keywords

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candidate for antitumor clinic usage in future.

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ACCEPTED MANUSCRIPT Introduction Bacterial superantigen Staphylococcal enterotoxins (SEs) are a class of

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immunostimulatory proteins secreted by Staphylococcusaureus and

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Streptococcus aureus [1]. In contrast to conventional antigens, SEs can bind to the region of major histocompatibility complex class II (MHC-II) molecules on antigen-presenting cells (APCs) outside the peptide groove

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and do not require processing or proteolysis, and then recognize the T-cell

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receptor (TCR) Vβ regions [2]. This trimolecular interaction between the SEs, MHC-II and TCR leads to massive proliferation of T cells.

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Ultimately, activated T cells release massive amounts of inflammatory

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cytokines such as interleukin-1 (IL-1), IL-2, gamma-interferon (IFN-γ)

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and tumor necrosis alpha (TNF-α) [3], which could activate the CTLs and NK cells to produce granzyme B (GzmB) and perforin (PRF1) [4-6]. It is

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well known that PRF1 and granzymes are released from cytotoxic granules to induce target-cell death [7, 8]. At molecular level, the granule exocytosis pathway partly accounts for cytotoxicity delivered by NK and CTLs [9]. Therefore, the characteristics of SEs have been extensively employed in several preclinical studies for cancer therapy [10, 11]. Staphylococcal enterotoxin C2 (SEC2) belongs to SEs family [12]. Our previous study found that SEC2 and its mutants exhibit antitumor effect in vitro and in vivo [13]. Furthermore, SEC2 has been employed in clinic as a promising drug to treat the malignant tumors [14]. In order to 4

ACCEPTED MANUSCRIPT increase the immune-stimulating and tumor-inhibiting activity of SEC2, based on previous reports, we designed and constructed a SEC2 mutant

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ST-4 (GKVTG102-106WWH) in the present study. We determined

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whether ST-4 had an enhanced immunocyte stimulation activity as well as the enhanced antitumor effect. Furthermore, the cellular and molecular consequences after ST-4 treatment were investigated to explore the

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mechanism of this procedure.

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Our results indicated that the amino acids substitution ST-4 contributed to the enhanced stimulation activity and antitumor effect. The enhanced

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antitumor effect was mediated by activated T cells and NK cells via the

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release of GzmB and PRF1 which induced apoptosis of tumor cells by

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mitochondrial apoptotic pathway. Materials and Methods

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Animals and Cell lines

Female wild-type BALB/c mice (6-8 week olds, 19 ± 2 g) were purchased from Chang Sheng Biotechnology Co., Ltd (Shenyang, China). All mice were maintained under specific pathogen-free conditions on a 12 h light-dark cycle, and with free access to autoclaved food and water. Experiments involving mice were approved by the institutional animal care and use guidelines.

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ACCEPTED MANUSCRIPT Mouse hepatoma cell line Hepa1–6 was obtained from American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium

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supplemented with 10% fetal bovine serum (FBS), at 37°C in a 5% CO2

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atmosphere.

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Reagents and antibodies

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Expression vector pET-28a(+) was purchased from Novagen (USA). Primers and peptides were synthesized from Sangon Biotech (Shanghai,

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china). The granzyme B inhibitor Z-AAD-CMK and perforin inhibitor

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Concanamycin A were obtained from Calbiochem (San Diego, CA, USA)

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and ApexBio Technology LLC (Boston, MA, USA), respectively. ELISA kits for granzyme B and perforin were purchased from Elabscience

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Biotechnology Co., Ltd (Wuhan, China). Monoclonal antibodies CD4-PE and CD8-FITC, CD3-APC and CD49b-FITC were from eBioscience and BioLegend (San Diego, CA, USA), respectively. FITC Annexin V apoptosis Detection Kit with PI was from BioLegend (San Diego, CA). SYBR Premix Ex TaqTM Kit, Primescript RT Master Kit and RNA-extracting reagent RNAiso plus were purchased from Takara Biotechnology Co. (Dalian, China). CFDA SE Cell Proliferation Assay and Tracking Kit, Caspase-3 and 9 Activity Assays Kit was from Beyotime (Haimen, Jiangsu, China). Primary antibodies BAX, Bcl-xL, 6

ACCEPTED MANUSCRIPT cytochrome c, GAPDH and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were purchased from (Proteintech Group, Inc., Wuhan,

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china).

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Construction, expression and purification of SEC2 mutant The recombinant expression vector pET-28a-SEC2 was used as the template for constructing mutated SEC2 gene [14]. The residues at

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position 102−106 of SEC2 were substituted for WWH by over-lap PCR.

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Primers were used for site-directed mutagenesis: ST-4R, 5′CAAGTTTTACCATGCCACCATACATTATCTTTGGATG-3′; ST-4F,

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5′-CATCCAAAGATAATGTATGGTGGCATGG-

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TAAAACTTGTATGTATGGAG-3′;

SEC2R,

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5 ' - C G G A A T T C G A G A G T C A A C C A G A - 3 ' ; a n d S E C 2 F, 5'-TCGCTCGAGTTATCCATTCTTTGTTG-3'. The primers were

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designed for amino acid substitution. The sequences of mutant residues are indicated in italics. The restriction enzyme sites for EcoR I and Xho I are underlined. PCR-generated fragments were digested with EcoR I and Xho I and ligated into plasmid pET28a(+) digested with the same en z yme s . Construct ed pl as mid was then t ransfo rme d int o E. coli BL21(DE3) separately and identified by DNA sequence analysis. Mutant protein ST-4 as well as wide type SEC2 were prepared as described earlier [14]. Briefly, transformed E. coli BL21(DE3) were cultured in LB medium. When OD600 reached 0.5-0.8, protein expression 7

ACCEPTED MANUSCRIPT was induced with 1.0 mM isopropyl β-D-thiogalactoside (IPTG) for 4 h at 30°C. Cells were harvested and disrupted by sonication on ice. The cell

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lysate was centrifuged at 12,000 rpm for 30 min. Then the supernatants

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were collected and loaded onto the Ni-saturated chelating sepharose column. After nonspecifically bound host proteins were washed off with washing buffer (50 mM NaH2PO4, 500 mM NaCl, 30 mM imidazole, pH

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7.9), SEC2 or ST-4 bound to the resin specifically was eluted with

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washing buffer containing 250 mM imidazole and dialyzed against phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM

by

sodium

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determined

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Na2HPO4, 1.4 mM KH2PO4, pH 7.4). Relative protein purity was dodecyl

sulphate-polyacrylamide

gel

staining.

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electrophoresis (SDS-PAGE) and Coomassie brilliant blue R-250

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CFSE proliferation assay According to the previous study, the splenocytes of BALB/C mouse had the good sensitivity to SEC2. We selected the BALB/C mouse as model animals. Mice splenocytes were prepared as described previously [11]. For the proliferation assay, freshly isolated mice splenocytes were labeled with

Carboxyfluorescein

diacetate,

succinimidyl

ester

(CFSE)

immediately. Briefly, cells were resuspended in RPMI 1640 with 10% FBS at a final concentration of 5 × 106 cells/mL, and CFSE solution was added for a final working concentration. The cells were incubated at 37°C 8

ACCEPTED MANUSCRIPT for 10 min, and added 10 mL RPMI 1640 with 10% FBS and washed once. Then, staining was fixed by adding 5 mL RPMI 1640 with 10%

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FBS, and the cells were incubated at 37°C for 5 min. Cells were then

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washed once and resuspended in the culture media.

CFSE labeled cells were plated in 96-well plates, stimulated with ST-4 at the concentrations of 100, 1,000 and 10,000 ng/mL for 72 h before the

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proliferation of splenocytes was determined by flow cytometry. RPMI

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1640 medium served as negative control. Cells were incubated at 37°C with 5% CO2 for 72 h. Splenocytes stained with CFSE were analyzed

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(version 7.6.2, Treestar).

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with BD LSRFortessa, and data were analyzed with FlowJo software

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Proliferation assay and flow cytometry The cells were maintained in RPMI 1640 medium supplemented with

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10% (FBS). Splenocytes were stimulated with series concentrations of 100, 1,000 and 10,000 ng/mL SEC2 or ST-4 in 96-well flat-bottomed plates at 1×106 cells/well in 0.2 mL culture medium for proliferation assay, or in 24-well flat-bottomed plates at 5×106 cells/well in 1 mL culture medium for flow cytometry assay. The plates were incubated for 48 or 72 h at 37°C in a humidified atmosphere containing 5% CO2, respectively. After incubation, cell proliferation was determined by MTS assay. Absorbance value was measured with a microplate reader at a test wavelength of 490 nm and a reference wavelength of 620 nm. The 9

ACCEPTED MANUSCRIPT proliferation index of splenocyte (PI

splenic lymphocytes)

was calculated as

follows: Abs value in experimental groups/Abs value in negative control

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groups.

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For flow cytometry assays, surface markers were determined by staining with fluorochrome-conjugated monoclonal antibodies. The panel consisted of PE anti-mouse CD4 for CD4+ T cell, FITC anti- mouse CD8

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for CD8+ T cell, and APC anti-mouse CD3 +/FITC anti-mouse CD49b for

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NK cells. Briefly, the stimulated splenocytes in 24-well plates were centrifuged and washed twice with cold PBS. Then cells (2×105 cells/tube)

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were suspended in 100μL cold PBS containing appropriately diluted

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antibodies according to the manufacture’s instruction and incubated for

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30 min at 37°C in dark. The flow cytometry were performed using BD LSRFortessa, and data were analyzed with FACSDiva software (BD

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Biosciences, San Jose, CA, USA). The proliferation index (PI) of splenic CD4+ and CD8+ T cells were calculated with the equation: (CD4+ or CD8+ T cells % tested) ⁄ (CD4+ or CD8+ T cells % control) × PI splenic lymphocytes. Competition assay The M1 and M2 peptides (with amino acid sequences within the TCR binding domain of SEC2: YFSSKDNVGKVTGGKT and ST-4: YFSSKDNVWWHGKT, respectively) were synthesized by Sangon Biotech (Shanghai, china). The different amino acids of synthetic peptides are indicated in italics. Splenocytes were pretreated with series 10

ACCEPTED MANUSCRIPT concentrations of 100, 1,000 and 10,000 ng/mL M1 or M2 peptides, and then stimulated with 100 ng/mL ST-4 in 96-well plates at 1 × 106

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cells/well in 0.2 mL culture medium for proliferation assay. After

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incubation for72 h, proliferation index was calculated according to above-described methods.

Quantitative real-time PCR (qRT-PCR)

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Splenocytes were harvested at 48 h after simulated with 1,000 ng/mL

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SEC2 or ST-4. Total RNA was isolate using RNA-extracting reagent RNAiso plus, and 0.5 μg of total RNA was reverse-transcribed using

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Primescript RT Master Kit according to the manufacturer’s instructions.

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qRT-PCR was performed on 25μL volume in duplicates using the SYBR

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Premix Ex TaqTM kit with ABI Prism 7000 (Applied Biosystems, Norwalk, CT). The thermal cycling parameters were as follows: initial

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denaturation at 94 °C (30 s), followed by 40 cycles at 94 °C (25 s), 60 °C (45 s) and 72 °C (45 s). Data analysis was performed by comparative Ct method of relative quantification using β-actin as endogenous control. The equation is following: △Ct = Ct(sample) - Ct(endogenous control); △△Ct = △Ct(sample) - △Ct(untreated); and fold change = 2-△△Ct. The primers for TCR Vβ, GzmB, PRF1 and β-actin were used as descried elsewhere [13, 15, 16].

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ACCEPTED MANUSCRIPT Cell growth inhibition assay in vitro Dilutions of SEC2 and ST-4 were added to 96-well plates separately at

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100, 1,000, or 10,000 ng/mL. Splenocytes were used as effector cells

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(5×105 cells/well). Hepa1-6 tumor cells were used as target cell at E:T ratios of 20:1 at least replicate wells [11]. The plates were incubated for 72 h at 37°C in a humidified 5% CO2 atmosphere. The blank wells

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(RPMI 1640 only), unsettled cell control wells (Hepa1–6 cells only), and

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lymphocytes-releasing wells (lymphocytes and proteins) were used as control. The negative control was BSA. For rescue assays, lymphocytes

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were pretreated with 100 µM/mL GzmB inhibitor (Z-AAD-CMK) or 100

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nM/mL PRF1 inhibitor (Concanamycin A) at 37°C for 2 h.

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Tumor cells growth inhibition induced by SEC2 and ST-4 was determined by MTS assay. Absorbance value was measured with a

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microplate reader at a test wavelength of 490 nm. Tumor growth inhibition (%) was calculated with the equation: [1 － (Abs value in protein-treated cells well －

Abs value in lymphocytes-releasing

wells)/(Abs value in unsettled tumor cells control wells － Abs value in blank control wells)] × 100.

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ACCEPTED MANUSCRIPT Transwell co-culture Transwell experiments were performed to determine whether soluble

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factors alone were sufficient to induce Hepa1-6 cell apoptosis. Heap1-6

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cells were plated in 12-well plates. 5 × 106 splenocytes in 300 mL were plated in each transwell chambers in presence of 100, 1,000, or 10,000 ng/mL of SEC2 and ST-4, and 2.5 × 105 Hepa1-6 cells in 1 mL were

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seeded into each well. Chamber inserts were placed on top of the wells.

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The membrane in the transwell chambers had a 0.4-μm pore size that prevents both cell–cell contact and cell migration but allows the diffusion

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of soluble factors. The plates were incubated for 72 h at 37°C in a

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Cytokine assay

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humidified 5% CO2 atmosphere.

After 72 h, the co-culture supernatants were collected, and the

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concentrations of GzmB and PRF1 in the supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kit according to the manufacture’s introductions. Western blotting and caspase-3, 9 activity Total cell lysates were prepared in RIPA buffer. For determining the cytochrome c release, Heap 1-6 cells with or without proteins treatment were harvested and washed once in cold PBS. The cytoplasm fraction and mitochondria fraction were separated using the Cell Mitochondria Isolation Kit (Beyotime) according to the manufacturer's instructions. The 13

ACCEPTED MANUSCRIPT resulting supernatant was used as cytosolic fraction. Equal amounts of protein (30 µg) were subjected to SDS-PAGE and transferred onto PVDF

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membrane (Millipore, Billerica, MA). Membrane was blocked with 5%

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skimmed milk for 1 h and subsequently incubated with specific antibodies. After washing with PBS containing 0.05% Tween-20 (PBST), the membrane was incubated with a HRP conjugated secondary antibody.

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Detection was performed by the enhanced chemical luminescence method

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(Pierce, Rockford, IL).

For caspase-3, 9 activity assay, the cell lysates (30 µg) were used to

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determine caspase-3, 9 activity using Kit according to the manufacture’s

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introductions. The specific caspase activity was normalized for total

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protein and then expressed as fold of the baseline caspase activity of control cells cultured in 1640 with 10% FBS.

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Apoptosis assays

After co-cultured in transwell for 72h, Hepa 1-6 cell apoptosis were determined using FITC Annexin V Apoptosis Detection Kit with PI according to the manufacture’s introductions. The flow cytometry was performed, and data were analyzed as mentioned above. Statistics analysis All data are shown as mean ± SD. Data were statistically analyzed using Student’s t tests. P values < 0.05 were considered to be statistically significant. 14

ACCEPTED MANUSCRIPT Results Mutagenesis, expression and purification of SEC2 mutant

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The residues at position 102−106 of SEC2 were substituted for WWH by

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over-lap PCR as described in materials and methods. The mutant protein ST-4 was expressed by engineered E. coli BL21 (DE3) induced with 1.0 mM IPTG at 30°C for 4h. After purification with Ni-saturated chelating

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purity of more than 95% (Fig. 1).

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sepharose, ST-4 protein shown as a single band on SDS-PAGE were of

Analysis of stimulation activity and TCR Vβ repertoires of ST-4

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To investigate whether ST-4 could induce an enhanced splenocyte

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proliferation, murine splenocytes or splenocytes labeled with CFSE were

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stimulated for 72 h with different concentrations of SEC2 or ST-4. The proliferation then was analyzed by MTS or flow cytometry. ST-4

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significantly induced the splenocyte proliferation in a dose-dependent manner. At each indicated concentration, the stimulation activity of ST-4 was significantly higher than that of SEC2 (P < 0.05, Fig. 2a). As shown in Fig. 2b, the percentage of splenocyte division stimulated by SEC2 was 6.5% (100 ng/mL), 16.1% (1,000 ng/mL) and 20.6% (10,000 ng/mL). ST-4 increased the percentage to 7.5% (100 ng/mL), 18.6% (1,000 ng/mL) and 24.0% (10,000 ng/mL). The stimulation activity of ST-4 showed the consistent results in MTS assay and CFSE assay. Furthermore, the control group only showed a slight proliferation 15

ACCEPTED MANUSCRIPT (4.0%, control group) (Fig. 2b). To determine whether amino acid substitution at position 102-106 of

amino

acids

at

position

94-109

of

SEC2

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the

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SEC2 results in the enhanced contact to T cells, two peptides sequencing

(M1:YFSSKDNVGKVTGGKT) and ST-4 (M2:YFSSKDNVWWHGKT) were synthesized for competitive assay. Splenocytes were pretreated with

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series concentrations of M1 or M2 peptides and then stimulated with 100

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ng/mL ST-4 for 72 h. Both M1 and M2 alone did not show any stimulation activity to splenocytes, but competitively inhibited the

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stimulation activity of ST-4 in a dose-dependent manner (P < 0.05, Fig.

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2c). It was noteworthy that the inhibition effect of M2 (44.75 ± 2.13%, PI:

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from 2.458 to 1.358) to the stimulation activity of ST-4 was higher than that of M1 (35.03 ± 2.26%, PI: from 2.458 to 1.597) at the concentration

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of 10,000 ng/mL, which implied that residues of WWH in ST-4 might lead to stronger contact to TCR than those of GKVTG in SEC2. We further investigated the distribution of TCR Vβ repertoires in splenocytes stimulated with SEC2 and ST-4 by qRT-PCR method. Among 24 TCR Vβ subgroups, Vβ 8.2 and 8.3 were specifically up-regulated by both ST-4 and SEC2. While the amplification levels of both Vβ 8.2 and 8.3 induced by ST-4 were significantly higher than that by SEC2 (P < 0.05, Fig. 2d).These results support the prediction that residues WWH may have higher affinity to TCR than GKVTG, which leads to the 16

ACCEPTED MANUSCRIPT enhanced stimulation activity of ST-4. Enhanced antitumor activity of ST-4 in vitro

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Tumor cell line Hepa1-6 was used as target cell and mixed with effect

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cell murine splenocytes in the presence of ST-4 or SEC2 for MTS assay. Compared with negative control BSA, both ST-4 and SEC2 showed significantly antitumor activities in vitro (P < 0.01) with a

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dose-dependent fashion (Fig. 3). Notably, tumor cell growth inhibition

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induced by ST-4 was markedly higher than that by SEC2 at the same concentration (P < 0.05, Fig. 3).

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Enhanced apoptosis on Hepa1-6 tumor cells by ST-4

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To confirm that the tumor cell growth inhibition was brought about by

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apoptosis, we detected apoptosis in Hepa 1-6 cells by phosphatidylserine externalization using the FITC Annexin V and PI double staining. We that

SEC2-induced

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found

Annexin-positive

and

apoptosis

PI-negative

[including (%)

and

early apoptosis: late

apoptosis:

Annexin-positive and PI-positive (%)] about 21.1% (100 ng/mL), 23.1% (1,000 ng/mL) and 27.8% (10,000 ng/mL). ST-4 increased the apoptosis to 24.8% (100 ng/mL), 27.9% (1,000 ng/mL) and 31.9% (10,000 ng/mL) (Fig. 4). The results of enhanced apoptosis (including early and late) induced by ST-4 were in line with enhanced tumour cell growth inhibition. We speculated that Hepal-6 cell apoptosis induced by ST-4 resulted in tumor cell growth inhibition. 17

ACCEPTED MANUSCRIPT Analysis of splenocyte phenotype stimulated by SEC2 and ST-4 To identify the component of CD4+, CD8+ T cells and NK cells stimulated

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by SEC2 and ST-4, splenocytes stimulated with protein for 48 h were

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used to perform marker antibody staining and flow cytometry. There were significant increased percentage of CD4+ positive events in ST-4 treated cell (36.9 ± 0.43%) compared with SEC2 treated cells (32.8 ± 1.0%) (P <

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0.05, Fig. 5a). However, no significant differences in the percentage of

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CD8+ positive events were observed between the cells treated with SEC2, ST-4 and control group. Then we calculated PI which indirectly evaluated

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the number change of positive CD8+ T cells. The results showed that the

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total amounts of CD8+ T cells in ST-4 and SEC2 treatment were,

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respectively, 3.19 and 4.89 fold that of the control group (Fig. 5b). Furthermore, the numbers of NK cells stimulated with ST-4 were

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significantly increased compared with SEC2, about 1.42 fold (P < 0.05, Fig. 5c).

Critical role of up-regulated GzmB and PRF1 in cytotoxicity induced by ST-4 for Hepa1-6 It has been widely reported that GzmB and PRF1 are critical for inducing cell death to eliminate tumor cell. We further tested the secretion levels of GzmB and PRF1 in co-culture supernatants at 72 h after SEC2 and ST-4 simulation by ELISA. Our results showed that both GzmB and PRF1 were slightly detected in control group, but markedly increased in protein 18

ACCEPTED MANUSCRIPT treated splenocytes in a dose-dependent manner (Fig. 6a). Furthermore, the levels of GzmB and PRF1 in ST-4 treatment were significantly higher

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than that in SEC2 treatment (P < 0.05, Fig. 6a).

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We further examined the levels of GzmB and PRF1 mRNA to determine the induction of gene transcription. The results indicated that mRNA levels of GzmB and PRF1 stimulated with ST-4 were

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significantly up-regulated compared to SEC2 (P < 0.05) with about

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1.9-fold and 2.9-fold, respectively (Fig. 6b). Our data implicate that ST-4 induced increased transcription levels followed by higher rates of protein

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synthesis of both GzmB and PRF1, which could also explain that ST-4

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potentiated apoptosis and growth inhibition on Hepa1-6 cells.

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To further analyze whether GzmB and PRF1 are key factors in killing tumor cells, we performed inhibition assay using GzmB inhibitor I

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Z-AAD-CMK and PRF1 inhibitor concanamycin A. In the Z-AAD-CMK treatment, about 34.0% and 29.1% tumor cells in SEC2 (83.4 ± 9.1%) and ST-4 (59.9 ± 8.2%) group were rescued compared with those without Z-AAD-CMK treatment of SEC2 (49.4 ± 4.2%) and ST-4 (30.8 ± 7.4%). The concanamycin A treatment in SEC2 and ST-4 group obtained the similar results, about 20.8% (70.2 ± 9.0% to 49.4 ± 4.2%) and 22.6% (53.4 ± 7.7% to 30.8 ± 7.4%), respectively (Fig. 6c). These data indicated that GzmB and PRF1 played critical roles in causing Hepa 1-6 cells death. 19

ACCEPTED MANUSCRIPT Analysis of mitochondrial apoptotic pathways induced with GzmB and PRF1

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To further evaluate the roles of GzmB and PRF1 in Hepa1-6 cell

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apoptosis induced by SEC2 and ST-4, key molecules in mitochondrial apoptotic pathways in Hepa1-6 cell were examined. As shown in Fig. 7a, we found that the expression of proapoptotic protein BAX and

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cytochrome c (Cyto C) were down-regulated, but expression of

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antiapoptotic protein Bcl-xL was up-regulated in the presence of the GzmB and PRF1 inhibitor. In agreement with these results, activation of

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caspase-3 and caspase-9, an event that occurs downstream from

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mitochondrial apoptotic pathways, was decreased when the GzmB and

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PRF1 inhibitor presenced (Fig. 7b). These results suggest that the mitochondrial apoptotic pathway is one of the mechanisms by which

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up-regulated GzmB and Prf1 from ST-4-stimulated splenocyte enhanced the Hepa 1-6 cell apoptosis. Discussion

SEs cause tremendous T lymphocytes proliferation, cytokines secretion, and CTL induction [17]. The predominant cytokines produced and released during superantigen activation are the IL-2, IFN-γ, TNF-α, Gzms and PRF1 [18-20], which intimately involved in the cytokine cascade during immune responses and antitumor effects. IFN-γ can activate CD8+ T cells, macrophages, and NK cells so that they become cytolytic [21]. 20

ACCEPTED MANUSCRIPT Thus SEs make an attractive approach to cancer immunotherapy as opposed to the direct killing mechanisms of both chemotherapy and

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radiation therapy [22]. In recent years, fusion protein containing a

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modified form of SEA and an antibody Fab fragment had been applied in the phase II trial for renal cell carcinoma [23]. In China, SEC2 has been employed in clinic as a promising drug to treat the malignant tumors [14].

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In order to further improve the immunostimulating and antitumor activity

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of SEC2 for clinical usage, one of the effective strategies is to optimize the SEC2 structure for better recognition to TCR. A mutant SEB (V26Y)

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with improved affinity for the Vβ 14.3.d chain exhibited significantly

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increased ability to simulate T cells [24]. Another reports showed that

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SEC3 variants were generated with up to a 150-fold increased affinity in TCR Vβ 8.2 by mutating five residues (GKVTG102-106WWH) in a

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flexible loop structure which are highly conserved among all three SEC subtypes (SEC1, SEC2, and SEC3) [25]. So we presumed that mutation on these five residues (GKVTG102-106) in SEC2 could increase the affinity to TCR followed by enhanced stimulation activity. Here, we show that the amino acids at position 102-106 of SEC2 play an essential role in stimulation activity, and up-regulation of GzmB and PRF1 induced the enhanced antitumor activity in a dose-dependent manner. In the present study, the residues at position 102−106 of SEC2 were substituted for WWH by over-lap PCR. We obtained an SEC2 mutant 21

ACCEPTED MANUSCRIPT named ST-4 which exhibited enhanced stimulation activity. The crystal structure of SEC2 had predicted that amino acids within this area

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implicated in the TCR interaction and crystal-packing contact [26]. Thus

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it is possible that change of the amino acids may increase the affinity of SEC2 to TCR, and elicits enhanced stimulation activity. Previous study had validated the speculation that increasing affinity of SEC3

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variants/TCR interaction caused a proportional increase in the ability of

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SEC3 to activate T cells [25]. These mutants affected the ability to form the trimolecular TCR-SEs-MHC II complex. Additionally, our results

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showed that expression of both Vβ 8.2 and 8.3 subgroups in T cells

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treated with ST-4 was significantly increased compared with those treated

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with SEC2, 1.46 and 2.97 fold, respectively. Results from qRT-PCR assays were generally consistent with previous reports with minor

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variation [25], and these discrepancies might be explained by the differences in the response to SEC2 among mice, techniques or dosage [27]. Moreover, the M2 peptide could inhibit the stimulation activity of ST-4 than the M1 peptide at the concentration of 10,000 ng/mL. This results therefore suggest that the substitution of amino acids at position 102-106 of SEC2 could offer a possibility to form hydrogen bond with the critical residues of TCR binding. Alternately, the conformational changes in SEC2 induced by the mutation may introduce another new contact with TCR and increase the TCR binding affinity of SEC2. 22

ACCEPTED MANUSCRIPT We observed that ST-4 induced enhanced cell apoptosis and exhibited markedly enhanced antitumor activity. T-cell stimulating potency by

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acting 1.4 fold CD4+, 1.6 fold CD8+ T cells and 1.4 fold NK cells,

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respectively, compared with SEC2 group. Previous studies indicated that C215Fab-SEA in the B 16 tumor model has established that mainly CD8+ T cells contribute to tumor therapy by tumoricidal cytokines in vivo [28].

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Whereafter, CD4+ and CD8+ T cells knockout assays found that CD4+ T

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cells were essential for infiltration into the tumor area, whereas CD8+ T cells were shown to perform effector functions [29]. Fehnigeret al.

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determined that NK cells activated by IL-15 required Gzmb and Prf1 for

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potent cytotoxicity in vitro [6]. Accordingly, we speculate that potentiated

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antitumor effect of ST-4 was caused by up-regulated GzmB and PRF1 which were secreted by the massive activated T cells and NK cells.

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Compared with SEC2, there was markedly up-regulated GzmB and PRF1 from ST-4-stimulated lymphocytes. The levels of GzmB and PRF1 mRNA are in-line with above results. Accordingly, the up-regulated GzmB and PRF1 may play a critical role in enhanced antitumor effect of ST-4. To further verify this hypothesis, we used GzmB inhibitor Z-AAD-CMK and PRF1 inhibitor concanamycin A to block this procedure and evaluate the interference to antitumor effect of ST-4. We observed that antitumor effect of ST-4 was significantly decreased in the presence of inhibitor GzmB or PRF1. 23

ACCEPTED MANUSCRIPT Previous results showed that a major pathway of cell-mediated cytotoxicity involves targeted release of perforin from cytoplasmic

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storage. Perforin-mediated killing is rapid and may eliminate certain

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tumor cells. In vivo studies in perforin KO mice have clearly demonstrated that perforin-mediated cytotoxicity plays a prominent role in

controlling

the

growth

of

fibrosarcoma

tumors

[9,

30].

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Perforin-deficient mice have normal numbers of CD8+ T cells and NK

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cells. However, these mice fail to mediate CD8+ T cell-dependent cytotoxicity against allogeneic fibroblasts tumor cells, and NK

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cell-mediated cytotoxicity against the YAC-1 target cells [9, 31].

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Although the apoptotic mechanisms induced by GzmB or/and PRF1 are

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not fully understood [32], these data strongly indicate that activated murine CD8+ T cells and NK cells require GzmB and PRF1 to acquire

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potent cytotoxicity against tumor targets in vitro [6]. Using Western blotting, we found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulate in the treatment with either ST-4 or SEC2. However, this tendency could be totally reversed by inhibitor of either GzmB or PRF1, which suggesting that mitochondrial events are also required to engage the apoptotic program. Previous study indicated that GzmB can promote activation of several members of the caspase family of cysteine proteases through 24

ACCEPTED MANUSCRIPT proteolytic processing of these proteins, and GzmB internalization into target cells requires perforin-mediated membrane pore formation [33-35].

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Others demonstrated that GzmB can also promote caspase activation

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indirectly through proteolysis of the Bcl-2 family protein Bid and BAX [36], and results in the release of mitochondrial cytochrome c into the cytosol [37]. Cytochrome c efflux from mitochondria then leads to the

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engagement of the apoptosome pathway and ultimately to programmed

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cell death [38].

Although in presence of GzmB or PRF1 inhibitor, growth of Hepa 1-6

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cell was only partially rescued, which indicated that besides GzmB and

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PRF1, there may be other cytokines that contribute to the antitumor

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activity as observed in previous reports and in the present study [16, 39-41]. We speculate that production of IFN-γ and TNF-α from

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splenocytes simulated by ST-4 maybe another factor in determining Hepa1-6 suppression [40]. The hypothesis could be supported by the previous study that addition of recombinant TNF-α to WiDr colon-carcinoma cells showed acts synergistically with IFN-γ to suppress tumor-cell growth [41]. In conclusion, enhanced immunostimulating activity of ST-4 accompanied by enhanced tumor growth inhibition. We provide evidences that the expression of TCR Vβ and competitive assay revealed that the substitution of residues 102-106 could increase the ability of TCR 25

ACCEPTED MANUSCRIPT binding and activated more CD8+ T cells and NK cells, which up-regulated the production of GzmB and PRF1. These cytotoxic

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granules could markedly enhance apoptosis by mitochondrial apoptotic

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pathway in Hepa1-6 cells. Accordingly, the ST-4 might be a good candidate in cancer immunotherapy and provide a new strategy for the clinic application of staphylococcus superantigens.

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Conflicts of interest

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The authors declare no financial or commercial conflict of interest. Acknowledgments

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This research was supported by grant from National Science &

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Technology Major Specific Projects of China for ‘Significant Creation of

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New Drugs’ (2012ZX09102301-013) and Natural Science Foundation of

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Liaoning Province (2015020693).

26

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Purity analysis of SEC2 and ST-4 by 15% SDS-PAGE. Lanes: (1)

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wild-type SEC2; (2) ST-4; (3) molecular size standard. Staining was

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performed with Coomassie Brilliant Blue

Fig. 2. Changes of stimulation activity and TCR Vβ repertoires of ST-4. a Analysis of splenocytes proliferation. Proliferation of murine splenocytes

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was determined by MTS after incubation for 72 h with SEC2 or ST-4 at

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the concentrations of 100, 1,000 and 10,000 ng/mL. b CFSE-labeled splenocytes were stimulated with ST-4 at the indicated concentrations

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before the proliferation of splenocytes was determined by flow cytometry.

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CFSE-labeled splenocytes without ST-4 were used as control. The data

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represent the percentage of the division cells and are the representative of three independent experiments c Competitive inhibition of M1 and M2

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peptides on stimulation activity of ST-4. Murine splenocytes were pretreated with indicated series concentrations of M1 or M2 peptides and then stimulated with 100 ng/mL ST-4, respectively. After 72 h incubation, splenocyte proliferation was determined by MTS assay. d Distribution of Vβ repertoires. Total RNA was collected and the levels of Vβ were identified by qRT-PCR after murine splenocytes had been stimulated with 1,000 ng/mL SEC2 or ST-4 for 48 h. The values are shown as fold-change of SEC2 or ST-4-induced mRNA compared to control group, utilizing the comparative Ct method, with β-actin used as endogenous control. Results 35

ACCEPTED MANUSCRIPT (mean ± SD) shown are from at least three independent experiments. The horizontal solid line indicates the control value 1. *P < 0.05, **P < 0.01.

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Fig. 3. Effects of SEC2 and ST-4 on inducing Hepa1-6 cells growth

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inhibition detected by MTS assay. Murine splenocytes were used as effector cells against Hepa1-6 target cells at E:T ratios of 20:1. The mixed cells were stimulated with ST-4 or SEC2 in indicated concentration and

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incubated for 72 h. BSA was used as the negative control. Results (mean

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± SD) shown are from one experiment representative of at least three independent experiments. *P < 0.05.

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Fig. 4. Enhanced apoptosis in Hepa1-6 cells induced by ST-4. Detection

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of apoptotic cells by AnnexinV and PI double staining. Hepa 1-6 cells

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were co-cultured with murine splenocytes and ST-4 or SEC2 in transwell for 72 h, and then harvested and stained with Annexin V and PI-labeling

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before analyzed by flow cytometry. A minimum of 104 events were acquired. Results from an individual experiment are shown and are representative of at least three independent experiments. Numbers indicate the percentage of cells in each quadrant Fig. 5. ST-4 increased the percentages of CD4+, CD8+ T cells, and NK cells. a, c The percentages of activited CD4+, CD8+ T cells, and CD3CD49b+ NK cells are shown on the flow cytometry plots. The resting and activated NK cells were gated on P2. Splenocytes were seeded at 5×106 cells/well in a 24-well plate and incubated with SEC2 or ST-4 at 36

ACCEPTED MANUSCRIPT concentration of 1,000 ng/mL. After 48 h, cells were harvested and stained for flow cytometry analysis. One representative out of three

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experiments is shown. Numbers indicate the percentage of cells in each

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quadrant. b The proliferation index (PI) of splenic CD4+ and CD8+ T cells. We calculated the PI which indirectly evaluated the number change of positive CD8+ T cells. Results (mean ± SD) shown are from one

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representative experiment of triplicate samples in three separate

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experiments

Fig. 6. Cytotoxic effects of up-regulated GzmB and PRF1 on Hepa1-6

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tumor cells in vitro. a After incubation for 72 h, the co-cultured

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supernatants were harvested and used for the determination of granzyme

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B (GzmB) and perforin (PRF1) by ELISA. b Murine splenocytes were treated with SEC2 or ST-4 (1,000 ng/mL). After incubation for 48 h, total

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mRNA was extracted and the transcription levels of GzmB and PRF1 were identified by qRT-PCR. All results (mean ± SD) shown are from three independent experiments. c The pre-treatment of murine splenocytes with 100 µM/mL of GzmB inhibitor I Z-AAD-CMK or 100 nM/mL PRF1 inhibitor concanamycin A for 2 h were co-cultured with Hepa 1-6 tumor cells in presence of SEC2 or ST-4 (1,000 ng/mL) for 72 h. Results (mean ± SD) shown are from one representative experiment of three independent experiments. *P < 0.05.

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ACCEPTED MANUSCRIPT Fig. 7. Analysis of GzmB and PRF1 effect on mitochondrial apoptotic pathway. a Western blotting analysis the expression of Bcl-xL, Bax and

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cytochrome c (Cyto C). Whole-cell extracts were prepared and subjected

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to Western blotting. The same blots were stripped and reprobed with GAPDH antibody to verify equal protein loading. b Caspase-3 and caspase-9 activation were determined by Activity Assays Kit. Results

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(mean ± SD) shown are from one experiment representative of at least

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three independent experiments. *P < 0.05.

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ACCEPTED MANUSCRIPT

Highlights

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 We obtained a SEC2 mutant ST-4 with enhanced superantigen and

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antitumor activity.

 Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation.

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 Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced

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tumor cells apoptosis.

 Enhanced tumor cells apoptosis induced by ST-4 via mitochondrial

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apoptotic pathway.

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