Immune reaction characteristics and the mechanism of anergy induced by recombinant enterotoxin a of Staphylococcus aureus

doi:10.4236/jbise.2010.36076

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J. Biomedical Science and Engineering, 2010, 3, 543-549

doi:10.4236/jbise.2010.36076 Published Online June 2010 (http://www.SciRP.org/journal/jbise/

JBiSE

Published Online June 2010 in SciRes. http://www.scirp.org/journal/jbise

Immune reaction characteristics and the mechanism of anergy

induced by recombinant enterotoxin a of Staphylococcus aureus

Shang Wu1,2*, Renli Zhang1*, Dana Huang1, Yijie Geng1, Shitong Gao1, Xiaoheng Li1 Zhangli Hu2**

1Shenzhen Centre for Diseases Control and Prevention, Shenzhen, China; *They contributed equally to this work;

2College of Life Science, Shenzhen University, Shenzhen, China; **Corresponding Author.

Email: [email protected]

Received 30 November 2009; revised 5 January 2010; accepted 15 January 2010.

ABSTRACT

To study immune reactions and the mechanism of

anergy induced by recombinant enterotoxin A (rSEA)

of Staphylococcus aureus. The gene encoding SEA

was cloned from standard strain of S. aureus and

high efficiently expressed in E. coli. After immuniza-

tion with purified rSEA, mice were examined for

production of specific antibody, subtype of IgG, cyto-

kine mRNA levels such as IFN-γ, IL-2 secretion and

T-cell surface PD-1 expression. Results showed that

high levels of specific antibodies were produced in

two weeks of primary immunization shot. During this

time, humoral immune reactions prevailed (IgG2a/

IgG1 < 1). During the early phase, Th1 type cytokine

mRNA is expressed at a higher level than Th2 type,

indicating cellular immune reaction prevailed. Splen-

ocyte IFN-γ secretion was significantly decreased af-

ter boosting immunization. The PD-1 expression was

detected by a flow cytometry examination in the sur-

face of T- lymphocytes which were induced by rSEA,

and the expression of PD-1 molecules increased along

with the number of boosting and the time after im-

munization.

Keywords: Staphylococcus Aureus; Enterotoxin A; Im-

mune Respose

1. INTRODUCTION

Superantigens are distinguished from ordinary antigens

by the ability that activates multiple T-cell clones. A very

small amount can very effectively initiate the activation

of immune system. Thus, superantigens have been con-

sidered to be widely applicable in tumor immune therapy.

Their efficacy has also been considered superior to ex-

ogenous cell factor. With the development of relevant

theory, superantigens have attracted much attention of

studies [1,2]. Earlier studies applied superantigens inde-

pendently in anticancer therapy. Later, they were used

after modification with targeted monoclonal antibody, or

as an enhancer of tumor vaccine, or gene vaccine or in

combination with other therapy. In the United States, a

superantigen Fab-SEA has been tested for anti-cancer

ability in phase I clinical studies. In China, superantigens

have been used to promoted leukocytes [3,4].

Most currently known exogenous superantigens are

toxins from bacteria. For example, the antigen studied in

the currently report, enterotoxin A of Staphylococcus

aureus, is one of these toxins. Superantigens that pro-

duced by bacteria upon infection may cause shock, fever,

dehydration, skin eruption, organ failure, even death.

The pathogenesis is that toxin superantigens stimulate

large number of T-cells to proliferate, induce the secre-

tion of cytokines from T-cell and APC cell, which result

in immune system disorder. Superantigens induced dis-

ease may occur through several ways: 1) Superantigen is

the directly cause of the disease, such as toxic shock and

food intoxication. 2) Superantigen enhances the effect of

the other infectious factors. 3) Superantigen induces

autoimmune reaction by activating large number of T

and B cells [5]. Up to date, there is no proof on the rela-

tionship between superantigen and some disease. How-

ever, the effects of superantigens in food intoxication,

toxic shock and some infectious disease are very clear

[6]. An understanding of the mechanism by which bacte-

rial superantigens activate T-cell and pathogenesis is

theoretical bases for the application of superantigens in

cancer immunotherapy. To this end, we studied the hu-

moral and cellular immune reactions to recombinant

SEA protein (rSEA), explored the relationship between

inhibitory lymphocyte receptors and anergy induced by

rSEA.

2. EXPERIMENTS

2.1. Materials and Methods

2.1.1. Materials

Staphylococcus aureus was provided by Shenzhen Cen-

544 S. Wu et al. / J. Biomedical Science and Engineering 3 (2010) 543-549

tre for Diseases Control and Prevention. Two strains of E.

coli, DH5α, BL21(DE3) and pET-28a were the collec-

tion in the author’s laboratory. Cloning vector pGEM-T

easy was purchased from TaKaRa.

Antibody FITC-CD3, PE-PD-1 and corresponding

negative control were purchased from BD Company.

ELISA kit was purchased from Shenzhen Biotech Lim-

ited. Trizol and reverse transcription kit were pur-

chased from TaKaRa Bio Inc. 1640 media, Lym-

pho-Spot TM serum-free media and mice IFN-γ

ELISPOT kit were purchased from Dakewei Biotech

Ltd. One hundred and eight BALB/c mice were pur-

chased from Guangdong Center of Experimental Ani-

mal, maintained according to standard clean protocol.

The mice used in the experiment were 15-20 g of body

weight, 6-8 weeks of age.

2.1.2. Preparation of rSEA

Primers were derived from GenBank SEA sequence

(AY827552): sea F: 5’GCC GCT AGC ATG AAA AAA

ACA GCA TTT ACA TTA C 3’ (underlined is NheI di-

gestion site); sea R: 5’CGC CGT CGA CTT AAC TTG

TAT ATA AAT ATA TAT CAA 3’ (underlined is Sal I

digestion site).

DNA template was from SEA producing standard

strain. PCR product was prepared with sea F and sea R

primers, separated with 1.5% agarose gel electrophoresis.

After purification, the PCR product was inserted in

pGEM-T easy vector. The recombinant plasmid was

propagated in DH 5α, selected with ampicillin on LB

plate.

An expression vector of SEA was constructed by sub-

cloning of the gene in pET-28a plasmid with E. coli

BL21 (DE3) host. A recombinant clone was cultured in

shaking incubator and induced by IPTG (final concen-

tration 1 mmol/L) for 6 hours. Bacteria were centrifuged,

lysed with ultrasound. After centrifugation, rSEA was in

the pellet. Subsequently, the pellet was resuspended in

8.0 mol/L urea, and purified by Ni2+ affinity chromatog-

raphy. The elution solution was imidazole (500 mmol/L).

The rSEA protein was refolded and further purified to a

high purity.

2.1.3. IgG Level Determination by ELISA Assay

Twenty BALB/c mice were assigned to either experi-

ment group or control group at random. Lyophilized

rSEA was resuspended in PBS and diluted to targeted

concentration, mixed with equal volume of adjuvant,

and injected subcutaneously into mice. The first injec-

tion was 100 µg, the second and third injection was 50

µg each at 2 weeks interval. Tail blood was collected

each week before and after injection for 8 continuous

weeks.

rSEA protein was diluted in embedding buffer to

10 µg/mL, aliquoted to multi-well plate (100 mL/well).

The plate was incubated at 4℃ overnight, and then

washed with PBST (PBST containing 0.05% Tween-200)

three times, blocked with blocking solution for 1 h at

37℃. Subsequently, serum from each experiment groups

were diluted 1:1000 in blocking solution, added to the

plate, incubated at 37℃ for 1 hour. The plated was then

washed with PBST for 3 times. HRP conjugated Rabbit

anti-mouse IgG (1:5000) was added to the plate and in-

cubated for 1 hour at 37℃, washed with PBST three

times. Finally, diaminobenzene substrate solution con-

taining hydrogen peroxide was added to the plate and

allowed to develop for 10 min in dark. The reaction was

stopped by addition of 2 mol/L H2SO4. Absorbance was

determined with spectrometry at 450 nm.

2.1.4. Subtype Antibody Level Determination with

ELISA

Serum antibody subtypes were determined at 3, 5 and 7

weeks after immunization. The method was the same as

described above, except the secondary antibody was

replaced by HRP conjugated mice IgG1 (1:1000) and

mouse IgG2a (100 µL/mL).

2.1.5. RT-PCR Analysis of Spleen Cytokine

Expression

Sixty BALB/c mice were assigned to two groups and

immunized with rSEA. Splenocytes were collected

from 5 mice in each group at 0 h, 2 h, 12 h, 24 h, 2 w

and 3 w post immunizations. Spleen total RNA was

extracted with Trizol according to manufacturer’s in-

struction. The mRNA levels of IL-2, IL-4 were deter-

mined with RT-PCR using specific primers. IL-2 prim-

ers were 5’- CTT CAA GCT CCA CTT CAA GCT-3’

(forward) and 5’-CCA TCT CCT CAG AAA GTC CAC

C-3’ (reverse). The amplicon was 198 bp. IL-4 primers

were 5’-CAT CGG CAT TTT GAA CGA GGT CA-3’

(forward) and 5’-CTT ATC GAT GAA TCC AGG CAT

CG-3’ (reverse). The IL-4 amplicon was 203 bp. The

expression of β-actin was used as internal control. The

primers for β-actin were 5’-CAT CCG TAA AGA CCT

CTA TGC CAA C-3’ (forward) and 5’-ATG GAG CCA

CCG ATC CAC A-3’ (reverse). The β-actin amplicon

was 238 bp. The thermal cycles of PCR were: pre de-

naturation at 94℃ for 5 min, 30 cycles of amplification

(94℃ 30 s, 55℃ 30 s and 72℃ for 1 min). RT-PCR

product was analyzed by 1% agarose gel electrophore-

sis [7].

2.1.6. Relative Quantification of Cytokine Expression

Gel image was scanned with UVI system, and bands

were quantified with UV band software. Band intensities

were determined using β-actin as internal control. The

relative expression levels (Ling, R.Y., et al.,) of cyto-

kines were determined by:

Relative expression level = (test gene band inten-

S. Wu et al. / J. Biomedical Science and Engineering 3 (2010) 543-549 545

sity)/(β-actin band intensity)×100%.

2.1.7. IFN-γ Detection by ELISPOT Assay

Twenty four mice were assigned to either control group,

or single immunization group or boost group (n = 8),

injected intraperitoneally with rSEA (100 µg/animal) or

PBS (control group). Mice were sacrificed by cervical

dislocation 24 h after the last injection, and splenocytes

were isolated with sterile procedure.

Lymphocyte preparation: spleen tissue was pressed

against 200 micron mesh, filtered and spun. The second

layer low density cells were collected and washed with

1640 medium. Cells were resuspended in Lympho-Spot

TM serum-free medium. Cell concentration was adjusted

to 2 × 106/mL and examined with trypan blue exclusion

assay. Cell viability was greater than 95%.

IFN-γ detection with ELISPOT pre-embedded kit: 1)

plates were seeded with splenocytes (1 × 105 cells/well),

which were stimulated with 5 µg/mL rSEA antigen.

Each sample was done with triplicates. ConA (5 µg/mL)

or medium was added to the positive or negative control

wells respectively. Cells were cultured at 37℃ for 36 h.

After wash, biotinated anti mouse IFN-γ was added to

the wells (100 µL/well), and the plate was incubated for

1 h at room temperature. 2) After washed, streptoavidin-

HRP was added to each well (100 µL/well) and incu-

bated for 1 h at room temperature. 3) Upon wash, HRP

substrate AEC was added to the plate. Color was devel-

oped in dark. The plate was washed with water and dried.

4) Spots were counted. The unit was defined as spots/105

spleen cells. Negative control has less than 10 spots/105

splenocytes. Test wells that had greater than 2-fold the

spot number of the negative control well was counted as

positive.

2.1.8. PD-1 Expression Determination by Flow

Cytometry

Sixty mice were assigned to either single immuniza-

tion group or boost group or control group (n = 20) and

injected with rSEA or PBS. Animals were sacrificed at

2 h or 24 h post last immunization. Splenocytes were

isolated as described above. Cells were stained with

FITC- conjugated anti-CD3 and PE-labeled PD-1. Af-

ter fixation, cells were examined with flow cytometer.

Cell concentration was adjusted to 2 × 106 cells/mL.

500 µL of the cell suspension was loaded to each of

two flow tubes. FITC Anti-Mouse CD3 (5 µL), PE

Anti-Mouse PD-1 (5 µL) or equal volume of respec-

tive control solution was added to the tubes. After mix

by shaking, the tubes were set in dark for 45 min at

room temperature. Cells were spun, washed with PBS

once and resuspended in 200 µL PBS, fixed with 500

µL paraformaldehyde (4%) and examined with flow

cytometry.

M 1 2

100

800

Figure 1. Amplification of SEA gene from Staphylococcus

aureus genomic DNA. M was 100 bp DNA Ladder, lane 1, 2

was PCR product of SEA gene.

2.1.9. Statistical Analysis

Data were analyzed with SPSS 11.5 software. Results

were presented as mean ±s. Comparisons between

groups were analyzed with ANOVA. Statistical signifi-

cances were inferred when p < 0.05 or p < 0.01.

3. RESULTS AND DISCUSSION

3.1. Cloning of SEA Gene

With the specific primers, we identified 2 strains, out of

10 wild type S. aureus, to be positive for producing en-

terotoxin A (Figure 1). The PCR product was inserted

into pGEM-T easy vector and propagated in DH5α. The

plasmid insert was sequenced, which confirmed that the

insert sequence was identical to AY827552. The size was

786 bp encoding 261 residues.

3.2. Expression, Purification and Refolding of rSEA

The recombinant plasmid pET-28-SEA was transformed

into E. coli BL21 cells and the transformants were in-

duced with IPTG for 6 h. Bacterial cells were disrupted

with ultrasound. Upon centrifugation, the recombinant

protein was identified in the precipitate, demonstrating

that rSEA was expressed as inclusion body. During Ni2+

affinity chromatography, rSEA was eluted at 500 mmol/L

imidazole. After purification and refolding, high purity

rSEA was obtained (Figure 2(a)).

Immunobloting analysis showed that the refolded SEA

was reactive with specific polyclonal antibody and the

molecular weight was as expected 31 kD. This result

demonstrated that the recombinant SEA has similar an-

tigenicity as the natural one (Figure 2(b)).

3.3. Humoral Immunity Induced by rSEA

In order to study the process of humoral immunity in-

duced by SEA, we examined specific IgG production in

sera from mice immunized three times with conventional

546 S. Wu et al. / J. Biomedical Science and Engineering 3 (2010) 543-549

M 1 2

M 1 2 3

kDa

116.0

66.2

45.0

35.0

25.0

18.4

(a) (b)

Figure 2. (a) SDS-PAGE analysis of recombinant SEA(rSEA)

preparation by affinity chromatography. M was molecular

weight markers, lane 1 was cell extract from pET28a-rSEA

transformed E.coli, Lane 2,3 was recombinant SEA of affinity

chromatography; (b) Western-blot analysis of purified rSEA

protein. M was molecular weight markers, lane 1 was negative

sera act as control sera, lane 2 was anti-rSEA sera act as first

antibody.

protocol. Results showed that specific antibody level in

the immunized mice (OD450 = 2.492 ± 0.082) was mark-

edly higher than that in the control mice (OD450 = 0.054

± 0.032) three weeks after the first immune shot (P <

0.01). There were no significant changes in antibody

levels before and after the second (week 3, 4) and third

shots (week 5, 6) in the immunized mice (P > 0.05). Two

weeks after the last shot, the antibody level was slightly

reduced, but not significantly (P > 0.05).

To evaluate the immunogenicity of rSEA as a candi-

date of cancer therapeutics, we assessed specific serum

antibody in immunized mice, investigated the process of

rSEA induced humoral immunization. The key steps of

humoral immunization are the activation and prolifera-

tion of B-lymphocytes, which require the stimulation of

secreted factor or exogenous antigen and the assistance

of CD4+ T cells. We performed ELISA analysis on the

mouse sera collected at different time point, discovered

that specific immunoreactions were strongly induced

within two weeks of immunization. (The immunized

animals produced significantly higher antibody levels

than the control animals and) the high antibody levels

were maintained for a long time. Compared with a single

shot immunization, boost immunization did not increase

rSEA specific antibody levels (Specific IgG induced by

rSEA can be used to destroy cancer cells through the

activation of complement and superantigen-dependent

cell-mediated cytotoxicity) [7].

3.4. IgG Subtype Induced by rSEA

Changes in specific IgG subtype levels were shown in

Figure 3. IgG1 levels were significantly different between

control group (C group) and test group (T group) three

weeks after immunization (P < 0.05). The differences

were even more pronounced 5 and 7 weeks after immuni-

zation (P < 0.01). IgG2a levels were different between the

two groups only at three weeks after immunization (P <

0.05). In the test group, specific IgG1 levels continuously

increased along with time and the number of immune

shots (Figure 3(a)). In contrast, IgG2a levels decreased

during the same period (Figure 3(b)). The ratio of

IgG2a/IgG1 did not change in the control mice at 3, 5 and 7

weeks (data not shown). However, this ratio showed a

trend of decrease in the test group (Figure 3(c)), which

(a)

(b)

(c)

Figure 3. Specific IgG subtypes induced by rSEA from mice

immunized. (a) IgG1; (b) IgG2a; (c) IgG2a/IgG1

S. Wu et al. / J. Biomedical Science and Engineering 3 (2010) 543-549 547

was significantly different from that of control group at

5 and 7 weeks (P < 0.05).

IgG1 (Th2) and IgG2a (Thl) are type markers of im-

munoreactions. The ratio of IgG2a/IgG1 indicates whether

the humoral immunoreactions are dominated by Th1 or

Th2. In the current study, we selected peripheral serum

from mice two weeks after immunization, assessed lev-

els of IgG1 and IgG2a with ELISA. Results showed that

IgG1 level was higher than IgG2a level after initial im-

munization, and that IgG1 exhibited a trend of increase

along with time and the increase in the number of shots

(Figure 4(a)), while IgG2a level showed a trend of de-

crease (Figure 4(b)). Thus, the ratio of IgG2a/IgG1was

always smaller than 1, and exhibited a trend of decline

(Figure 4(c)). These data indicated that rSEA-induced

immunity was dominated by Th2 reaction and the domi-

nance tends to be enhanced by boost shots.

3.5. Effects of rSEA on Splenocyte Cytokine

mRNA Levels

Messenger RNA levels of IFN-γ, IL-4 and β-actin in

(a)

-0.2

0.2

0.4

0.6

0.8

1.2

0h2h12h 24h 48h

Ti me

mRNA Relative Amount

IFN- γ

IL- 4

(b)

Figure 4. (a) RT-PCR results of cytokine in spleen of immu-

nized mice with rSEA, PBS acted as control of immunization,

and fragment of -action was as a RT-PCR control; (b) Analy-

sis of Cytokine mRNA expression in spleens of immunized

mice at different time.

splenocytes were analyzed with RT-PCR. The PCR

products were the expected sizes 198 bp, 203 bp and

238 bp (Figure 4(a)). Before immunization (0 h), IFN-γ,

IL-4 mRNA was not detectable. After immunization,

mRNA levels increased. Levels of mRNA in immunized

groups were significantly higher than that in PBS group

at 2, 12 and 24 h (P < 0.01). There was no significant

difference between the two groups in the mRNA levels

24 h after immunization. At 2 and 12 h after immuniza-

tion, IFN-γ mRNA levels were higher than IL-4 in the

immunized grouping (P < 0.01). In contrast, at 24 h after

immunization, IL-4 mRNA level was higher than IFN-γ

mRNA (P < 0.05) (Figure 4(b)).

We investigated mRNA levels of two cytokines after

immunization with semi-quantitative RT-PCR. Results

showed that both cytokines were greatly increased after

induction with rSEA. At 2 h post immunization, IL-2

mRNA was higher than IL-4 mRNA level and reduced

soon after. In contrast, IL-4 mRNA was low at 2 h after

immunization and gradually increased, peaked at 12 h

and then gradually decreased. Therefore, during immune

reaction, Th1 type cytokine proliferated earlier than Th2

type cytokine, but the level rapidly reduces to become

lower than Th2 cytokine level. This kinetic process is

consistent with the change in serum IgG subtype [8,9].

Microphages (MΦ) are very important immune cells

with unique anti-cancer effect. They provide immune

surveillance, antigen presentation and effector functions.

Through antigen presentation, MΦ activates T-cell and

enhances specific anti-cancer immunity. MΦ also non-

specifically destroys cancer cell upon contact. Activated

MΦ releases many cytokines and bioactive factors that

regulate cancer immunity [10]. IFN-γ secreted by T cells

and NK cells is the most potent MΦ activator. However,

IFN-γ production during tumor genesis is insufficient,

leading to insufficient MΦ activation. Superantigens

have powerful immune activation ability that can induce

the release of large amount of cytokines. This point has

been proven by previous experiments [11,12]. To further

study the relationship between rSEA and IFN-γ secretion,

we analyzed splenocyte IFN-γ secretion in immunized

mice with ELISPOT technique. Our results showed that

splenocyte can secret IFN-γ at high frequency after pri-

mary immunization, (which is significantly different

from the control splenocytes). This cellular immune re-

action is rSEA specific, because splenocyte stimulated

by other antigens produced little IFN-γ. Boost shot did

not enhance antibody production (P > 0.05), suggesting

rSEA as a superantigen may induce anergy after primary

immunization [13].

3.6. Effects of rSEA on Splenocyte IFN-γ

To further explore immunoreactions to rSEA, we as sessed

548 S. Wu et al. / J. Biomedical Science and Engineering 3 (2010) 543-549

changes in IFN-γ secretion by splenocytes with ELIS-

POT assay. As shown in Figure 5, little specific IFN-γ

were produced without stimulators. In contrast, IFN-γ

was produced in the three groups of mice stimulated

with rSEA or ConA. Mice in single shot group and

boosted group produced more IFN-γ than that in control

group (P < 0.01). There were no differences in IFN-γ

levels between the single shot group and the boost group

(P > 0.05).

tion of immune reactions. The mechanism has been

widely investigated. Studies have shown that interaction

between PD-1 and its complementary PD-L leads to the

inhibition of T-cell proliferation and the attenuation of

IL-2, -10 and IFN-γ secretion. This mechanism is very

important for organ transplant, autoimmune disease and

cancer immunity. An examination of splenocyte IFN-γ

secretion with ELISPOT assay revealed that boost shot

did not enhance cellular immunity, suggesting possibility

of anergy after rSEA immunization.

Regulation of immunity includes initiation and termi-

nation of immune reactions, dependent of internal and

external signal. External signal is transmitted through

surface receptors. There are stimulatory and inhibitory

receptors that positively or negatively regulate cell acti-

vation. Inhibitory receptors of the B7 family include

cytotoxic T lymphocyte associated antigen 4 (CTLA-4),

programmed death-1 (PD-1) and B and T lymphocyte

attenuator (BTLA) [14]. These three inhibitory receptors

belong to the CD28 family. Upon binding with different

members of B7 family, these receptors can inhibit activa-

3.7. T-Cell PD-1 Expression Induced by rSEA

PD-1 expression in T-cell was examined 2 h and 24 h

after a single shot immunization or boost immunization.

Splenocytes were stained with FITC-anti-CD3 and PE-

anti-PD-1, fixed and examined with flow cytometer.

Results showed that T-cell surface PD-1 expression in

the rSEA immunized mice were significantly higher than

that in the control mice (P < 0.01). Significant differ-

ences were observed between 2 h and 24 h in the expres-

sion of PD-1 in the single shot and boost shot immuniza-

tion. Also, significant differences were also observed in

PD-1 expression between single shot and boost shot (P <

0.91) at 2 h and 24 h (Figure 6).

PBS control

rSEA stimulation

ConA stimulation

First

immunization

Before

immunization

Second

immunization

We examined the expression of PD-1 in immunized

splenocytes with flow cytometry to interrogate its rela-

tionship with immunized time and the number of shots.

Results showed rSEA immunized mice had higher PD-1

expression and boost shots further enhanced PD-1 ex-

pression. PD-1 levels in boosted group were higher than

that in the non-boosted group. Also PD-1 levels at 24 h

after immunization were higher than that at 2 h. This

reaction was rSEA-specific, since the PD-1 level in con-

trol group did not change over time. These results ex-

plained unresponsiveness of IFN-γ level to boost shots,

suggesting that inhibitory receptor PD-1 attenuated

T-cell proliferation and IFN-γ secretion [15].

Figure 5. ELISPOT analysis of IFN-γ in mice spleen immu-

nized with rSEA at different stage of immunization.

Figure 6. Expression of PD-1 molecular on spleen T cells of mice immunized with rSEA, FCM showed different expres-

sion of PD-1 molecular on 3d ,6d and 9 d.after immunized.

JBiSE

S. Wu et al. / J. Biomedical Science and Engineering 3 (2010) 543-549 549

4. CONCLUSIONS

Superantigens are powerful activator of T-cell. Distinct

from ordinary antigens, superantigens can directly attach

to the outer groove of MHC-II and Vβ domain of TCR

on T-cell surface, without the processing by antigen

presentation cells. Even a trace amount of superantigen

can activate 5%-20% of T-cells. Therefore, superanti-

gens have been tried in cancer immune therapy to pro-

mote internal anticancer immunity. Good results have

been obtained from these trials. However, it has also

been found that superantigen may induce apoptosis and

inability after activating T-cells, leading to the attenua-

tion of response to additional stimulation. This property

of tolerance induction limits the efficacy of superanti-

gens. Therefore, we have used rSEA to immunized mice,

studied the characteristics of rSEA induced specific hu-

moral immunity and cellular immunity and the mecha-

nism of anergy. These studies could provide foundations

for further studies of superantigens as tumor suppressors.

rSEA has superantigen properties and that it can in-

duce powerful humoral and cellular immune responses.

However, boosting immunization with rSEA caused an-

ergy through PD-1 mediated inhibition.

5. ACKNOWLEDGEMENTS

The authors acknowledge research funding from the National Natural

Science Foundation of China (Grant No. 30770340,30470281 ), the

national major program of Science and technology for water pollution

control and restoration in china (Grant No. 2009ZX07423-003) and

Shenzhen Grant Plan for Science and Technology.

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