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.
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
bp
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
kD
33
24
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
¥
β
(a)
-0.2
0
0.2
0.4
0.6
0.8
1
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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