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Introduction
A popular model to explain the requirement of co-signals in T-cell activation is the two-signal
hypothesis[1]. In this model, signal one is derived from the T Cell Receptor after triggering by antigenic peptide presented by major histocompatibility
complex (MHC) molecules and signal two is delivered through costimulation. Integration of the two signals induces the
optimal activation of T cells. Lack of a co-stimulatory signal leads to decreased responses and, in some cases, even induces
tolerance or anergy (an unresponsive status of T cells to antigens). However, this two-signal model (or `missing signal
model¡¯) has been recently challenged by the discovery of new co-inhibitory pathways and their crucial roles in the control of
T-cell activation. The co-inhibitors described so far function in at least three distinct co-signalling pathways: the
CD80/CD86-CTLA4 axis, the B7-H1/B7-DC-PD1 axis, and B7-H4 and
BTLA[2].
Chen et al identified B7-H4 (B7S1, B7X), which is a B7-family member with co-inhibitory
function[3-5]. B7-H4 has approximately 25% homology in the extracellular portion with other B7-family members. Despite mRNA expression in various human
tissues, immunohistochemistry analysis does not reveal positive staining for protein in any tissues or organs from healthy
human individuals[6], indicating a tight control of B7-H4 protein expression, possibly at the posttranslational level.
Cell-surface B7-H4 expression can be induced on human T cells, B cells, monocytes and Dendritic Cells(DCs)
after in vitro stimulation. It is reported that B7-H4¡¯s unidentified receptor is also expressed on activated T cells. In mice, B7-H4 was found
to be expressed constitutively by B220+ B cells in the spleen and to be downregulated after
activation[3]. B7-H4 protein is expressed in many cancers such as ovarian, breast and lung
cancer[7]. B7-H4 may be responsible for the escape of tumor cells
from tumor-specific T-cell responses in cancer patients. Immobilized B7-H4-immunoglobulin inhibited T-cell proliferation,
prevented them from entering the cell cycle and reduced the secretion of interleukin (IL)-2, IL-4, IL-10
andinterferon (IFN)-g in response to CD3/CD80 stimulation. Together, these studies reveal an important role for B7-H4 as a coinhibitor of T-cell
immunity.
In order to understand the costimulatory function of B7-H4 on cellular immunity, human B7-H4 gene was cloned and
expressed in the E coli system, to analyze the role it plays in T-cell modulation
in vitro. In this report, we first described
methods for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein
from E coli. Further studies on the target protein GST/hB7-H4 indicated its biological function of inhibiting T-lymphocyte proliferation
and cytokine secretion, which may be important evidence for the hB7-H4 down-regulatory
feature.
Materials and methods
Materials The E coli strain TOP10 and expression host BL21-RIL was purchased originally from Novagen (La Jolla, CA,
USA). Expression vector pGEX-5X-3 (GST fusion protein vector) was provided by Amersham Pharmacia (Piscata-way, NJ,
USA). All restriction endonucleases, T4 DNA ligase,
rTaq & Pyrobest DNA polymerase, pMD18-T Vector and DNA
molecular weight marker were obtained from TaKaRa Biotech (Dalian, China). Protein molecular weight marker is a product of
Fermentas (Burlington, Ontario, Canada). Gel Extraction Mini Kit, Plasmid Mini Kit, RT-PCR Mini Kit were purchased from
Shanghai Watson Biotechnologies Inc (Shanghai, China). Anti-CD3 monoclonal antibody was from Sigma (St Louis, MO,
USA). BM Chemiluminescence Western Blot Kit (Mouse/Rabbit) was obtained from Boehringer (Mannheim, Germany).
IL-2 and IFN-g ELISA kit were from R&D Systems (Minneapolis, MN, USA). Protein concentrations were estimated by the
Bradford method using Bio-Rad protein assay. All other reagents and apparatus were of high quality available from
commercial sources.
Cloning of hB7-H4 cDNA and construction of expression
vector Using human cDNA FLJ22418 as a template, the
full-length hB7-H4 cDNA was obtained by PCR. The PCR amplification was carried out with the following primers: B7-H4-A: 5¡¯-
ACG CTG CAG ACC ATG GCT TCC CTG GGG CAG -3¡¯; B7-H4-B: 5¡¯-
GCG GAT CCT TAT TTT AGC ATC AGG TAA G -3¡¯. The
PCR product was then cloned into pMD18-T vector. Confirmed by sequencing, the recombinant vector containing the
sequence encoding entire human B7-H4 protein was conserved as a template for future work, and named as
pMD18-T/hB7-H4. Two other primers were constructed to facilitate cloning of the extracellular domain of hB7-H4 from pMD18-T/hB7-H4 as
following: B7-H4-ECD-A: 5¡¯- TTG AAT TCT ATT GGG GAG GAT GGA
-3¡¯(EcoR I restriction endonuclease site, as underlined);
B7-H4-ECD-B: 5¡¯- TTA CTC GAG CCC TGT TGC TTT GGC -3¡¯
(Xho I restriction endonuclease site, as underlined). Digested
with EcoR I and Xho I, the PCR product was cloned into the corresponding region of pGEX-5X-3 vector. Identified by
restriction enzyme digestion and sequencing, the correct recombinant prokaryotic expression vector was named as
pGEX-5X-3/hB7-H4[8].
Expression of GST/hB7-H4 fusion protein The
E coli strain BL21-RIL, transferred with the recombinant vector
pGEX-5X-3/hB7-H4, was cultured in 10 mL LB medium containing 100 µg/mL ampicillin and 2% glucose at 37
oC overnight. The next day, 2 mL overnight bacteria culture was added to 200 mL fresh medium, and incubated in two 500-mL conical flasks at 37
oC, 225 r/min. To ensure adequate aeration, flasks were filled to only 20%-25% capacity. When
the OD600 value reached 0.6-0.8, the recombinant protein was induced by the lactose analog
isopropyl-b-D thiogalactoside (IPTG) to the final concentration
of 1 mmol/L. Five hours later, cells were harvested by centrifugation and rinsed three times in 1×PBS.
Refolding and purification of the recombinant protein
Preparation of extracts to be identified by
SDS-PAGEThe expressed fusion protein was resuspended in 6 mL
dual-boiling water (Add 1-10 mmol/L DTT (1,4-Dithiothreitol), 1 mmol/L PMSF (Phenylmethyl Sulfonyl Fluoride)). Adding DTT
prior to cell lysis can significantly increase binding of some GST fusion proteins to Glutathione Sepharose. The addition of
lysozyme (0.1 volume of a 10 mg/mL lysozyme solution in 25 mmol/L Tris-HCl, pH 8.0) may help to improve the efficiency of
sonication. After being sonicated at a high setting (duty time=10 s, rest time=10 s) of 480 W in ice until the viscosity
disappeared, the sample was centrifuged at
5000×g for 10 min. Cell disruption is evidenced by partial clearing of the
suspension or may be checked by microscopic examination. Frothing was avoided as this may denature the fusion protein.
Oversonication can also lead to co-purification of host proteins with the GST fusion
protein[8]. The supernatant was saved and the pellet was resuspended in 12 mL washing buffer [20% (
v/v ) Glycerol, 2% (v/v) Triton X-100, 2 mol/L urea, 50
mmol/L NaCl, 50 mmol/L EDTA, 100 mmol/L Tris-Cl (pH 8.0)]. After 10 min at room temperature, the samples were centrifuged at 5
000×g for 10 min at 4 oC. Following three cycles of washing, the
precipitate was denatured in 8 mol/L urea with 50 mmol/L
Tris-Cl, 100 mmol/L NaCl, 2 mmol/L EDTA, 5 mmol/L DTT, 1 mmol/L PMSF (pH
8.9) overnight at 4 oC. The supernatant and deposit collected respectively by centrifugation for 15 min at 12
000×g were analyzed by SDS-PAGE (12%).
Purification by GST-affinity chromatography After high-speed centrifugation, the supernatant was dropped into 200
mL renaturation solution (1 mmol/L reduced and 0.2
mmol/L oxidized glutathione, 1 mol/L urea, 50 mmol/L
Tris-Cl, 100 mmol/L NaCl, 1 mmol/L PMSF, 50 mmol/L EDTA, pH
8.0) with constant agitation at 4 oC for 48 h. The temperature of renaturation was then changed to room temperature. Another
12 h later, the renaturation solution containing the fusion protein was dialyzed for 24 h at 4
oC against 1×PBS (pH 7.3), which was replaced every 6 h. The renatured protein was then loaded onto the 1×PBS pre-equilibrated GST affinity chromatography
column (Glutathione Sepharose 4B, Amersham Pharmacia) at a rate of 1 mL/min. Following another equilibration, the bound
GST-fusion protein was then eluted with elution buffer (50 mmol/L Tris-HCl, 10 mmol/L reduced glutathione, pH 8.0).
Decreasing the flow rate during purification or further elution with higher concentrations of glutathione (20-50 mmol/L) at times
improved yield. The pooled fractions containing GST/hB7-H4 recombinant proteins were filtered through sterile 0.22 µm
membrane. Protein concentration was determined by the Bradford method using bovine serum albumin as standard. The
protein was also subjected to SDS-PAGE to determine the purity of the target
protein[9-12].
Identification by SDS-PAGE and western blot
The total bacterial protein was loaded to sodium dodecyl
sulfate-polyacrylamide (SDS) gel electrophoresis (12%), transferred onto the nitrocellulose filters, and stained with rabbit-anti human
polyclonal antibodies against hB7-H4, which was prepared in our laboratory (data not shown). To eliminate
non-specific reaction, polyclonal antibodies
were cross-adsorbed with anti-GST antiserum
with E coli proteins (BL21-RIL harboring GST-mock) using CNBr-activated Sepharose
4B[8]. The protein band was visualized by using the BM Chemiluminescence
Western Blotting Kit according to the manufacturer¡¯s instructions.
T-cell proliferation and cytokine assay Peripheral blood mononuclear cells were isolated from healthy peripheral human
blood by Ficoll gradient centrifugation. Monocytes were removed by plastic adherence for 2 h in RPMI-1640 Medium
(Roswell Park Memorial Institute)+10% fetal calf serum (FCS) at 37
oC. CD3+ T-cells were collected by passing over a nylon
wool column (>90%)[13]. For T-cell proliferation assay, a 96-well flat-bottom plate was precoated with CD3 mAb (0.3
µg/mL) in PBS at 4 oC overnight. Purified T cells were then seeded into a 96-well plate
(2×104 /well) in the presence of various
concentrations of GST/hB7-H4. The negative control was a corresponding dose of soluble GST (sGST) protein, which was
prepared in the same way as GST/hB7-H4 with a high purity (>90%). After incubation for 96 h in a 37
oC, 5% CO2 incubator, the supernatant of T cells was harvested immediately before the addition of
[3H]thymidine.[3H]thymidine 1 µCi (1 Ci=37 GBq)
was added to each well and the cells were incubated for another 16 h before harvesting. All the measurements were performed
in triplicate[12]. The concentrations of IL-2 and
IFN-g in culture supernatant were detected with ELISA kits according to the
manufacturer¡¯s instructions.
Statistical analysis All data presented as mean±SD. Student¡¯s
t-test was used to determine the statistical significance.
P<0.05 was considered statistically significant.
Results
Cloning and construction of expression vector of hB7-H4
With PCR, an 870 bp fragment was amplified from cDNA
FLJ22418 (Figure 1A). Confirmed by DNA-sequencing (Bioasia, Shanghai, China), the obtained sequence was entirely
identical to the published sequence of human B7-H4 in the Genbank Database (Accession
No AY280972). The 567 bp-extracellular region of hB7-H4 was cloned from the recombinant vector pMD18-T/hB7-H4 (Figure 1B) to constitute the
recombinant prokaryotic expression vector pGEX-5X-3/hB7-H4, which was further identified by restriction
enzyme EcoR I and Xho I (Figure 1B).
Expression and purification of recombinant protein GST/hB7-H4
After being transformed with pGEX-5x-3/hB7-H4,
E coli BL21-RIL was induced to express fusion protein GST/hB7-H4. The electrophoretic mobility of the expressed protein
approximated to a molecular weight of 21 kDa plus the 26 kDa GST protein, which is in agreement with the molecular weight
47 kDa calculated based on its deduced amino acid sequence. And the expressed protein constituted
52% of the total bacterial protein after being induced with 1
mmol/L IPTG for 5 h at 37 oC
(Figure 2A). The insoluble protein
following denaturation and renaturation was purified through GST affinity chromatography column. The elution profile was
monitored and analysis by SDS-PAGE showed that the purity reached 95% (Figure 2B, C).
Fusion protein identified by western
blot With recombinant GST/hB7-H4 as antigen, Western blot showed that the
polyclonal antibodies against hB7-H4 could recognize a protein with approximately 47 kDa, which is in accordance to the
molecular weight of GST/hB7-H4. Lysates of uninduced BL21-RIL harboring GST/hB7-H4 and sGST protein respectively
were set as negative control. As expected, no obvious band was detected. This result confirmed that the fusion protein had
the same immunodomain as hB7-H4 (Figure 3).
Inhibitory effects of T cells costimulated by GST/hB7-H4
in vitro The proliferation of T cells was determined by
[3H]thymidine assay after a 96-h incubation. The results showed that GST/hB7-H4 protein could obviously inhibit CD3mAb
activated T cell (0.3 µg/mL) (Figure 4A). At the same time, it reduced the production of IL-2 and
IFN-g of anti-CD3-stimulated T cells in a dose-dependent manner
in vitro (P<0.05, unpaired
t-test) (Figure 4B, C). These data were representative of three
independent experiments.
Discussion
B7-H4, the latest member of the B7 superfamily, was only identified recently. Mounting data showed that hB7-H4 might
represent a novel regulatory means for cell-mediated immunity. In our research, we found that immobilized B7-H4 could
inhibit the proliferation of T cells experiencing Ag stimulation. In addition, B7-H4 was known to suppress the production of
Th1-derived cytokines such as IL-2 and IFN-g from the naive T cell. Preliminary data
indicated that engagement by B7-H4 on receptors on activated T cells had a minimal effect on programmed cell death but had a profound effect on cell cycle arrest, by
which most of the T cells activated by anti-CD3 are arrested in the
G0/G1 phase[3-5]. Therefore, B7-H4 may play a role in the
negative regulation of T-cell immunity in peripheral tissues.
The work presented in this paper demonstrated that hB7-H4 could be expressed in bacteria and readily purified in
reasonable quantities for research on its biofunctions. In theory, the GST/hB7-H4 protein could be specially digested with
thrombin to delete the GST part. But because of economic problems, we did not do so. Our results showed that the
recombinant protein GST/hB7-H4 maintained the bioactivity to inhibit T-cell proliferation and IL-2 secretion. This indicated
that we had a convenient tool to study hB7-H4 and its functions in regulating immune responses. The full-length cDNA of
human B7-H4 cloned by PCR from cDNA fragments would facilitate the preparation of the hB7-H4 monoclonal antibody for
further study. In our study, the efficiency of the recombinant protein expression was time- dependent reaching the highest
level at the 5 h after adding IPTG. Meanwhile, we gained a lot of experience in purification and refolding of proteins. We first
found GST/hB7-H4 protein could obviously reduce IL-2 secretion, which suggested hB7-H4 might have effect on
T-cell-mediated immune response. Our results showed that the special antibody against hB7-H4 could partly reverse the inhibition
of T cells mediated by GST/hB7-H4 fusion protein. The putative counter-receptor of hB7-H4 could be rapidly expressed on
T cells after stimulation by PHA or anti-CD3 mAb for 6 h (data not shown).
In conclusion, our study demonstrated that the prokaryote expression system could be used to generate hB7-H4 protein
with natural spatial conformations and biological functions, which provided an efficient and economical way for the
preparation of this target protein, and received clues on how to clarify the mechanism of hB7-H4 in immune responses.
Acknowledgement
We Thank Dr Hiroko HATA (Laboratory of Genome Structure Analysis, Human Genome Center, Institute of Medical
Science, University of Tokyo, Japan) for his generous gift of clone HRC08590, which contains full-length human B7-H4
cDNA.
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