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https://www.abbs.info e-mail:[email protected] ISSN 0582-9879 |
Expression of Recombinant Human ICOS and
in Vitro Characterization of Its Bioactivity on B Lymphocytes
DENG Zhong-Bin#,
LU Chang-Ming#, HUANG Wei-Da1, SHEN Li-Qin, ZHU Wei, MA
Hong-Bing, FAN Pan-Sheng, ZHANG
Xue-Guang*
( Biotechnology
Research Institute, Soochow University, Suzhou 215007, China; 1
Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai
200433,China)
Abstract Inducible
costimulator (ICOS) is a novel costimulatory molecule expressed in activated T
cell and has critical regulation effect on special immune response. In this
study, the cDNA encoding human ICOS was cloned from activated tonsil cells via
RT-PCR, and was expressed in E. coli on pET28 expression vector. The
recombinant ICOS protein expressed from E. coli showed a molecular
weight of 14 kD on SDS-polyacrylamide gel electrophoresis and was further
confirmed by Western blot. In presence of IL-10, the purified rhICOS
significantly increased in vitro B cell growth stimulated by pokeweed
mitogen (PWM), and enhanced the secretion of IgG from B cells..
Key words costimulatory
molecules; ICOS protein; E. coli; antibody
A costimlatory signal
is required for proliferation and differentiation of lymphocytes delivered
through T-cell and antigen presenting cells (APCs) interaction. Stimulation of
T cells by antigen in absence of costimulatory signal can cause unproductive
T-cell response［1, 2］. CD28 is a most extensively
characterized costimulatory molecule on T cells and interacts with CD80 or CD86
on APCs. Recently, new members of the CD28-B7 family have been identified. The
inducible costimulator (ICOS), one of such molecules expressed on activated T
cells, has been shown to play an important role in the regulation of T cell
activation and differentiation. The open reading frame of ICOS mRNA encodes a
new protein of 199 amino acids. ICOS is a homodimeric protein with molecular
mass 55 －60 kD［3－6］. The ICOS
ligand, B7 homologous protein (B7h) /B7-related protein 1 (B7RP-1) /GL50/ligand
of ICOS, hereafter designated as B7h, is constitutively expressed on B cells
and is inducible on monocytes and dendritic cells at low levels. B7h expression
in these APCs and fibroblasts can be induced by proinflammatory cytokines such
as IFN-γ and TNF-α［7－11］.
Functional studies of ICOS have suggested that ICOS enhances T cell cytokine
production, upregulates CD40 ligand expression, and promotes Ig production by B
cells［3, 12, 13］. Earlier studies of the ICOS
blockade［14－16］ and ICOS-deficient mice［17－19］ suggested a predominant role of
ICOS costimulation in both Th2 humoral immune responses and formation of
germinal center (GC)［20］. Ligation or
cross-linking of ICOS on activated T cells by ICOS mAb or GL50 fusion protein
strongly enhance the production of cytokines including IL-4, IL-5, especially
IL-10［21, 22］. It is well known that IL-10 may
induce the differentiation of B cells. But most of current knowledge about ICOS
was obtained from studies on mice. It is crucial to explore how ICOS-GL50
signal affect on human humoral immune response.
In this study, we reported the cloning of human ICOS cDNA and expression of a
recombinant ICOS protein. Then the effects of rhICOS on proliferation and
production of B lymphocyte-dependent antibody were also evaluated.
1 Materials
and Methods
1.1 Materials
1.1.1 Plasmid, E. coli strains Cloning vector pET-28a and the
Escherichia coli strains BL-21 and TOP10 were purchased originally from
Stratagene (USA), Pharmacia (Sweden) and Invitrogen (San Diego, USA).
1.1.2 Reagents All restriction endonucleases and T4 DNA ligase were
purchased from TaKaRa Biotech (Dalian, China). Yeast extract and peptone were
from Oxford (USA). Polyclonal rabbit anti-human ICOS antibody was obtained from
Santa Cruz. ELISA kit for quantitative analysis of IL-10 was purchased from
R&D (UK). BM Chemiluminescence Western blot kit (Mouse/Rabbit) was obtained
from Boehiringer Mannheim (Germany).
1.2 Methods
1.2.1 Cloning of ICOS cDNA RNA
was prepared from PHA-activated (1 μg, 5 d) peripheral blood T cells blasts and
human tonsillar using Trizol (Life Technologies) according to manufacturer’s
instructions. Based on the published human ICOS full-length gene sequence, two
oligonucleotide primers (sense, 5′-GTTGGATTCATGAAGTCAGGCCTCTG-GT-3′; antisense,
5′-GAGAATTCTTATAGGGTCAC-ATCTG-3′) were synthesized (Songon Biotech, Shanghai).
cDNA was prepared from 1－3 μg RNA, Superscript II reverse transcriptase (Takana Shuzo,
Shanghai), random primed according to manufacturer’s instructions. RT-PCR was
used for the cloning of ICOS cDNA from the random primed cDNA in 100 μl
reaction mixture containing 1 μg cDNA, 2.5 u Taq DNA polymerase, diluted
buffers, nucleotide and ICOS specific primer sets. Reactions was amplified for
33 cycles, with an amplification profile of 94 °C, 45 s; 58 °C, 45 s; 72 °C, 60
s. The PCR product was then cloned into pMD18-T Vector and the single band was
detected by 1.2% agarose gel electrophoresis. The sequence of the recombinant
was confirmed by the dideoxy chain-termination DNA sequencng method of double
stranded DNA (Bioasia, Shanghai).
1.2.2 Construction of expression vector The
primers for PCR were synthesized by DNA synthesizer (Sangon) based on human
ICOS extracellular gene sequence. Forward primer: 5′-GAGTTCCATGGGAGAAATCAATGGTTCTG-3′,
containing NcoI restriction site; reverse primer: 5′-GAGAAGCTTAGAACTTCAGCTGGCAACA-3′,
containing HindIII restriction site. The pMD18/ICOS was used as
template. A typical condition for PCR was 45 s at 94 °C, 45 s at 58 °C and 60 s
at 72 °C for 34 cycles. The amplified cDNA fragments of ICOS digested with
NcoI and HindIII were ligated into the pET-28-A vector that had been
previously digested with the same enzymes. The recombinant construct was then
transformed into the E. coli strain BL-21 by the CaCl2 method.
1.2.3 Expressing rhICOS protein in E. coli A single colony from E. coli BL-21 harboring the pET-28/ICOS
construct was inoculated with 2 mL LB culture media containing 1% glucose and
100 μg kanamycin for overnight at 37 °C, the colony was expanded by inoculating
with 400 mL LB media at 37 °C till the A600 value of LB medium reached
to 1.0 IPTG (1 mmol/L IPTG for 5 h) was then added to induce the specific
protein expression. The recombinant E. coli was harvested by
centrifugation at 5000 r/min for 5 min. The pellets were washed twice with PBS,
resuspended in solution A (50 mmol/L Tris・HCl, pH 8.0, 1 mmol/L EDTA, 1 mmol
DTT, 1 mmol/L PMSF) and disrupted by super-sonication at 100 W for 30 min with
10 s of pulse and 10 s of pulse off time. Inclusion bodies were obtained by
centrifugation at 8000 r/min for 10 min, then washed with 1mol/L urea and 5%
Triton-X100 and dissolved in dilution buffer (6 mol/L Gu・HCl, 50 mmol/L,Tris・HCl
pH 8.0). The supernatants were collected by centrifugation at 3000 r/min for 7
min, and the renaturation was done by dialysis to reduce guanidium
hydrochloride gradually.
1.2.4 Purification of rhICOS The
supernatant protein was concentrated by ammonium sulfate precipitation at 70%
saturation. The precipitate was dissolved and dialyzed overnight at 4 °C
against 30 mmol/L phosphate buffer, pH 7.5. The dialysate was further
concentrated by ultrafiltration with a molecular cut-off of 5000. The concentrated
sample was loaded onto a Q-Sepharose HP column (Pharmacia) at a flow rate of 60
ml/h, which was preequilibrated with 30 mmol/L phosphate buffer, pH 7.5. The
column was developed with a linear 0 to 1 mol/L gradient of NaCl in 30 mmol/L
phosphate buffer, pH 7.5. The peak fractions were pooled and analyzed by
SDS-PAGE, followed by Western blotting. The purified rhICOS was stored at －20 °C for further studies.
1.2.5 SDS-PAGE and Western blotting Purified
proteins were analyzed by 12% SDS-PAGE according to the standard procedure.
After SDS-PAGE, the gel was immersed in the transfer buffer (0.24% Tris, 1.153%
glycine, 5% methanol, pH 8.8), and the proteins were transferred to
nitrocellulose filter by electrophoresis on 40 V for 10 h. The filter was incubated
1 h in the blocking buffer (5% blocking reagent, B.M.) at room temperature.
After being washed three times (10 min each) with TBS-Tween buffer (10 mmol/L
Tris・HCl, pH 7.5, 500 mmol/L NaCl， 0.05％ Tween 20),
the filter was incubated with the anti-ICOS conjugate for 1 h at room
temperature, and then was washed three times (10 min each) with TBS-Tween
buffer. After briefly rinsed with Dig-buffer III (100 mol/L Tris・HCl, pH 9.5,
100 mmol/L NaCl, 50 mmol/L MgCl2), the specific proteins were colorized by BCIP/NTP
(Sigma).
1.2.6 Effect of rhICOS on proliferation of B cells and secretion of IgG The subpopulations
(CD4,CD8,CD19) of tonsillar monocytes obtained from the cell suspension by
Ficoll-hypane isolation were analyzed with flow cytometry and immunofluorescence.
Cells (3×105 cells/well) were cultured in the 24-well plates with
addition of PWM (4 mg/L), and other supple-ments (5 mg/L control protein, 100
μg/L IL-10, 5 mg/L rhICOS protein and rhICOS+IL10, respectively) as desired.
After incubating for 10 d at 37 °C, the supernatants were collected and used
for the measurement of IgG level.
2 Results
2.1 Cloning of hICOS cDNA and
construction of expression vector
A pair of primers was designed to amplify the ICOS DNA full length. Fig.1(A)
showed that a 600 bp gene fragment was obtained by RT-PCR from total RNA
isolated from PHA-activated peripheral blood and tonsil T cells. The PCR
product was cloned between EcoRI and BamHI sites of pMD18 vector. The enzyme
and sequence analysis showed that the gene fragment consists with other
published results ［Fig.1(B)］. The hICOS extracelluar domain was cloned into the expression
vector PET-28 by a pair of primers containing NcoI and HindIII.
After digestion, about a 386 bp DNA fragment was cleaved off from the
recombinant plasmid ［Fig.1(C)］. The result of sequence coincided with the database.
Fig.1
Agrose gel electrophoresis of products of PCR and endonuclease digestion
(A) Amplification of hICOS cDNA by RT-PCR. 1, ICOS DNA; 2, DNA marker.
(B) Enzyme analysis of the recombinant hICOS by EcoRI and BamHI.
1, hICOS DNA digested with the enzymes; 2, DNA marker.(C) PCR and enzyme
analysis of the recombinant (PET-hICOS). 1, extracellular fragment of ICOS DNA;
2, hICOS DNA digested with the enzymes; 3, DNA marker.
2.2.2Expression and purification of
rhICOS
For expression, E.coli was transformed with the
construct and induced by IPTG. When the final concentration of inducer IPTG
reached to 1 mmol/L, a specific protein with the same molecular weight of
rhICOS (14 kD) was detected by SDS-polyacrylamide electrophoresis and staining
of Coomassic brilliant blue (Fig.2). About 20% recombinant hICOS in total
protein was gained by SDS-PAGE. Approximately 20－25 mg of the recombinant protein determined by ELISA was obtained
from 1 ml of bacterial culture. The protein was further purified and confirmed
as a single band by phenyl-sepharose CL-4B affinity chromatography and SDS-PAGE
［Fig.3(A)］. Western blot analysis confirmed
that the protein was identical with rhICOS ［Fig.3(B)］.
Fig.2
Isolation of recombinant from E.coli BL-21
1, uninduced
BL-21/PET28-hICOS; 2, induced BL-21/PET28-hICOS; 3, uninduced BL-21/PET28; 4,
induced BL-21/PET28; 5, protein molecular weight marker.
Fig.3
SDS-PAGE analysis of hICOS purification (A) and Western blot analysis of hICOS
(B)
(A) SDS-PAGE for fractions during
purification. 1, total cellular E.coli BL-21; 2, the pellet precipitated
by ammonium sulfate; 3, purified rhICOS protein; 4, standard protein.(B)
Western blot analysis of hICOS. 1, purified hICOS protein; 2, negative control.
2.3 Effect of rhICOS on proliferation of
B cell and secretion of IgG
The ELISA results demonstrated that rhICOS alone failed to enhance the
secretion of IgG of B cell stimulated by PWM compared with the control （t=0.089， P>0.05）. When IL-10 was added with rhICOS,
IgG level was increased remarkably at day 4 compared with that after the
addition of rhICOS alone （t=3.015，
P<0.01） (Fig. 4). Interestingly, although
rhICOS couldn’t enhance the secretion of IgG, it promoted the proliferation of
B cells in presence of IL-10 compared with control group（t=1.735， P<0.05）(Fig.5). These date indicated that
ICOS/GL50 signal synergized with IL-10 to affect on humoral immune response.
Fig.4 Effect of rhICOS
on the production of T lymphocyte-dependent antibody
B cells were stimulated by PWM in the
presence of 5 mg/L of purified PET (A) or rhICOS (B) or IL-10 (C) or
rhICOS+IL-10 (D). IgG levels were measured by ELISA at day 10. Data are expressed
as the mean value of five duplicated wells (x±s).
Fig.5
Effect of rhICOS on the proliferation of B cells
Total number of B cells was counted at day
4. Data are expressed as the mean value of colonies per well from five
duplicated plates (x±s). □, rhICOS+IL10; ■, rhICOS; △, IL-10; ▲, PET.
3 Discussion
Inducible
costimulator (ICOS), a third member of CD28 family, has been shown to express
only on activated T cells. It belongs to immunoglobulin gene superfamily and
shares about 39% similarity with CD28［3, 5］. Blocking
GL50-ICOS pathway by ICOS-Ig, Th2-type cytokine production in lung inflammation
and airway reactivity was reduced in vivo, which may provide new
insights into for therapeutic intervention［23, 24］. So purified
recombinant protein can provide very useful material for further study. In this
paper, we cloned and expressed ICOS protein in E. coli. The specific
purified recombinant protein was identified by Western blot. Then bioactivity
of recombinant ICOS protein was further evaluated in the production of T
lymphocyte-dependent antibody. Our findings showed that ICOS-GL50 signal, like
CD40-CD40L signal, could not exert a direct affect on IgG secretion of B cells
stimulated by PWM in vitro. IgG secretion was increased significantly
when rhICOS and IL-10 were added to the cultures together. These results
suggested that interaction of rhICOS with GL50 expressed on B cells may be
involved in the regulation of antibody in presence of IL-10. It has been
reported that ICOS-GL50 signal can remarkably upregulate the expression of
IL-10, a crucial growth factor for B cell, which can not only directly promote
antibody secretion but also upregulate the expression of IL-2R［25, 26］ on B cells, and then in turn
synthesize IL-2 to upregulate antibody secretion. Thus, ICOS-GL50 signal
transduction can promote the proliferation of B cells and secretion of
antibody.
Previous study
shows that an important role of activated T cells is providing help for B cell
activation. Recently, the date suggests that ICOS-deficient animals fail to
generate germinal centers and have impaired IgG1, IgE and IgG2a production
following primary and subsequent immunization with T-dependent antigens［17－19］. However, McAdam
and colleagues showed that the Ig class switching deficiency in ICOS－/－ mice could be rescued by CD40 stimulation, suggesting that
GL50-ICOS interactions were important for T-cell-B-cell collaboration in
generating humoral responses. Indeed, ICOS signaling can induce CD40L
expression on T cells［3,19］. Moreover,
although GL50 is constitutively expressed, its expression may be downregulated
following antigen-receptor signaling and CD40 signaling rescues GL50
expression, suggesting that GL50 is only induced following proper interaction
of the B cell with its cognate T cell［22］. These
observations show critical cross-regulation of GL50-ICOS and CD40-CD40L
signaling. The relations and the mechanism of cooperation between the two
signal pathways in humoral immunity need to be further explored.
In summary, we have successfully constructed a plasmid for human ICOS
expression in E. coli. The recombinant ICOS protein has full biological
activity after purification, and might have important basic research and potential
applied value. Other characteristics of ICOS as its effect on the maturation of
DCs and antibody production in vitro, especially its relation with
CD40L, are being investigated in our laboratory.
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________________________________________
Received: March 18, 2003 Accepted: May 8, 2003
This work was supported by a grant from the National Natural Science Foundation
of China (No. 2001CB51003)
# These two authors contributed equally to this work
*Corresponding author: Tel, 86-512-65125011; Fax, 86-512-65104908; e-mail, [email protected]