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ISSN 0582-9879               ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(7): 601-605  CN 31-1300/Q

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]