Expression and function of inducible costimulator on peripheral blood T cells in patients with systemic lupus erythematosus

Jia-Hui Yang, Jun Zhang, Qing Cai1, Dang-Bao Zhao1, Jian Wang, Ping-E. Guo, Li Liu, Xing-Hai Han1 and Qian Shen

Department of Laboratory Diagnosis and 1 Department of Rheumatology, Changhai Hospital, Second Military Medical University, Shanghai, P. R. China.

Correspondence to: Q. Shen, Department of Laboratory Diagnosis, Changhai Hospital, Second Military Medical University, 174# Changhai Road, Shanghai 200433, P. R. China. E-mail: yjhsmmu{at}yahoo.com.cn


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To investigate the role of inducible costimulator (ICOS) in the pathogenesis of SLE, we assessed its expression on peripheral blood CD4 and CD8 T cells and functional roles in patients with systemic lupus erythematosus (SLE).

Methods. Expression of ICOS on peripheral blood CD4 and CD8 T cells and ICOS ligand (ICOSL) on peripheral blood CD19 B cells from patients with SLE, patients with rheumatoid arthritis (RA) and healthy volunteers were determined by two-colour flow cytometry. The functional costimulatory effects of ICOS on peripheral blood mononuclear cells (PBMC) were assessed by T-cell proliferative responses, cytokines, anti-double-stranded DNA (anti-dsDNA) antibody and total IgG production.

Results. Peripheral blood CD4 and CD8 T cells expressing ICOS were significantly increased in patients with SLE compared with patients with RA and healthy subjects. Peripheral blood CD19 B cells expressing ICOSL in SLE were markedly reduced compared with RA. Proliferative responses of anti-CD3/ICOS costimulation were significantly higher than those of anti-CD3/hamster IgG (HIgG) in healthy subjects, but not in patients with SLE. Anti-CD3/ICOS-stimulated SLE PBMC secreted similar levels of IL-10 and IFN-{gamma} but a significantly lower level of IL-2 than healthy PBMC. Anti-CD3/ICOS-mediated costimulation significantly enhanced the production of anti-dsDNA antibodies and total IgG in patients with SLE.

Conclusion. Hyperexpression of ICOS on peripheral blood CD4 and CD8 T cells from patients with SLE contributed to the dysregulated T-cell proliferation, T-cell activation and pathogenic autoantibody production, which showed that the abnormality of ICOS costimulation may play an immunopathological role(s) in the pathogenesis of SLE.

KEY WORDS: Inducible costimulator, Systemic lupus erythematosus, Cytokines, Anti-dsDNA antibodies, Flow cytometry


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Lupus is a chronic autoimmune inflammatory disease with complex clinical manifestations. Human patients and murine models of lupus manifest a wide range of immunological abnormalities. The most consistent and pervasive of these are (i) the ability to produce pathogenic autoantibodies, (ii) lack of T- and B-lymphocyte regulation, and (iii) defective clearance of autoantigens and immune complexes. Pathogenic memory T cells are thought to be produced on disease initiation following presentation of self antigens by dendritic cells activated by danger signals. These cells then activate naive autoimmune B-cell precursors via cytokines and costimulatory molecules. During this time, B cells undergo class switching and somatic mutation, resulting in the production of high titres of high-affinity immunoglobulin (Ig) G autoantibodies. Furthermore, the interaction of T and B cells via costimulatory molecules may generate anti-apoptotic signals in patients with lupus, leading to increased numbers of autoreactive B cells producing pathogenic autoantibodies [1].

Two signals are required from antigen-presenting cells (APCs) for optimal activation of naive antigen-specific T cells [2, 3]. The first signal is provided by specific antigen recognition through the interaction of major histocompatibility complex molecules and the T-cell receptor (TCR)–CD3 complex. The second signal (called a costimulatory signal) is delivered to T cells by costimulatory molecules expressed on APCs. The most studied costimulatory signal is that of CD28, which is constitutively expressed on most peripheral CD4 T cells and half of CD8 T cells, and responds to its counter-receptors, B7-1 (CD80) and B7-2 (CD86), on APCs [4, 5]. Cytotoxic T lymphocyte antigen 4 (CTLA4), the second member of CD28 family, is another receptor for B7-1/B7-2, and its expression is rapidly up-regulated following T-cell activation. CTLA4 has a higher affinity for B7-1/B7-2 than CD28, and its engagement delivers negative signals. Thus, CTLA4 might inhibit T-cell responses by out-competing CD28-mediated signalling [6, 7].

Recently, a third member of the CD28 family, the inducible costimulator (ICOS), has been identified. Like CD28, ICOS enhances T-cell proliferation and cytokine secretion; enhancement of cytokine production is characterized by high levels of IL-4, IL-10 and IFN-{gamma}, but a low level of IL-2. In addition, ICOS is expressed on the T cells in germinal centres. These studies indicate a role for ICOS in T-cell help for B-cell activation, antibody production and antibody switching/maturation [8, 9]. But ICOS has its own characteristics in the following respects: (i) ICOS is only expressed on activated T cells and memory T cells, so its function at the effector phase is more dominant than that of CD28; (ii) It is reported in mice that ICOS is expressed at high levels by Th2 cells and at low levels by Th1 cells; it strongly increases the production of IL-10 but weakly induces IL-2; and (iii) ICOS has its own specific ligand, ICOSL (also known as B7h, B7RP-1, LICOS and B7H2), which is constitutively expressed on B cells and monocytes. ICOSL is also induced on non-lymphoid cells by inflammatory cytokines, such as TNF-{alpha} and IL-1 [10, 11], and regulates the production of many important inflammatory cytokines, including IFN-{gamma}, IL-4, IL-10 and IL-13 [12, 13].

Until now, there has been only one report about ICOS expression and its possible role(s) in patients with systemic lupus erythematosus (SLE) [14]. But in a murine model it was shown that administration of anti-ICOSL monoclonal antibody (mAb) before the onset of renal disease significantly delayed the onset of proteinuria and prolonged survival; in addition, ICOSL blockade after the onset of proteinuria prevented disease progression and improved renal pathology [15]. In this study, we demonstrate (i) higher levels of expression of ICOS on peripheral blood (PB) CD4 and CD8 T cells from patients with SLE, and (ii) a functional role for ICOS in SLE patients, including effects on T-cell proliferation and cytokine production, and on T-cell-dependent autoantibody production. Together, these results suggest a possible role for ICOS in the pathogenesis of SLE.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and controls
Peripheral blood samples were obtained from 79 patients with SLE [20 hyperactive (hAS), 17 females and three males, ages 20–78 yr; 26 moderately active, all females, ages 23–70 yr; 33 stable (SS), all females, ages 19–57 yr] at the department of Rheumatology of Changhai Hospital. Approval for the study was granted by the local ethics committee, and informed consent was obtained from all patients and healthy volunteers. Among the active SLE patients, 10 cases were also observed during active and stable stages. All of these SLE patients met the American College of Rheumatology (ACR) criteria for the diagnosis of SLE [16] and the SLE Disease Activity Index (SLEDAI) was used as the measure of disease activity [17] (SLEDAI score ≥10 means hyperactive; 6 ≤ SLEDAI score<10 means lower active; SLEDAI score <6 means stable). As a normal control, 56 healthy age- and sex-matched normal volunteers (N) were used. As a disease control, 30 patients with active rheumatoid arthritis (RA) (22 females and eight males, ages 22–80 yr) were enrolled. RA patients satisfied the 1987 ACR criteria for RA [18]. Active RA was diagnosed according to the ACR criteria for remission [19].

When the experimental study was performed in patients with SLE, 42 patients were receiving only low-dose steroids (<10 mg/day), 18 were receiving low-dose steroids (<10 mg/day) and hydroxychloroquine, six were receiving low-dose steroids (<10 mg/day) and cytotoxic drugs, and 13 patients were not receiving any medications. Patients who were receiving steroids were asked not to take this medication for at least 24 h before blood was obtained from each patient.

Reagents and culture medium
Phorbol 12-myristate 13-acetate (PMA), ionomycin (IO) and pokeweed mitogen (PWM) were purchased from Sigma-Aldrich (St Louis, MO, USA). IL-10 was obtained from PeproTech (Rocky Hill, NJ, USA). hCTLA4– hFc soluble protein was provided by Prof. Ya-Jun Guo (International Joint Cancer Institute of Second Military Medical University, Shanghai, China). RPMI-1640 culture medium (Gibco, Carlsbad, CA, USA) was supplemented with 10% calf serum, 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 µg/ml).

Monoclonal antibodies
Anti-CD3 (12-F6) was kindly provided by Prof. Ya-Jun Guo (International Joint Cancer Institute of Second Military Medical University). Anti-CD28 mAb (CD28.6, mouse IgG2a) and anti-ICOS mAb (C398.4A, Armenian hamster IgG) were obtained from eBioscience (San Diego, CA, USA). Hamster IgG (HIgG) was obtained from Biolegend (San Diego, CA, USA). The following conjugated mAbs were used. CYC-conjugated anti-CD4 mAb (RPA-T4, mouse IgG1), CYC-conjugated anti-CD8 mAb (HIT8a, mouse IgG1), fluorescein isothiocyanate (FITC)-conjugated anti-CD28 (CD28.2, mouse IgG2a), FITC-conjugated anti-CD19 (HIB19, mouse IgG1) and PE-conjugated mouse IgG were purchased from BD PharMingen (San Diego, CA, USA). Phycoerythin (PE)-conjugated anti-ICOS (C398.4A, Armenian hamster IgG) and PE-conjugated anti-ICOSL (MIH12, mouse IgG1) were obtained from eBioscience. PE-conjugated HIgG (HTK888) was purchased from Biolegend.

Flow cytometry
To detect expression of ICOS and CD28 on PB CD4 and CD8 T cells, and ICOSL on PB CD19 B cells from patients with SLE, patients with RA and healthy subjects, 1 x 106 cells were stained with appropriate PE-conjugated anti-ICOS mAb, anti-ICOSL mAb or isotype-matched mouse IgG, hamster IgG and CYC-conjugated anti-CD4 mAb, anti-CD8 mAb or FITC-conjugated anti-CD19 mAb on ice for 30 min. After lysis of erythrocytes and washing, the fluorescence intensity on the cell surfaces was analysed using FACSCalibur (Becton Dickinson, Mountain View, CA, USA). Samples of 1 x 104 cells were acquired and all the data were analysed with CellQuest software (Becton Dickinson, Rutherford, NJ, USA).

Expression and purification of hICOS–mFc soluble protein
The soluble recombinant protein was expressed in a mammal expression system and purified with protein A column (Sigma, St Louis, MO, USA). Briefly, the coding portion of the extracellular domain of human ICOS (amino acids 20–140) and mouse IgG Fc were isolated by reverse transcription–polymerase chain reaction, and the amplified ICOS product was ligated into pGL-3 basic vector with KpnI and SmalI, and the amplified mouse IgG Fc was subsequently ligated into the pGL-3-basic hICOS vector with SmalI and XbaI. The fusion gene was then subcloned into the mammal expression vector pSecTag2/Hygro A (Invitrogen, Carlsbad, CA, USA). The pSecTag2/Hygro A-hICOS– mFc vector was then transfected into CHO cells and the stable clones were selected with hygromycin B (600 µg/ml). The hICOS– mFc fusion protein was recovered from the filtered supernatants of the transfected CHO cells using a protein A column according to the manufacturer's recommendations (Sigma). HPLC showed that the purity of the hICOS– mFc fusion protein was more than 90%, and the protein could specifically bind with ICOSL residing on the surface of Raji and Daudi cells (data not published).

Isolation and culture of peripheral blood mononuclear cells
Heparinized peripheral venous blood was obtained from patients with SLE, patients with RA and healthy subjects. PBMCs were isolated by centrifugation on Ficoll–Hypaque (density 1.077) gradients and resuspended in RPMI-1640 with 10% calf serum.

Kinetics of ICOS expression after anti-CD3 stimulation in vitro
Purified PBMCs (1 x 106/well) from hAS patients and healthy subjects were cultured in 24-well microtitre plates and stimulated with soluble anti-CD3 (1 µg/ml). At the indicated time, cells were collected and analysed by flow cytometry.

T-cell proliferation assay
Purified PBMCs (2 x 105/well) from SLE patients with very active disease and healthy subjects were cultured in 96-well microtitre plates and stimulated with anti-ICOS mAb (3 µg/ml), anti-CD28 mAb (3 µg/ml) or control HIgG (3 µg/ml) in the presence of suboptimal soluble anti-CD3 (200 ng/ml) at 37°C for 72 h. After incubation, cells were pulsed for 16 h with [3H]-thymidine (1 µCi/well), harvested on glass fibre filters, and counted for radioactivity (in counts per min, c.p.m.) in a liquid scintillation system.

In order to test the inhibiting effects of hICOS– mFc soluble protein and hCTLA4– hFc soluble protein on T-cell proliferation, purified PBMCs (2 x 105/well) were cultured in 96-well microtitre plates and stimulated with suboptimal soluble anti-CD3 (200 ng/ml) plus control HIgG (3 µg/ml) in the absence or presence of various concentrations of hICOS– mFc soluble protein or hCTLA4– hFc soluble protein at 37°C for 72 h.

Cytokine assay
Purified PBMCs from very active SLE patients and healthy subjects were cultured in 24-well microtitre plates and stimulated with PMA (20 ng/ml) and IO (1 µmol/l) at 37°C for 48 h. Cells were collected and washed twice with phosphate-buffered saline, and then cultured in 96-well microtitre plates (2 x 105/well) and stimulated with suboptimal soluble anti-CD3 (200 ng/ml) plus anti-ICOS mAb (3 µg/ml) or anti-CD28 mAb (3 µg/ml), or control HIgG (3 µg/ml). At indicated times after stimulation, supernatants were collected and the concentrations of IL-2, IFN-{gamma} and IL-10 were determined by a sandwich enzyme-linked immunosorbent assay (ELISA; Jingmei Biotech Company, Shenzhen, China) according to the manufacturer's recommendations.

Total IgG and T-cell-dependent anti-dsDNA antibody production
Purified PBMCs (2 x 105/well) from SLE patients with high levels of serum anti-dsDNA antibodies and from healthy subjects, were cultured in 96-well microtitre plates and stimulated with suboptimal PWM (2 µg/ml) and IL-10 (20 ng/ml) plus anti-ICOS mAb (3 µg/ml), or anti-CD28 mAb (3 µg/ml) or control HIgG (3 µg/ml). Five days later, supernatants were collected and the concentrations of anti-dsDNA antibodies and total IgG were detected by ELISA (EUROIMMU, Medicinische Labor diagnostika and Bethyl, Montgomery, TX, USA, respectively). In order to test the inhibiting effects of hICOS– mFc soluble protein and hCTLA4– hFc soluble protein on the production of anti-dsDNA antibodies, purified SLE PBMC (2 x 105/well) were cultured in 96-well microtitre plates and stimulated with suboptimal PWM (2 µg/ml) and IL-10 (20 ng/ml) plus HIgG (3 µg/ml) in the absence or presence of various concentrations of hICOS– mFc soluble protein or hCTLA4– hFc soluble protein.

Statistical analysis
Results are expressed as mean ± S.D. Expression levels of ICOS on cell surfaces between different patient groups were compared using the non-parametric Kruskal–Wallis U rank sum test, and P values less than 0.05 were considered significant. Proliferation and cytokine production by T cells and anti-dsDNA antibody production by B cells in patients with SLE and healthy subjects were compared using the non-parametric Mann–Whitney rank sum test, and P values less than 0.05 were considered significant. Proliferation and cytokine production by T cells and anti-dsDNA antibody production by B cells from patients with SLE or healthy subjects with and without the experimental treatments were compared by one-way analysis of variance, and P values less than 0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Expression of ICOS on PB CD4 and CD8 T cells
To assess the contribution of ICOS– ICOSL interaction in SLE, we first analysed the expression of ICOS on PB CD4 and CD8 T cells from patients with SLE by two-colour flow cytometry (Fig. 1A). The proportions of PB CD4 and CD8 T cells expressing ICOS were significantly increased in all groups of SLE patients, compared with those in patients with RA and healthy subjects (CD4, 34.68±16.26%, 34.64±12.78%, 33.99±15.66% vs 22.07±8.95%, 24.81±8.87%, P<0.05; CD8, 18.85±8.03%, 18.77±5.66%, 15.65±5.36% vs 7.49±3.56%, 10.00±3.68%, P<0.05) (Fig. 1B), but there were no significant differences between SLE patients with very active, moderately active and stable disease. The mean fluorescence intensity (MFI) of ICOS expression among CD4ICOS double-positive T cells in patients with very or moderately active SLE and ICOS MFI among CD8ICOS double-positive T cells in patients with very active SLE were significantly higher than those in normal individuals (Fig. 1B).




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FIG. 1. Expression of ICOS, ICOSL and CD28 molecules on freshly isolated PB cells from normal controls (N) (n = 56), hyperactive patients (hAS) (n = 20), lower active patients (lAS) (n = 26), stable SLE (SS) (n = 33) and active RA patients (RA) (n = 30). (A) Representative data showing the increased proportion of ICOS+ cells on PB CD4 (upper) or CD8 T cells (lower) from SLE patients with high disease activity compared with those from normal controls. (B) Proportions and MFI of ICOS-expressing CD4 or CD8 T cells were significantly increased in SLE patients, especially patients with very high active disease activity. (C) Proportions of CD28-expressing CD4 or CD8 T cells were reduced in hAS patients. (D) In the individual SLE patients studied longitudinally (n = 10), proportions of PB CD4 (left) or CD8 T cells (right) expressing ICOS were significantly increased in the active phase. (E) Proportions and MFI of ICOSL-expressing CD19 B cells were markedly decreased. *P < 0.05 vs normal controls, {Delta}P < 0.05 vs RA patients.

 
In contrast, CD28 was constitutively expressed at high levels on PB CD4 and CD8 T cells from all individuals. However, both PB CD4 and CD8 T cells expressing CD28 were significantly reduced in patients with very active disease compared with normal individuals (Fig. 1C). In addition, when individual patients were examined when they had active disease or were in remission, the proportions of PB CD4 and CD8 T cells expressing ICOS were significantly increased in the active phase (CD4, 39.85±13.56% vs 23.56±9.27%, P<0.05; CD8, 30.29±14.71% vs 15.28±6.04%, P<0.05) (Fig. 1D). The percentages of ICOSL-expressing CD19 B cells and ICOSL MIF among CD19 B cells were significantly decreased in all groups of SLE patients compared with RA patients (percentages, 32.68±11.33%, 38.24±19.66%, 40.36±23.60% vs 65.85±19.19%, P<0.05; MIF, 12.24±1.22, 13.67±3.86, 13.22±3.12 vs 20.60±6.60) (Fig. 1E).

Induction of ICOS expression on PBMCs
To evaluate the kinetics of ICOS expression, purified PBMCs from very active SLE patients and healthy subjects were stimulated with soluble anti-CD3 and the levels of expression of ICOS on PB CD4 and CD8 T cells were determined by two-colour flow cytometry. After anti-CD3 stimulation, ICOS on PB CD4 and CD8 T cells from patients with SLE was rapidly induced as early as 6 h and persisted to 96 h. In contrast, ICOS on PB CD4 and CD8 T cells from healthy subjects was significantly increased only after 12 h (CD4 T cells) or 24 h (CD8 T cells) of stimulation, and, most importantly, ICOS expression on CD4 and CD8 T cells at any time (except 48 h) was significantly lower than that on cells from patients with SLE (Fig. 2).



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FIG. 2. Induction of ICOS on PB CD4 (left) or CD8 T cells (right) from hAS patients (n = 5) and normal controls (n = 5) by soluble anti-CD3 (1 µg/ml) stimulation for 0, 6, 12, 24, 48, 72 and 96 h in vitro. proportions of CD4 T cells (left) and CD8 T cells (right) expressing ICOS from SLE patients were higher than those of normal controls.

 
Costimulation of T-cell proliferation by ICOS
To determine whether ICOS has functional costimulatory activity, we stimulated freshly isolated PBMCs from very active SLE patients and healthy subjects with anti-ICOS, anti-CD28 or control HIgG in the presence of suboptimal concentrations of anti-CD3. As shown in Fig. 3A, anti-ICOS in the presence of anti-CD3 only enhanced proliferative responses of PBMCs from healthy subjects, but failed to enhance responses of PBMCs from very active SLE patients, compared with stimulation by anti-CD3 and HIgG. In contrast, anti-CD28 in the presence of suboptimal soluble anti-CD3 significantly enhanced proliferative responses of PBMC from the two groups.



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FIG. 3. (A) Proliferative responses of PBMCs obtained from normal controls (n = 5) and hAS patients (n = 5) were induced by control HIgG (3 µg/ml), anti-CD28 mAb (3 µg/ml) or anti-ICOS mAb (3 µg/ml) in the presence of suboptimal soluble anti-CD3 mAb (200 ng/ml). The proliferative responses of PBMCs from SLE patients with high disease activity by ICOS costimulation were significantly lower (*P<0.05 vs normal controls). (B, C) The inhibiting roles of hICOS–mFc soluble protein (B) and hCTLA4–hFc protein (C) in the proliferative responses of PBMCs obtained from normal controls (n = 5) and hAS patients (n = 5). The blockade of PBMC proliferative responses occurred in a hICOS–mFc dose-dependent manner in normal controls but not in hAS patients compared with anti-CD3/HIgG stimulation. The blockade of PBMC proliferative responses occurred in a hCTLA4–hFc dose-dependent manner in normal controls and hAS patients. (D) The role of double-blocking ICOS and CD28 costimulation in the proliferative responses of PBMCs from normal controls (n = 5) and hAS patients (n = 5). The proliferative responses of PBMCs from hAS patients and healthy subjects were further reduced compared with single blockade of ICOS or CD28 costimulation.

 
To further determine whether the ICOS– ICOSL interaction affects activation of PBMCs, we firstly blocked ICOS– ICOSL interactions with hICOS– mFc soluble protein in the presence of suboptimal soluble anti-CD3 and HIgG. As shown in Fig. 3B, the proliferative responses of PBMCs from healthy subjects, but not SLE patients, were significantly reduced in a dose-dependent manner compared with CD3/HIgG stimulation. hICOS– mFc soluble protein down-regulated ICOS-mediated proliferative responses of normal PBMCs at a concentration between 5 and 20 µg/ml. As a control, when we blocked CD28 costimulation with hCTLA4– hFc soluble protein, the proliferative responses of PBMCs from SLE patients and healthy subjects were both significantly reduced (Fig. 3C). When we simultaneously blocked ICOS and CD28 costimulation, the proliferative responses of PBMCs from SLE patients and healthy subjects were further reduced compared with blockade of either ICOS or CD28 costimulation alone (Fig. 3D).

Cytokine production
To address the contribution of ICOS to cytokine production in SLE, we measured the amounts of IL-2, IFN-{gamma} and IL-10 produced by PBMCs from very active SLE patients and healthy subjects. As previously reported, PBMCs from normal subjects stimulated by anti-CD3 and anti-ICOS produced similar amounts of IFN-{gamma} and IL-10, and lower amounts of IL-2 compared with those stimulated with anti-CD3 and anti-CD28. PBMCs from patients with SLE stimulated by anti-CD3/ ICOS produced comparable amounts of IFN-{gamma} and IL-10 but significantly lower amounts of IL-2 compared with those from healthy subjects (Fig. 4). In addition, we compared the kinetics of IL-2, IFN-{gamma} and IL-10 production by anti-CD3/ICOS stimulation in patients with SLE and healthy subjects. As shown in Fig. 4, the peak concentrations of IFN-{gamma} and IL-2 were observed 24 h after anti-CD3/ICOS stimulation in healthy subjects, but at 12 h in patients with SLE. The peak concentrations of IL-10 were observed 48 h after anti-CD3/ICOS stimulation in both patients with SLE and healthy subjects. Thus, these results indicated that signalling through ICOS costimulated the production of various cytokines by both Th1 and Th2 cells, but that the level of the costimulatory effect varied amongst the different cytokines.



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FIG. 4. Kinetic cytokine production of PBMCs obtained from normal controls (n = 7) and hAS patients (n = 7). First, PBMCs were prestimulated by PMA (20 ng/ml) plus IO (1 µg/ml) for 48 h, then they were further stimulated with control HIgG (3 µg/ml), anti-CD28 mAb (3 µg/ml) or anti-ICOS mAb (3 µg/ml) in the presence of suboptimal soluble anti-CD3 mAb (200 ng/ml). The production of IFN-{gamma} and IL-10 after ICOS costimulation was comparable in normal controls (n = 7) and hAS patients (n = 7), but the production of IL-2 after ICOS costimulation in hAS patients was significantly lower than that in normal controls (*P<0.05 vs normal controls).

 
Total IgG and anti-dsDNA antibody production
To address the effects of ICOS on the production of IgG and pathological autoantibodies in patients with SLE, we determined the amounts of IgG and anti-dsDNA antibodies produced by freshly isolated PBMCs from SLE patients known to have high levels of serum anti-dsDNA antibodies, and healthy subjects after in vitro stimulation with anti-ICOS, anti-CD28 and/or HIgG in the presence of PWM and IL-10. As shown in Fig. 5A, PBMCs from patients with SLE stimulated by PWM/IL-10/ICOS produced even higher amounts of anti-dsDNA antibodies compared with patients stimulated with PWM/IL-10/CD28. In healthy subjects there were no detectable amounts of anti-dsDNA antibodies before and after stimulation. In contrast, ICOS-mediated costimulation significantly increased the production of IgG, to a similar degree as CD28, in both SLE patients and healthy subjects (Fig. 5B).



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FIG. 5. (A, B) Anti-dsDNA antibody and total IgG production of PBMCs obtained from normal controls (n = 6) and SLE patients with high levels of serum anti-dsDNA antibodies (n = 6). PBMCs were induced by control HIgG (3 µg/ml), anti-CD28 mAb (3 µg/ml) or anti-ICOS mAb (3 µg/ml) in the presence of PWM (2 µg/ml) and IL-10 (20 ng/ml) for 5 days. Anti-dsDNA antibody and IgG production of PBMCs from patients with SLE were significantly increased by ICOS costimulation (*P<0.05 vs HIgG control). (C, D) The inhibiting effects of hICOS–mFc soluble protein (C) and hCTLA4–hFc (D) soluble protein on the production of anti-dsDNA antibodies from SLE PBMCs (n = 5). The blockage of anti-dsDNA antibody production occurred in a hICOS–mFc (C) or hCTLA4–hFc (D) dose-dependent manner compared with PWM and IL-10 plus control HIgG. (E) The role of double-blocking of ICOS and CD28 costimulation with hICOS–mFc and hCTLA4–hFc soluble proteins in anti-dsDNA antibody production obtained from SLE PBMCs (n = 5). The anti-dsDNA antibody production of PBMCs from SLE patients with high levels of serum anti-dsDNA antibodies was further reduced compared with single blockade of ICOS or CD28 costimulation.

 
To further determine the effects of the ICOS–ICOSL interaction on the production of anti-dsDNA antibodies, we blocked the interaction with hICOS–mFc fusion protein. As shown in Fig. 5C, hICOS–mFc fusion protein blocked anti-DNA antibody production in a dose-dependent manner by PBMCs from SLE patients known to have high levels of serum anti-dsDNA antibodies. As a control, when we blocked CD28 costimulation using hCTLA4– hFc soluble protein, anti-dsDNA antibody production of PBMCs from SLE patients with a high level of serum anti-dsDNA antibodies was also significantly reduced (Fig. 5D). When we simultaneously blocked ICOS and CD28 costimulation with hICOS– mFc and hCTLA4– hFc soluble proteins, the production of anti-dsDNA antibodies was only about 60% of that when either ICOS or CD28 costimulation is blocked alone (Fig. 5E).


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In this study we demonstrated that a recently described costimulatory molecule, ICOS, was markedly up-regulated in PB CD4 and CD8 T cells from patients with SLE, whereas ICOSL expression on B cells was down-regulated, which is in accordance with results published recently by Hutloff et al. [14]. In the individual patients who were studied longitudinally, proportions of PB CD4 and CD8 T cells expressing ICOS were significantly higher in active disease than in stable disease. In vitro experimental results showed that ICOS had a similar costimulatory role to CD28 in enhancing the production of IFN-{gamma} and IL-10 from SLE patients with high disease activity and control PBMCs, but failed to enhance T-cell proliferation and IL-2 production from SLE patients with high disease activity. Most importantly, we also demonstrated that the ICOS– ICOSL interaction markedly enhanced the production of total IgG and pathogenic anti-dsDNA antibodies by B cells from SLE patients with high levels of serum anti-dsDNA antibodies. Our present results suggest that ICOS up-regulation on PB CD4 and CD8 T cells from patients with SLE might play an immunopathological role in chronic SLE inflammation.

Consistent with a previous study showing that anti-ICOS antibody could enhance T-cell proliferation mediated through the TCR, anti-CD3/ICOS stimulation enhanced PBMC proliferation to a comparable extent as anti-CD3/CD28 stimulation in healthy subjects. Our results also showed that although ICOS expression on SLE PB CD4 and CD8 T cells was markedly higher than on normal PBMCs, both at baseline and after CD3 stimulation, the proliferative responses of SLE PBMCs were significantly lower than those of normal PBMC. These paradoxical results of PBMC proliferation via anti-CD3/CD28 stimulation have been observed previously [20, 21]. They were attributed to the effects of chronic inflammation down-regulating the T-cell receptor {zeta} chain, thereby inducing resistance against anti-CD3 stimulation in T cells. On the other hand, we also speculated that the key signalling molecules in ICOS-mediated T-cell proliferation might be defective in SLE patients. We suggest that the higher expression of ICOS on PB CD4 and CD8 T cells in SLE may, to some extent, represent compensation for the defect in TCR signalling.

Studies have shown that ICOS-mediated costimulation was more effective than CD28 in the production of IL-10, comparable in effect on the production of IFN-{gamma}, and less effective in IL-2 production [22–25]. Our studies showed that ICOS-mediated costimulation was comparable to CD28 in the production of IFN-{gamma} and IL-10, but ICOS-mediated production of IL-2 was severely defective in SLE patients with high disease activity compared with that of healthy subjects, which might partly explain the defects of ICOS-mediated T-cell proliferative responses in SLE patients with high disease activity, because IL-2 is the most potent cytokine for T-cell proliferation. IL-10, which is an important cytokine in SLE, is a potent stimulator of B-lymphocyte proliferation and differentiation, and also induces activated B cells to secrete large amounts of IgG, IgA and IgM [26, 27]. Our studies show that ICOS can markedly enhance the production of IL-10, to a similar extent as CD28, which indicates that ICOS might activate B cells and enhance the production of autoantibodies through IL-10 in SLE patients.

To provide further evidence for the possibility of the contribution of ICOS to the pathogenesis of SLE, we examined the costimulatory role of ICOS in the production of anti-dsDNA antibodies and total IgG from SLE PBMCs and control PBMCs. Anti-dsDNA antibodies are found in 50–80% of SLE patients and are thought to be pathogenic and exclusive to lupus. It has been proved that ICOS play a role in T-cell help for B-cell activation, antibody production and antibody class switching/maturation. Hutloff et al. [14] found that the expression of ICOS on CD4 as well as CD8 T cells was increased in SLE patients, while ICOSL was down-regulated on a high proportion of PB memory B cells, which was in accordance with our results. They inferred that this ICOSL down-regulation on B cells was a sign of recent interaction with ICOS+ T cells in vivo; thus, ICOS is one of the forces driving the formation of memory B cells and plasma cells in SLE. Our results showed that ICOS could enhance the production of anti-dsDNA antibodies and total IgG in SLE patients, but could only increase the production of total IgG in healthy individuals. This indicates that ICOS-mediated costimulation indeed has a role in the production of pathogenic autoantibodies. Precisely how ICOS enhances autoantibody production in SLE is unclear; it may be via IL-10 or other costimulatory molecules, such as CD40L. Further studies will be required to determine the relative importance of ICOS–ICOSL interactions in the pathogenesis of SLE, and the most suitable interventions.


    Acknowledgments
 
We thank Prof. Jian-Ming Yang for review of the manuscript and critical comments, and we are grateful to Prof. Ya-Jun Guo for providing the mAb specific for CD3 (12-F6) and hCTLA4–hFc soluble protein. Supported by a grant from the Hi-tech Research and Development Program of China 2002AA214091 and Shanghai Natural Science foundation grant 3ZR14026.

No conflict of interest has been declared by the authors.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 22 January 2005; revised version accepted 20 May 2005.



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