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. 2010 Feb 8;7(2):133–142. doi: 10.1038/cmi.2009.120

Blockade of CD28 by a synthetical peptoid inhibits T-cell proliferation and attenuates graft-versus-host disease

Na Li 1,4, Faliang Zhu 1,4, Fei Gao 1, Qun Wang 1, Xiaoyan Wang 1, Haiyan Li 1, Chunhong Ma 1, Wensheng Sun 1, Wenfang Xu 2, Chaodong Wang 3, Lining Zhang 1
PMCID: PMC4001891  PMID: 20140006

Abstract

CD28 is one of the costimulatory molecules crucial for T-cell activation and thus has become an attractive target for therapeutic immunomodulation. Conventional strategies for blocking CD28 activity using monoclonal antibodies, Fab fragments, antagonistic peptide and fusion proteins, have apparent disadvantages such as inherent immunogenicity, unwanted Fc signaling, poor tissue penetration and bioinstability. Recent research has been directed toward the creation of non-natural, sequence-specific biomimetic oligomers with bioinspired structures that capture the amino-acid interface of the targeted proteins. One such family of molecules is the poly-N-substituted glycines or peptoids, which have close structural similarity to peptides but are essentially invulnerable to protease degradation. To screen for peptoids that specifically target CD28, we first designed and chemically synthesized 19 candidate peptoids based on molecular modeling and docking. Using the phage-displaying system that expresses the extracellular domain of the CD28 homodimer and contains the core B7-binding motif, a peptoid (No. 9) with a molecular formula of C21H29N3O7, was identified to display the highest binding activity to CD28. This peptoid not only inhibited the lymphocyte proliferation in vitro, but suppressed immunoresponses against alloantigens in vivo, and attenuated the graft-versus-host disease in a mouse bone-marrow transplantation model. These results suggested that peptoids targeting CD28 are effective agents for blocking the CD28-mediated costimulation and suitable for development of novel therapeutic approaches for diseases involving this pathway.

Keywords: CD28, peptoid, phage display

Introduction

Optimal activation of T cells requires two signals: the antigen-specific signal delivered via T-cell receptor (TCR) and costimulatory signals provided by the cross-linking of costimulatory molecules.1 Among these, signals mediated via the CD28/B7 pathway appears to be the most critical.2, 3, 4 CD28 ligation with specific antibodies (Abs) or natural ligands expressed on professional antigen-presenting cells (APCs), B7-1 and -2, delivers ‘positive' signals to T cells which results in augmented T-cell proliferation, cytokine production, clonal expansion and effector function.5, 6 In contrast, blockade of this pathway results in the induction of antigen-specific unresponsiveness, or anergy, both in vitro and in vivo.7, 8 However, several other T cell–APC interactions can also provide costimulation. One such interaction is mediated by cytotoxic T-lymphocyte antigen-4 (CTLA-4), which is structurally related to CD28, but it inhibits rather than enhances T-cell activation.9 Importantly, CTLA-4 and CD28 bind to identical ligands on APCs (i.e., B7-1 and -2).10

Several different strategies for blocking the CD28–B7 interactions have been explored to seek for potential immunotherapies for autoimmune diseases and transplantation rejection. For example, B7 blockade can ameliorate clinical symptoms of experimental allergic encephalomyelitis (EAE)11 and inhibit intestinal allograft rejection.12 However, B7 blockade can also result in disease exacerbation, presumably by interfering with regulatory B7–CTLA-4 interactions.13, 14 Indeed, given the complexity of B7–CD28/CTLA-4 interactions, B7-mediated costimulation is not necessarily equivalent to CD28-mediated costimulation. Therefore, direct manipulation of CD28 and CTLA-4 may be preferable to B7 blockade. Perrin et al. have directly targeted T-cell CD28 with specific monoclonal antibodies (mAbs) both during initial Ag priming and after the onset of clinical signs of EAE, and found that CD28 blockade ameliorated EAE and attenuated the clinical course.15 However, the extensive applications of the Ab-based antagonism to clinical treatment are limited by apparent disadvantages such as inherent immunogenicity, unwanted Fc signaling, poor tissue penetration and bioinstability. In recent years, antagonistic peptides have gained momentum as therapeutic agents. Their potential high efficacy combined with minimal side effects made them widely considered as lead compounds in drug development, and at present, peptide-based therapeutics exists for the treatment of a wide variety of human diseases, including osteoporosis, diabetes, infertility, etc.16 Nonetheless, there are still some limitations for peptide drugs including short half-life, rapid metabolism, vulnerable to protease and poor bioavailability.17

Recent research has been directed toward the creation of non-natural, sequence-specific biomimetic oligomers with bioinspired structures that capture both the amino-acid sequence patterning and three-dimensional folds of natural proteins.18, 19, 20 These oligomers may eventually serve as useful peptide replacements with better in vivo stability than that of the natural molecules. Several different families of abiological oligomers have been proposed as novel mimics of natural molecules. One such family of molecules is the poly-N-substituted glycines or ‘peptoids', which have close structural similarity to peptides but are essentially invulnerable to protease degradation and hence are biostable.21 In the current study, we sought to screen for synthetical peptoids that specifically target the crucial costimulatory molecule, CD28, and test their efficacy in blocking the CD28-dependent T-cell activation both in vitro and in vivo.

Methods

Computational design and synthesis of CD28-binding peptoids

The CD28-binding peptoids were designed based on the three-dimensional structure of the CD28 molecule on the basis of docking protocol by using Tripos molecular modeling packages Sybyl 7.0 (Tripos Incorporation, St. Louis, MO, USA). First, a three-dimensional structure of the human CD28 molecule was built by the Sketch module in Sybyl 7.0 and optimized by using the Tripos force field. The docking position of peptoid molecules was established inside the binding pocket containing the core-binding motif, MYPPPY, for B7. Then the CD28-ligand-binding geometry was optimized by flexible docking using the FlexiDock module in Sybyl 7.0. The atomic charges were recalculated by using Kollman all-atom for the protein and Gasteiger–Hückel for the ligand. The interaction energy was calculated using van der Waals, electrostatic and torsional energy terms of the Tripos force field. Further optimization was carried out on the FlexiDock-generated CD28-ligand complexes by using energy minimization and molecular dynamics.

Peptoid oligomers were synthesized on 50 µmol of Rink amide resin (NovaBiochem, San Diego, CA, USA) at a substitution level of 0.47 mmol/g. The synthesis were performed according to the improved method by Kirshenbaum et al.22 Briefly, after removal of the first Fmoc group, the following 90-min monomer addition cycle was performed by a robotic synthesizer and repeated until the desired length was obtained. The amino resin was bromoacetylated by adding 830 µl of 1.2 M bromoacetic acid in N,N-dimethyl formamide and 200 µl of N,N-diisopropyl carbodiimide. The mixture was agitated for 40 min at 35 °C, drained and washed with N,N-dimethyl formamide (three times with 2 ml). Next, 0.85 ml of a 1 M solution of a primary amine in dimethyl sulfoxide was added to introduce the side chain. The mixture was agitated for 40 min at 35 °C, drained and washed with N,N-dimethyl formamide (four times with 2 ml). After the last coupling, the peptoid resin was cleaved and lyophilized as described.21

Amplification of cDNA of the heavy and the light chains of human CD28 by reverse transcription-polymerase chain reaction

The total RNA was isolated from Jurkat cell (Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Shanghai, China) using Trizol reagents. First-strand cDNA was synthesized using oligo (dT) primers and avian myeloblastosis virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The extracellular region of the human CD28-coding gene was amplified by PCR using two pairs of primers: one for the heavy chain of CD28 (CD28H) (forward: 5′-CCGCTCGAGAACAAGATTTTGGTGAAGCAG-3′ reverse: 5′-GACACTAGTGGGCTTAGAAGGTCCGGG-3′), another for the light chain of CD28 (CD28L) (forward: 5′-ATCGAGCTCAACAAGATTTTGGTGAAGCAG-3′ reverse: 5′-TGCTCTAGAGGGCTTAGAAGGTCCGGG-3′). The thermal cycling conditions included an initial denaturation at 95 °C for 4 min, followed by 30 cycles of reaction as: denaturation at 95 °C for 50 s, annealing at 65 °C for 50 s (for CD28H) or at 60 °C for 50 s (for CD28L) and extension at 72 °C for 1 min, and an final extension at 72 °C for 10 min.

Construction of a phagemid expressing the heavy and the light chains of human CD28

The PCR product for CD28H was first digested with XhoI and SpeI and cloned into the pComb3HSS phagemid at the XhoI/SpeI site, to make an initial pComb3HSS-CD28H construct. After transformation and expanding in the XL1-Blue Escherichia coli (Stratagene, La Jolla, CA, USA), phagemid DNA of the construct was isolated and digested with SacI and XbaI, at which sites the PCR product for CD28L that was digested with the same restriction enzymes was subcloned. After another round of transformation and expansion, the phagemid DNA for the final construct expressing both CD28H and CD28L (designated as pComb3HSS-CD28), was prepared for the subsequent experiments.

Displaying of the heavy and the light chains of human CD28 on phages

In order to display the CD28 homodimer on phage, 1 ml (1012 plaque-forming units) of helper phage VCSM13 (Stratagene) was added to a 20 ml culture of E. coli XL1-Blue transformed with pComb3H-CD28 and incubated on a shaker overnight at 37 °C. The mixture culture was centrifuged at 4000g for 15 min and 4% polyethylene glycol 8000 and 3% NaCl were added into supernatant. After incubation on ice for 30 min, the pComb3H-CD28 phages in supernatant was spun down at 14000g for 5 min, resuspended in 2 ml of phosphate-buffered saline (PBS) and stored at 4 °C. To detect titters of the phages, E. coli XL1-Blue in mid-log phase (the optical density at 600 nm is ∼0.5) were infected at room temperature for 20 min by a series of dilution (10–6–10–10) of CD28 phages and transferred onto SB/Amp+ plates. The plates were incubated overnight at 37 °C and clones on the plate that has <100 clones were counted. Titers of the phages were calculated by multiplying the number of clones and the dilution factor for the plate, and recorded as clone-forming units.

Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA

ELISA was performed to measure the CD28-phage binding activity to the anti-CD28 Ab and its ligand, B7-1. In doing this, 96-well plates were coated with 200 µl of 1 µg/ml anti-CD28 Abs or 150 µl of 1 µg/ml recombinant human B7-1, in 0.1 M NaHCO3 (pH 8.6) at 4 °C overnight, blocked with blocking buffer for 2 h at 4 °C and washed for three times with PBS/Tween. Twofold serial dilutions of the CD28 phages in 200 µl of PBS was added into the well and incubated at 37 °C for 1 h. The wells were washed and 200 µl of diluted horseradish peroxidase/anti-M13 (Pharmacia, Stockholm, Sweden) was added for incubation at 37 °C for another 45 min. Finally, 200 µl of O-phenylenediamine substrate solution was added to each well and incubated at room temperature for 15 min. Optical densities were read at 490 nm by a PowerWave XS microplate spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT, USA). To screen for peptoids that bind specifically to CD28, or test dosage-dependent binding activity between phage-displayed CD28 and peptoids, competitive ELISA was conducted. CD28-displaying phages were incubated with different dosages (5, 10, 15 and 20 µg) of each of the candidate peptoids or a selected peptoid in PBS for 1 h at 37 °C, then the phage–peptoid mixture was added into the 96-well plates coated by anti-CD28 mAbs and subjected to the subsequent ELISA steps as described above. All the ELISA experiments were triplicated in three wells.

Flow cytometry analysis

To indirectly detect CD28 expressed in phages, the CD28-expressing or blank phages were premixed with 1×106 Jurkat cells at 37 °C for 45 min and then stained with FITC-conjugated mouse anti-human CD28 mAbs at room temperature for 15 min. To directly detect the binding of the phage-displayed CD28 to B7-1 (CD80), the CD28-expressing or blank phages were premixed with 1×106 Raji cells at 37 °C for 45 min and then stained with FITC-conjugated rabbit anti-human CD80 Abs. A total of 10 000 events were collected and analyzed with the CXP program in an FC-500 flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA). Histogram profiling was used to display the percentages of CD28- and CD80-positive subsets of the respective cells.

Assay of lymphocyte proliferation of human peripheral blood mononuclear cells (PBMCs) stimulated by the anti-CD28 Ab and inhibited by the CD28-binding peptoids

The proliferation assay was employed to detect the effects of CD28-binding peptoids on the CD28-dependent lymphocyte proliferation. Mononuclear cells were separated from human peripheral blood by Ficoll density gradient centrifugation. Mononuclear cells in concentration of 2×105/ml in RPMI-1640 were added to the wells in 96-well plates and stimulated by phytohemagglutinin (PHA, 50 µg/ml), PHA (50 µg/ml)+CD28 Ab (4 µg/ml), or PHA (50 µg/ml)+CD28 Ab (0.4 µg/ml)+peptoids (5, 10 and 25 µg) for 56 h at 37 °C in an incubator filled with 5% CO2. For an additional 72 h of incubation, 1 µCi of 3H-thymidine was added to each well. The cells were then collected and the radioactivity was measured by a liquid scintillation counter. Experiments for each stimulation condition were repeated for three times.

Assay of lymphocyte proliferation of mouse splenocytes by stimulation with anti-CD28 Abs and by mixed lymphocyte reaction

Prior to assaying of lymphocyte proliferation in mice, splenocyte suspension was prepared. First, C57BL/6 mice were killed and their spleens were removed and placed in 5 ml of RPMI-1640 medium. The spleens were then squeezed to push the splenocytes to run into the medium through a sterile 200-hole copper mesh. Cells were centrifuged at 1200g for 5 min at 4 °C. Red blood cells were removed by lysing with 5 ml of red blood cell lysis buffer for 10 min and washing with RPMI-1640 medium for three times. Finally, cells were resuspended with RPMI-1640 medium supplied with 10% of fetal bovine serum and adjusted to a concentration of 1×106/ml. Measurements of proliferation stimulated by anti-human CD28 and suppressed by CD28-targeting peptoids were performed using the same method as in the above proliferation assay for human PBMCs.

For mixed lymphocyte reaction, lymphocytes suspensions were prepared from C57BL/6 mice for responder cells and from BALB/c mice for stimulator cells, as described above. For making responder cells, mitomycin C was added into the 1×106/ml of splenocytes to final concentration of 25 µg/ml. After treatment at 37 °C for 30 min, cells were spun down and washed twice, and then resuspended by 1 ml of RPMI-1640 supplied with 15% of fetal bovine serum, counted and adjusted to 4×106/ml. Splenocytes for stimulators was precultured in an incubator at 37 °C with 5% CO2 for 2 h. The responders and stimulators were then mixed at a ratio of 1:1 and cultured with or without the human CD28-targeting peptoids, as described above, except that 0.5 µCi/well of thymidine was added 16 h before the radioactivity measurement.

Mouse bone-marrow transplantation, peptoid treatment and phenotype observation

The mouse model of bone-marrow transplantation was established using C57BL/6 mice as the donors and BALB/c mice as the recipients. Briefly, the donor C57BL/6 mice was killed and fixed with 70% alcohol for 15 min. After separating the thigh and shin bones, the femurs was removed and placed in 5 ml of RPMI-1640 medium, with which marrow was flushed out from the medullary cavity. To remove bits of bone, the solution was put through a sterile 200-hole copper mesh and collected in a 50 ml tube. The volume was put to 50 ml with medium and then centrifuged at 2000g for 10 min at 4 °C. The cell pellet was washed twice and resuspended with the serum-free medium, and cells were counted and adjusted to a density of 5×108/ml. In addition to bone-marrow cells, splenocytes were prepared from the donors' spleens with a similar procedure and adjusted to a density of 2×108/ml. The bone-marrow cells and splenocytes were then mixed with a ratio of 1:1 as transplantation preparation. Four to six hours prior to transplantation, the BALB/c recipient mice were irradiated with a dosage of 6.5 cGy. For transplantation, 0.1 ml of the transplantation preparation was injected into the tail veins of the recipient mice. Since the second day of transplantation, recipients were intraperitoneally injected daily with either 100 µg/ml of No. 9 peptoid or 0.1 ml of fetal bovine solution. Symptoms such as diarrhea, viability, hairspring and back arching were observed daily and the weight was measured twice a week. Phenotypic severity was assessed and scored as 0, 1 and 2, which stood for normal, mild and severe phenotypes, respectively. The survival rate was recorded three times a week.

Histological study

Liver and colon tissues were obtained from the recipient mice at the most phenotypically severe stage. Serial paraffin slices were prepared and hematoxylin and eosin staining was performed. Microscope observation was compared between the tissues derived from peptoid-treated and non-treated transplant recipients.

Statistical analysis

All statistical analyses were performed using Student's t-test. P<0.05 was considered statistically significant.

Results

Synthesis of sequence-specific peptoid oligomers-targeting human CD28

Based on the three-dimensional structure of the human CD28 molecule and its essential motif for binding to B7, MYPPPY (Figure 1a), and by molecular docking (Figure 1b), we created a family of 19 oligomeric peptoid products containing a specific sequence of diverse side-chain moieties. Reactive side-chain functionalities were protected by trifluoroacetic acid-labile groups (Boc foramines and t-butylesters for carboxylic acids) that were removed during cleavage of the peptoid from the resin. Molecular weights were confirmed by electrospray mass spectrometry and were uniformly in agreement with expected values. The structures and molecular weights of the peptoids were shown in Tables 1 and 2. According to the structure, these peptoids were divided into two serials: the first 10 peptoids adopted piperidine diketone structure as the lead compound and epimino as the backbone (Table 1), and the remaining nine chose hydroxyproline as the backbone (Table 2).

Figure 1.

Figure 1

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Computational design and modeling of peptoids binding to the MYPPPY motif of CD28. (a) Three-dimensional structure of CD28 and the MYPPPY motif built by the sketch module and optimized by using the Tripos force field in the Tripos molecular modeling packages Sybyl 7.0. (b) Docking of No. 9 peptoid with CD28.

Table 1. Structures and molecular weights of the first serial of peptoids.

graphic file with name cmi2009120t1.jpg

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Table 2. Structures and molecular weights of the second serial of peptoids.

graphic file with name cmi2009120t2.jpg

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The phage-displayed extracellular domain of the human CD28 homodimer binds to B7-1 with high affinity

To screen for CD28-targeting peptoids, we first constructed the CD28 homodimer expression system using the phagemid DNA as a vector. The resulting pComb3H-CD28 construct expresses both the heavy (CD28H) and the light (CD28L) chains of CD28, which were fused with phage gpIII protein. The insertion of sequences of CD28 light and heavy chains were confirmed both by restriction digestion and sequencing. After transforming into the VCSM13-sensitive XL1-Blue competent cells and incubation with the VCSM13 helper phage, the fusion protein comprised of the extracellular domain of the CD28 homodimer, and the gpIII protein was expressed and displayed onto the surface of progeny phages. By infecting XL-Blue bacteria with the CD28-expressing phages and counting the colony-forming units positive colonies, we obtained the CD28-expressing phages with a titer of 5×109 clone-forming units per ml. To confirm the expression of CD28 on phages, its binding activity to anti-CD28 Abs was assayed both by flow cytometry and ELISA. For flow cytometry analysis, the CD28-expressing phages were premixed with the Jurkat cells and the CD28 molecule on Jurkat cells was detected with anti-CD28 Abs by flow cytometry. As shown in Figure 2, when mixed with the CD28-expressing phages, the CD28-stained Jurkat cells decreased from 96.6% (Figure 2a) to 79.2% (Figure 2b), whereas no obvious change was observed when mixed with blank phages (Figure 2c). These results confirmed the existence of CD28 on the surface of phages, which, in a concentration-dependent manner, compete with the CD28 on the surface of Jurkat cells to bind to the anti-CD28 Ab. In ELISA assay, phage-displayed CD28 directly bound to the anti-CD28 Ab in a concentration-dependent manner, reaching a titer of 1:256 (Figure 2d). Similar flow cytometry analysis and ELISA assay were performed to examine whether the phage-displayed CD28 maintains its binding activity to B7-1 (CD80). In flow cytometry analysis, mixing with CD28-expressing phages decreased the rate of CD80-positive Raji cells from 31.1% (Figure 2e) to 18.9% (Figure 2f), whereas no obvious change (30.6%) (Figure 2g) was observed when mixed with blank phages, suggesting that the CD28 expressed by the phages can compete with the anti-CD80 Ab to bind to CD80 expressed in Raji cells. Similarly, in ELISA assay, CD28-expressing phages bound to B7-1 in a concentration-dependent manner, reaching a titer of 1:512 (Figure 2h).

Figure 2.

Figure 2

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Confirmation of expression of the CD28 homodimer on phages and its binding activities to B7-1 (CD80) by flow cytometry analysis and ELISA. For testing the expression of CD28, Jurkat cells were either directly stained with FITC-conjugated mouse anti-human CD28 mAbs (a), or premixed with the CD28-expressing (b) or blank (c) phages before staining. For testing the binding activities of CD28-expressing phages to CD80, Raji cells were either directly stained with FITC-conjugated mouse anti-human CD80 mAbs (e), or premixed with the CD28-expressing (f) or blank (g) phages before staining. Flow cytometry analysis was performed and histogram profiling was used to display the percentages of CD28- or CD80-positive cells. For ELISA, twofold serial dilutions of CD28-expressing phages were added into the CD28 mAb- (d) or recombinant CD80-coated (h) wells and incubated for 1 h. Horseradish peroxidase was added for coloration. mAb, monoclonal antibody.

Identification of a peptoid that binds to the phage-displayed CD28 homodimer with high affinity and specificity

To screen for high affinity CD28-binding peptoids, a competitive ELISA assay was performed between the anti-CD28 mAb and the peptoids. As shown in Figure 3a, when CD28-displaying phages was mixed with 10 µg of the Nos. 1–19 studied peptoids, but not irrelevant poptoids, varying degrees of their binding activities to the coated anti-CD28 mAb were inhibited, and the inhibitory effect was comparable with B7-1, a natural CD28 ligand. Among the 19 peptoids, peptoids Nos. 7 and 9 displayed the highest inhibition, even higher than B7-1. However, only the inhibitory activity of peptoid No. 9 displayed both peptoid- (Figure 3b) and CD28-concentration-dependent (Figure 3c) manners, indicating that only No. 9 peptoid binds to CD28 with high affinity and specificity.

Figure 3.

Figure 3

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Screening for peptoids that bind specifically to CD28 and dosage-dependent binding activity of peptoid No. 9 to CD28. CD28-displaying phages were preincubated with 10 µg of the candidate peptoids (Nos. 1–19), irrelevant peptoids or B7-1, and added to wells coated by anti-CD28 mAbs for competitive ELISA. CD28-displaying phages without peptoids and VCSM13 blank phages were set for controls (a). For measuring the dosage-dependent binding activity between phage-displayed CD28 and peptoid No. 9, mixture of phage-displayed CD28 and varying concentrations of peptoid No. 9 (b) or of peptoid No. 9 and varying dilutions of CD28-displaying phages (c), were added to wells precoated by anti-CD28 mAbs, and binding activities of the mixtures to CD28 mAbs were recorded as OD values. mAb, monoclonal antibody; OD, optical density.

The peptoid No. 9 inhibits proliferation of human and mouse T lymphocytes

To examine whether the peptoid No. 9 inhibits the CD28-mediated lymphocyte proliferation in vitro, human PBMCs were stimulated by either PHA or CD28 Ab alone, or along with peptoid No. 9 at incremented concentrations. As indicated by 3H-TdR incorporation, the proliferative activity stimulated by anti-CD28 and PHA was significantly inhibited by peptoid No. 9 (P<0.01) (Figure 4a). Interestingly, due to the identical CD28-binding motif of mouse CD28 to its human counterpart, on which the peptoid design was based, this peptoid also inhibited the CD3 Ab- and CD28 Ab-stimulated proliferation of lymphocytes derived from mice (P<0.05) (Figure 4b). Moreover, in the mixed lymphocyte reaction experiment, the proliferation of responder lymphocytes from C57BL/6 mice stimulated by APCs from BALB/c mice was significantly suppressed by addition of 10–20 µg of peptoid No. 9 (P<0.01) (Figure 4c), indicating an inhibitory effect of the peptoid on the proliferation triggered by alloantigen stimulation.

Figure 4.

Figure 4

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Inhibitory effects of peptoid No. 9 on proliferation of human (a) and mouse (b) T lymphocytes and on mixed lymphocyte reaction triggered by alloantigen stimulation (c). (a) Human PBMCs were stimulated either by PHA+anti-CD28 alone, or by a combination with peptoid No. 9 at incremental concentrations. 3H-TdR were added and radioactivities were measured (*P<0.01 compared with PHA+CD28 mAbs alone group). (b) Mouse splenocytes were stimulated either by CD3 Ab+CD28 Ab alone, or by a combination with peptoid No. 9 at incremental concentrations. 3H-TdR were added and radioactivities were measured (*P<0.05 compared with PHA+CD28 mAbs alone group). (c) Splenocytes derived from BALB/c and C57BL/6 mice as stimulators and responders, respectively, were mixed at a ratio of 1:1 and cultured for 56 h with or without the human CD28-targeting peptoid No. 9. 3H-TdR was added and radioactivities were measured (*P<0.01 compared with the BALB/c+C57 alone group). Ab, antibody; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin.

The peptoid No. 9 attenuates the graft-versus-host disease (GVHD) in mice by suppressing alloantigen-induced immunoinflammation

The CD28-dependent costimulatory is crucial for alloantigen-induced T-cell activation and clonal expansion and plays important roles in the pathogenesis of acute and chronic GVHDs. To test whether the CD28-binding peptoid No. 9 can interfere with the immunoresponses against alloantigens and alleviate the inflammation in vivo, bone-marrow transplantation was conducted using C57BL/6 mice as the donors and BALB/c mice as the recipients. One to eight days after transplantation, symptoms such as diarrhea, loss of weight, decrease in viability, back bending and developmental retardation occurred in the recipient mice. The mice in the GVHD group without peptoid treatment had the most severe symptoms and their phenotypical severity scores increased quickly from day 1 and reached the peak (score 10) at day 5 and maintained for up to 4 weeks. In contrast, the GVHD mice received a daily intraperitoneal injection with 100 µg/ml of peptoid No. 9 reduced the severity by nearly two scores from day 5 to day 18 (Figure 5a). Survival rate observation revealed that the recipient mice without peptoid treatment started to die from day 3. The mortality was 70% on day 14 and went up to 100% on day 28. In contrast, the recipients received peptoid No. 9 treatment started to die on day 7 and the mortality on day 14 was declined to 25%, although it also reached 100% on day 28 (Figure 5b). Histological study on day 19 when symptoms were severe showed that pathological changes were prominent in liver and colon tissues. Changes included bleeding, necrosis, mild edema, etc. The results of H&E staining (Figure 5c) showed that lymphocyte infiltration was prominent in both liver (Figure 5c-1) and colon (Figure 5c-3) tissues of the recipient mice without peptoid treatment, whereas the inflammation was significantly suppressed in those pre-treated with 10 µg of the peptoid No. 9 (Figure 5c-2 and -4). Collectively, these results suggested that the peptoid No. 9 attenuates the GVHD in mice by suppressing alloantigen-induced immunoinflammation.

Figure 5.

Figure 5

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Effects of peptoids on clinical severity and survival of the graft-versus-host-disease (GVHD) mice. Since the second day of transplantation, phenotypical severity scores were recorded daily for the mice irradiated but without transplantation (irradiation control) and recipient mice for bone-marrow transplantation without peptoid treatment (GVHD) or with intraperitoneal injection of peptoid No. 9 (a). In addition, survival status of recipient mice with different treatments were recorded daily and shown by survival rate curves (b). Histopathological changes of liver (c-1 and c-2) and colon (c-3 and c-4) post-bone-marrow transplantation in control and peptoid-treated GVHD mice (H&E staining, ×40). 1 and 3: non-treated; 2 and 4: peptoid-treated. H&E, hematoxylin and eosin.

Discussion

By chemical synthesis and screening with the phage-displayed CD28 homodimer, we first identified a peptoid (No. 9) that targets the CD28 molecule with high affinity and specificity, and effectively abrogates the in vitro proliferation of T cells. In addition, treatment with this peptoid inhibits the immunoresponses triggered by allogenic transplantation and ameliorates the phenotypes of the GVHD in a mouse model. These results suggested that the CD28-targeting peptoid can effectively block the CD28-dependent T-cell response to antigens both in vitro and in vivo. Because the human CD28 shares 60% identity in amino acids with its mouse counterpart and the B7-binding motif of human CD28, MYPPPY, for which the design of peptoids was based on, was identical to its counterpart in mouse, the experiments in mice using the peptoids against human CD28 showed evident bioactivity both in vivo and ex vivo. These results suggested that peptoids directed against the costimulatory system may therefore become a novel strategy for blocking the activation of effector T cells. Moreover, as the peptoid is a relative small molecule and synthetically accessible, it has many advantages such as desirable tissue penetration, little immonogenicity and cost effectiveness, and holds promise to develop novel therapeutic approaches to a large proportion of diseases in which T-cell activation needs to be tuned down.

CD28 is one of the costimulatory molecules that are critical to drive clonal expansion, survival and differentiation of activated T cells into distinct functional subsets. The activation of T cells by T-cell-receptor stimulation in the absence of CD28 costimulation generally resulted in anergy and apoptosis of responding T cells. In the transplant setting, without enough costimulation conferred by CD28–B7 interaction, alloantigen-mediated stimulation of T cells might be aborted and rejection might be prevented.23 In selected rodent transplant models, blocking CD28 costimulation induced long-lasting allografts survival, and in some cases, donor-specific tolerance.24 Our study for donor tolerance using the screened peptoid-targeting CD28 provided further evidence for the effectiveness of this blocking strategy. Moreover, the method we used for blocking the costimulation was directed against CD28, but not B7, thus avoiding the interference by the CTLA4-mediated reverse effects which are usually seen in the B7 blockade studies. However, further studies are required to test whether the peptoid also binds to CTLA-4 and, if so, whether the CTLA-4-peptoid interaction influences the CD28-blocking efficiency. Further, cautions should be taken before applying it to immunotherapy, because blocking CD28/B7 pathway has not been universally effective in generating tolerance in rodent transplant models nor has it been able to induce tolerance in non-human primates.25 Most recent studies have demonstrated that the original simplistic belief that T-cell costimulatory molecules are uniquely required for robust T-cell activation requires drastic revision. Rather, it is the integration of both positive and negative costimulatory signals by T cells during and after their initial activation, dictated by their temporal and spatial expression patterns, that ultimately determines the fate and the functional status of the T-cell response.26 Thus, modulation of T-cell costimulation in attempts to promote allograft tolerance is more complex than simply blocking primary positive costimulatory interactions.27 In this view, the peptoid, like many other CD28-antagonizing agents, needs to be further studied with respect to its therapeutic efficacy in various animal models and to the more complex mechanisms underlying its inhibition of T-cell proliferation and prevention of donor rejection.

Developing antagonizing molecules for a specific target usually requires a simple, effective and high-throughput screening system. In our study, we first developed a phage-displaying system that expressed the core-binding motif of CD28 to B7. The phage-displayed extracellular domain of the CD28 homodimer not only possessed the natural spatial conformation of CD28, but maintained high affinity with its ligand, B7. The displaying system includes a phagemid called Comb3HSS, which was first constructed by Barbas et al.28 and has been successfully employed to display differentiation antigens (CDs) such as CD99 and CD147.29, 30, 31 However, all the reported phage-displayed CDs were single-stranded. In contrast, we showed here that this system can also display double-stranded CDs. Thus, construction of CD molecule-displaying system using the method that we established will provide useful tools for identifying important CDs-interacting molecules and for studies of the mechanisms underlying immunoresponse in more detail.

In summary, we established a simple, rapid and high-throughput screening system for CD28-targeting peptoids. Using this tool, we first identified a peptoid that binds to CD28 with high affinity and specificity. This peptoid can not only effectively inhibit T-cell proliferation in vitro, but also attenuate the GVHD through suppressing the alloantigen-induced immunoresponse in vivo. Although the peptoid-based strategy holds promise to develop novel immunomodulation approaches, further studies are needed to confirm its efficacy in various animal models and elucidate its complex mechanisms underlying the immunoregulation.

Acknowledgments

This work was supported by grants from the Natural Science Foundation of China (No. 30628015 and No. 30872309) and National 973 Program (No. 2006CB503803).

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