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The Journal of Immunology, 2001, 167: 578-585.
Copyright © 2001 by The American Association of Immunologists

A Retro-Inverso Peptide Mimic of CD28 Encompassing the MYPPPY Motif Adopts a Polyproline Type II Helix and Inhibits Encephalitogenic T Cells In Vitro1

Mythily Srinivasan*, Richard M. Wardrop{dagger}, Ingrid E. Gienapp{dagger}, Scott S. Stuckman{dagger}, Caroline C. Whitacre{dagger},§ and Pravin T. P. Kaumaya2,*,{ddagger},§

Departments of * Microbiology, College of Biological Sciences; {dagger} Molecular Virology, Immunology, and Medical Genetics; {ddagger} Obstetrics and Gynecology, College of Medicine and Public Health; and § Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210 response unit(s); MBP, myelin basic protein; CD, circular dichroism/dichroic; {theta} = mean residue molar ellipticity; SPR, surface plasmon resonance; L-CD28, CD28 free peptide; EL-CD28, end group-blocked CD28; RI-CD28, end group-blocked retro-inverso CD28; RL-CD28, end group-blocked reverse L CD28; D-CD28, end group-blocked D-CD28; LNC, lymph node cell(s).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Complete activation of T cells requires two signals: an Ag-specific signal delivered via the TCR by the peptide-MHC complex and a second costimulatory signal largely provided by B7:CD28/CTLA-4 interactions. Previous studies have shown that B7 blockade can either ameliorate experimental autoimmune encephalomyelitis by interfering with CD28 signaling or exacerbate the disease by concomitant blockade of CTLA-4 interaction. Therefore, we developed a functional CD28 mimic to selectively block B7:CD28 interactions. The design, synthesis, and structural and functional properties of the CD28 free peptide, the end group-blocked CD28 peptide, and its retro-inverso isomer are shown. The synthetic T cell-costimulatory receptor peptides fold into a polyproline type II helical structure commonly seen in regions of globular proteins involved in transient protein-protein interactions. The binding determinants of CD28 can be transferred onto a short peptide mimic of its ligand-binding region. The CD28 peptide mimics effectively block the expansion of encephalitogenic T cells in vitro suggesting the potential usefulness of the peptides for the treatment of autoimmune disease conditions requiring down-regulation of T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell activation requires two signals; the first signal is Ag specific and is delivered via the TCR by the peptide-MHC complexes on the APCs. The second costimulatory signal is provided largely via the CD28 molecule on the T cell surface. Upon binding the B7 ligands on the APC, CD28 transduces signals that mediate sustained proliferation and survival of Ag receptor-activated T cells (1). Subsequently, activated T cells express CTLA-4, a homolog of CD28 that also binds the B7 ligands on the APC and serves as a negative regulator. Antigenic activation in the absence of CD28 costimulation is known to induce a state of unresponsiveness in T cells (2, 3).

The critical role played by B7/CD28:CTLA-4 costimulatory interactions in determining the fate of immune responses (activation vs anergy/apoptosis) makes it an attractive target for therapeutic immunomodulation (4). Administration of mAbs to B7 or CTLA-4 Ig fusion protein, both of which abrogate B7:CD28 interactions, have been shown to ameliorate autoimmune diseases in animal models including experimental autoimmune encephalomyelitis (EAE),3 diabetes, and systemic lupus erythematosus (5, 6, 7, 8). However, the results of these studies were variable and dependent upon the timing of administration. Prevention of CTLA-4 engagement has been shown to augment T cell responses in vitro and exacerbate disease in vivo (9, 10). Anti-CD28 Fab that specifically blocks B7:CD28 interactions has been shown to suppress the encephalitogenicity of myelin basic protein (MBP)-primed T cells (11). Thus, a therapeutic strategy aimed at selectively blocking the B7:CD28 interactions while retaining the B7:CTLA-4 pathway is likely to be more efficient in suppressing T cell responses.

In recent years, mutagenesis and protein structural studies have been adopted to develop rationally designed small peptide or nonpeptidyl molecules as antagonists for protein-protein interaction. Structurally engineered peptide analogs of the binding epitope(s) of a protein have been known to block functional interactions both in vitro and in vivo (12, 13). Hence, we addressed the question of whether a minimal peptide sequence derived from the ligand-binding region of CD28 can be designed to selectively block B7:CD28 interactions.

The CD28 molecule on the T cell surface is a member of the immunoglobulin superfamily with an extracellular IgV-like domain, which provides a stable platform for the display of specific determinants for recognition reactions (14, 15). Sequence alignment studies of the IgV fold revealed a rigorous conservation of a hexapeptide motif MYPPPY in the complementarity-determining region 3 (CDR3)-like region of CD28. The localization of the motif in the solvent-exposed CDR and conservation across species strongly suggest the presence of a candidate ligand-binding epitope in this region. The hydrophobic motif forms a loop that is conformationally constrained due to the presence of adjacent proline residues (16, 17). Similar proline-rich sequences in other globular proteins, such as the proteins involved in the Ras pathway, are known to form part of an adaptor system bringing together proteins for transient interactions (18).

A unique feature of the B7:CD28/CTLA-4 interaction that can be exploited for selective blockade of CD28 signaling is the differential kinetics of binding, with CTLA-4 exhibiting a faster on rate and higher affinity for B7 ligands than CD28 (14, 19). Thus, we hypothesized that a mimic of the ligand-binding region of CD28 could selectively block B7:CD28 interactions by competing with cell surface CD28 for binding B7 ligands. In contrast, cell surface CTLA-4 could theoretically overcome this competition due to its higher affinity for B7 ligands.

We have identified a 20-residue peptide sequence consisting of the polyproline motif and the flanking residues derived from the solvent-exposed region of the mouse CD28 molecule as a short peptide mimic of the ligand-binding epitope of CD28. To design a functionally stable peptide that mimics the putative bioactive topology as well as to circumvent the problem of rapid proteolytic degradation we used a retro-inverso modification of the synthetic CD28 peptide incorporating D-amino acids (20).

In this study, the design, synthesis, and structural and functional characterization of a synthetic CD28 peptide and its retro-inverso isomer (end group-blocked retro-inverso CD28, RI-CD28) are described. By circular dichroism (CD), it is shown that the synthetic CD28 peptides adopt a polyproline type II (PP II) helical conformation, and surface plasmon resonance (SPR) studies indicate that the peptides compete with CD28 Ig for binding the B7-1 ligand. To test whether the peptides have an inhibitory potential, the biological assays were performed using T cells from transgenic mice that carry the V{alpha}4/V{beta}8.2 TCR specific for the encephalitogenic epitope of MBP, MBP Ac1–11. Our results indicate that exposure to CD28 peptide analogs inhibits expansion of encephalitogenic T cells accompanied by decreased IL-2 production. The binding properties of these peptides, their preliminary structural features, and their functional efficiency in blocking T cell activation indicate that the synthetic CD28 peptide mimics act as B7:CD28 antagonists and may be potentially useful for down-regulating T cell responses in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide synthesis and purification

Peptides of the CD28 CDR3-like region were synthesized by solid phase peptide synthesis following 9-fluorenylmethoxycarbonyl-t-butyl chemistry with dicyclohexylcarbodiimide/1-hydroxybenzotriazole as coupling reagent on a fully automated peptide synthesizer (model 396-5000 Multiple Peptide Synthesizer; Advanced ChemTech, Louisville, KY) as described previously (21). The CD28 free peptide (L-CD28) was assembled on 4-methylbenzhydrylamine resin with 4-(hydroxymethyl) phenoxyacetic acid as the linker. The end group-blocked peptides were assembled on F-moc-2,4-dimethyloxy-4'-(carboxymethyloxy)-benzylhydrylamine (Rink amide) resin (Advanced ChemTech) as peptide amides. Immediately after the final deprotection step, the free NH2 group of the terminal amino acid residue was acetylated with 3 mmol acetylimidazole in dimethyl formamide. The completion of acetylation was confirmed by a negative Kaiser ninhydrin test. With respect to the parent peptide, the retro-inverso peptide was assembled in reverse order of amino acids with F-moc-D-amino acid derivatives. The crude peptides were purified by semipreparative reversed phase HPLC using a C18 column (Vydac, Hesperia, CA). Analytical HPLC was performed using a VIDAC C18 column using a linear gradient of 60% acetonitrile in water containing 0.1% trifluoroacetic acid. The identity of peptides was finally confirmed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Mice

Female B10.PL mice (6–8 wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME). TCR-transgenic mice (B10.PL background, H-2u), which express a TCR{alpha}{beta} (V{alpha}4/V{beta}8.2) specific for MBP peptide NAc1-11, were generated as described previously (22, 23). The transgenic mice were bred and maintained under specific pathogen-free conditions at The Ohio State University.

Antigens

MBP was extracted from guinea pig spinal cords (Harlan Sprague Dawley, Indianapolis, IN) as described previously (24). MBP NAc1-11 (Ac-A-S-Q-K-R-P-S-Q-R-H-G-COOH) peptide was synthesized as described above.

Circular dichroism

CD measurements were recorded on an Aviv model 62A DS spectrometer equipped with a thermostatic temperature controller and microcomputer (Aviv Associates, Lakewood, NJ) as described previously (21). CD spectra were recorded in a quartz cell of 0.1-cm pathlength. Each spectrum was obtained by averaging 1 nm/s in the 190- to 270-nm wavelength range, using a bandwidth of 1.0 nm and a response time of 1 s. CD28 peptides were dissolved in PBS, pH 7.4, in 50% trifluoroethanol or in 4–6 M CaCl2 at a 412 µM concentration for CD measurements. The CD spectra were recorded at a range of temperatures between 5°C and 90°C. Raw CD signals (in millidegrees) were converted to mean residue molar ellipticity ({theta}) in deg·cm2/dmol using the formula [{theta}]MRW = [{theta}]obs/10lcn, where {theta}obs is the observed ellipticity, l is the pathlength in centimeters, c is the molar concentration of peptide, and n is the number of residues in the peptide (21).

SPR experiments

Binding experiments were performed by SPR, which measures biomolecular interactions in real time on a BIAcore instrument (Pharmacia Biosensor, Uppsala, Sweden). All experiments were performed at 37°C using HBS-EP buffer (25 mM HEPES, pH 7.4, 150 nM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20).

CD80 Ig (extracellular domain of murine CD80 fused with the constant region of mouse IgG1 heavy chain, a gift from Jay Fine, Schering-Plough Institute, Kenilworth, NJ) at 35 µg/ml in 10 mM sodium acetate buffer, pH 4.2, was coupled to a research grade CM 5 sensor chip using a standard amine-coupling procedure (25). This typically resulted in immobilization of 1422–1660 response units (RU) of CD80 Ig on the sensor chip. Following coupling, noncovalently bound ligand was removed by washing twice with 5 mM NaOH. Kinetic analysis was performed by injecting the analytes (L-CD28, end group-blocked CD28 (EL-CD28), RI-CD28, end group-blocked reverse L-CD28 (RL-CD28), and end group-blocked D CD28 (D-CD28) peptides) at 0.375–18.5 µM concentrations in HBSS-EP, pH 7.4, for 300 s with a flow rate of 10 µl/min. The analytes were also injected at the same concentration and injection times over an empty flow cell with nothing immobilized. Data analysis was performed with BIAevaluation and BIAsimulation software version 2.1 (Pharmacia Biosensor). Before kinetic analysis, data were adjusted to a zero baseline by subtracting the background responses obtained by injection of the analytes through a control flow cell with no ligand immobilized. Kd, (units: s-1), Ka, (units: M-1s-1), and the affinity constant were determined as described previously (26).

Competitive kinetic analysis

Competitive binding assays were performed as described previously using purified anti-mouse IgG1 Fc (31437zz; Pierce, Rockford, IL) to indirectly immobilize CD80 Ig (27). Initially, the affinity constant of the interaction between the CD28 Ig or CTLA-4 Ig and CD80 Ig was determined as described (26). For competitive kinetic analysis, 100 µl of a 3.2 µM CD28 Ig solution was mixed with 100 µl of EL-CD28 or RI-CD28 peptides (51.6–503 µM in HBS-EP) and injected over the surface of CD80 Ig as secondary analyte for 5 min at a flow rate of 5 µl/min. For competition between CTLA-4 Ig and the CD28 peptides, CTLA-4 Ig at a constant concentration of 0.8 µM mixed with increasing concentrations of EL-CD28 or RI-CD28 peptides (6.18–1237 µM) were used as analytes. The CD80 Ig surface was regenerated between injections by washing for 3 min with 5 mM NaOH. The mixtures were also injected on an empty flow cell with no protein immobilized, as a control. The observed response Rt is the sum of the contributions of R1 (CD28 Ig or CTLA-4 Ig) and R2 from the two analytes (EL-CD28 peptide or RI-CD28 peptide) (27).

Proliferation analysis

CD4+ T cells were purified by positive selection from lymph nodes (inguinal, axillary, brachial, cervical, popliteal), mesenteric lymph nodes, and spleens of 6- to 8-wk-old V{alpha}4/V{beta}8.2 TCR-transgenic mice. Briefly, single cell suspensions were prepared using a lympholyte M gradient, followed by washing and incubation with magnetic bead-conjugated anti-mouse CD4 (L3T4) (Miltenyi Biotec, Auburn, CA) and by positive selection using magnetic selection columns. Purity of the CD4+ cells was >90% as assessed by staining with FITC-labeled anti-mouse V{beta}8.2 mAb (PharMingen, San Diego, CA). During the selection process, T cells maintain a naive phenotype with no evidence of T cell activation as measured by proliferation in culture. Purified CD4+ T cells (5 x 104 cells/well) were cultured together with splenocytes from B10.PL mice as APCs (1 x 105 cells/well) in RPMI 1640 containing 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 5 x 10-5 M 2-ME in round-bottom 96-well plates with or without MBP NAc1–11 (0.1–100 µg/ml) for 72 h, including a final 18-h pulse with [3H]thymidine. Cultures contained synthetic CD28 peptide analogs at different concentrations in triplicate wells. Cultures were harvested onto glass-fiber mats using a Skatron harvester (Skatron, Sterling, VA) and counted by liquid scintillation on an LKB Betaplate (LKB Instruments, Gaithersburg, MD). The means of triplicate wells were determined and the results are expressed as {delta} cpm (mean cpm of cultures with Ag - mean cpm of cultures with medium alone) ± SE.

Cytokine ELISPOT assay

ELISPOT analysis was performed as described previously (28). Unifilter 350 plates (Polyfiltronics, Clifton, NJ) were coated overnight at 4°C with rat anti-mouse IL-2 (clone JES6-1A12) (PharMingen) at 2 µg/ml. The plates were washed with PBS and blocked with 1% BSA in DMEM (Life Technologies, Gaithersburg, MD) for 2 h at room temperature. Single cell suspensions of CD4+ lymph node cells (LNC) specific for MBP-NAc1–11 from TCR-transgenic mice were washed and resuspended in HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 1% L-glutamine and 50 µg/ml gentamicin (Life Technologies, Grand Island, NY). CD4+ LNC (5 x 106 cells/ml) were added to the plates in triplicate with or without 40 µg/ml MBP in the presence of CD28 peptides or control peptides at 50, 75, and 120 µM concentrations under the conditions as specified for the proliferation assays. After 24 h of culture at 37°C, the plates were washed with PBS and thereafter with PBS containing Tween 20 (1:2000) (PBST). Biotinylated anti mouse-IL-2 (clone JES6-5H4; PharMingen), 2 µg/ml was then added and the plates were incubated at 4°C overnight. After washing with PBST and PBS, goat anti-biotin Ab conjugated to alkaline phosphatase (Vector Laboratories, Burlingame, CA) diluted to 1/1000 in PBST containing 1% BSA was added for 2 h at room temperature. The spots were visualized by adding 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium phosphatase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Image analysis of ELISPOT wells was performed on an Immunospot Image Analyzer (Zeiss, Oberkochen, Germany). Frequencies are expressed as the number of MBP-responsive cells per million ± SEM.

Statistical analysis

For in vitro proliferation and ELISPOT analyses, a one-way ANOVA with Tukey’s post hoc test was performed to determine the differences between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Peptide design, synthesis, and structural characterization

Mutagenesis studies have shown that truncation or substitution of the conserved 99MYPPYLDN106 sequence in the CDR3-like loop region of CD28 abrogated CD28 binding to B7 ligands (29, 30). Molecular modeling of the CD28 extracellular domain using the solution structure of CTLA-4 as a template predicted several charged residues conserved only in CD28 (95K, 97E, 109R) that may modulate ligand binding (16). Based on these observations, we defined a 20-residue linear peptide that comprised the polyproline motif and the delineated flanking sequence.

To mimic the end groups of the ligand-binding region of the parent CD28 molecule, the amino terminus was acetylated and the carboxyl terminus of L-CD28 was obtained as an amide. In addition, this modification would likely stabilize the secondary structure of the CD28 peptide that may be important for its functional interaction.

We also synthesized a retro-inverso isomer of the CD28 peptide, which exhibits increased resistance to proteases. The retro-inverso modification involves the reversal of all amide bonds within the peptide backbone achieved by reversing the direction of the sequence and inverting the chirality of each amino acid residue by using D-amino acids. The goal of this topochemical approach is to create an analog such that the reversed amide bonds (NHCO) in the modified peptide retain both the planarity and conformational restrictions of peptide bonds (CONH). As such, the spatial orientation of the side chains remains closely related to that of the original peptide (31).

As controls for the retro-inverso peptide, D-CD28 and RL-CD28 were also synthesized. The sequence of all CD28 peptides used in this study is shown in Table IGo.


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  		Table I. Amino acid sequences of synthetic CD28 peptides and control peptidesa

 
Conformation of CD28 peptide analogs in solution

To gain an understanding of the conformational characteristics of the CD28 peptides, we studied the CD spectrum in aqueous solution. CD is a powerful analytical method for the characterization and quantification of peptides and protein secondary structures (e.g., {alpha} helix, {beta} sheet, etc.). The CD spectrum of L-CD28 showed a large minimum at 202 nm ({theta} = -62.73 x 103 deg·cm2·dmol-1) and a slight maximum at 221 nm ({theta} = -3.5 x 103 degt·cm2·dmol-1) (data not shown) at 25°C. The CD spectrum of the EL-CD28 peptide presented a strong {theta} minimum at 205 nm and a weak maximum at 221 nm although still in the negative ellipticity region at 25°C (Fig. 1GoA). Compared with the free L-CD28 spectrum, the intensity of ellipticity near 200 nm was considerably enhanced in the EL-CD28 peptide, suggesting stabilization of the helical secondary structure (data not shown). The retro-inverso CD28 peptide made up of D-enantiomers presented a mirror image-like spectrum of the EL-CD28 peptide. At 25°C, the RI-CD28 peptide exhibited a {theta} maximum at 205 nm, a weak minimum at 215 nm, and a weak maximum at 223 nm (Fig. 1GoB). These observations are consistent with the CD spectrum of polypeptide sequences reported to prefer a PP II-type helical structure (32). Because it is known that the secondary structures of the peptides are sensitive to higher temperatures, we studied the CD signal of the CD28 peptides at an elevated temperature. When the temperature was raised to 90°C, there was a decrease in the {theta} of the EL-CD28 peptide (Fig. 1GoA), in the {theta} maximum of RI-CD28 peptide (Fig. 1GoB), and a shift to the right. A similar destabilizing effect of elevated temperature has been observed in other proline-rich peptides due to a transition from the PP II helical structure to a disordered random coil conformation (33). The CD spectrum of EL-CD28 peptide in 6 M CaCl2 at 25°C exhibited a dramatic decrease in the intensity of the {theta} minimum at 205 nM ({theta} = -28 x 103 deg·cm2·dmol-1) (Fig. 1GoA). Similarly, the CD spectrum of the retro-inverso CD28 peptide in 6 M CaCl2 showed a dramatic decrease in molar ellipticity maximum at 205 nM ({theta} = 1.10 x 104 deg·cm2·dmol-1), complete loss of both the molar ellipticity minimum at 215 nM, and the maximum at 223 nM (Fig. 1GoB). These observations reflect the destabilizing effect of CaCl2 on the PP II helical conformation adopted by the retro-inverso CD28 peptide. The CD spectrum of the six residue-free peptides comprising the MYPPPY motif alone showed a weak minimum ({theta} = -8.6 x 101 deg·cm2·dmol-1) at 208 nm (data not shown). This suggests that the length of the CD28 peptide and the side chain interactions with the flanking residues are important in the formation of a PP II helix.



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  		FIGURE 1. CD spectra of EL-CD28 showing {theta} minimum (A) and RI-CD28 showing the {theta} maximum (B) at 412 µM in PBS 50% trifluoroethanol at 25°C (-), at 90°C ({triangleup}), and in 6 M CaCl2 ({circ}). {theta} is expressed in degrees cm2·dmol-1.

 
Binding of CD28 peptide analogs to CD80 Ig

Sensograms from direct kinetic analyses of EL-CD28 and RI-CD28 peptides are represented in Fig. 2Go, A and B, respectively. Both peptides reached equilibrium binding very rapidly (12–20 s) and in the washing phase dissociated rapidly (10–12 s). The background responses following injection of EL-CD28 and RI-CD28 peptides over an empty flow cell with no protein immobilized were equivalent and very similar to the responses obtained following injections of control peptides (data not shown). The RU observed at the beginning and end of the experiments were similar, indicating that the bound CD80 Ig was stable. Dissociation of L-CD28, EL-CD28, and RI-CD28 peptides from bound CD80 Ig was analyzed from the buffer flow phase of the sensogram. Kd and Ka for each curve were determined and were quite consistent over the entire range of concentrations used. A linear regression plot of the rate of change in the response against RU was plotted using these values of Ka and Kd for each CD28 peptide analyte. The slope of this plot was then plotted against the concentration of the peptide to yield a Kd of 2.44, 2.34, and 2.53 µM for L-CD28, EL-CD28, and RI-CD28, respectively, for binding to CD80 Ig (data not shown, Fig. 2Go, E and F). A short linear six-residue peptide encompassing the polyproline motif alone (MYPPPY) did not bind CD80 Ig consistent with the lack of PP II helix formation observed by CD studies (data not shown). This observation is not without foundation, because it is well known that small linear peptides do not exhibit defined conformation in solution.



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  		FIGURE 2. Affinity and kinetics of EL-CD28 and RI-CD28 binding to CD80 Ig. EL-CD28 (A) or RI-CD28 (B) at varying concentrations (375 nM to 18 µM) were injected for 300 s at 10 µl/min through a flow cell with CD80 Ig (1488/1512 RU) or no protein (Control, data not shown) immobilized. The binding at each concentration as measured in RU is shown. Injection phase analysis ({blacksquare}) and the residual ({diamondsuit}) of a representative binding curve following injection of EL-CD28 (6 µM) (C) or RI-CD28 (6 µM) (D) over immobilized CD80 Ig are shown. A plot of rate of change in the RU (dR/dt) against RU was calculated from the sensograms (data not shown), and the slope of this plot was plotted against concentration of EL-CD28 or RI-CD28. Ka of the interaction between EL-CD28 (E) or RI-CD28 (F) and CD80 Ig as measured from the slope of this plot is indicated. The dissociation rate constant is obtained from the intercept. The dotted line represents the trend. The data yield Kd of 2.34 and 2.53 µM for the interaction between CD80 Ig and EL-CD28 (E) or RI-CD28 (F), respectively.

 
A competitive binding assay was performed by injecting a mixture containing constant concentration of mouse CD28 Ig or mouse CTLA-4 Ig and increasing concentrations of CD28 peptides over immobilized CD80 Ig. Fig. 3Go, A and B, are representative sensograms obtained from the competitive experiments using the RI-CD28 peptide and CD28 Ig or CTLA-4 Ig, respectively. The top curve is the interaction of CD28 Ig (3.2 µM) or CTLA-4 Ig (0.8 µM) alone with the CD80 Ig. In Fig. 3GoA, the response level gradually decreases as the concentration of the peptide increases, indicating that the RI-CD28 peptide competes with CD28 Ig for binding CD80 Ig. A similar decrease in RU was also observed when increasing concentrations of EL-CD28 peptide were competed with CD28 Ig (3.2 µM) for binding CD80 Ig (data not shown). In contrast, no appreciable decrease in RU was observed when increasing concentrations of the RI-CD28 peptide (51.6–804 µM) were competed with CTLA-4 Ig for binding the CD80 Ig (Fig. 3GoB). Similar results were observed for the EL-CD28 peptide (data not shown). However, at a concentration of 1237 µM or 3 mg/ml both the RI-CD28 peptide and the EL-CD28 peptide exhibited a decrease of 189 and 168 RU, respectively (Fig. 3GoB and data not shown). This suggests that at higher concentrations, the EL-CD28 and the RI-CD28 peptides may compete weakly with the CTLA-4 fusion protein for binding the CD80 ligand.



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  		FIGURE 3. Competition between RI-CD28 and CD28 Ig (A) or CTLA-4 Ig (B) for binding CD80 Ig. An overlay of sensograms obtained from injection of a mixture of constant concentration mCD28 Ig (3.2 µM) (A) or mCTLA-4 Ig (0.8 µM) (B) and RI-CD28 at the indicated concentration at 5 µl/min over a flow cell with bound CD80 Ig. The top curve represents the binding of CD28 Ig alone (A) or CTLA-4 Ig alone (B) in the absence of the competing peptide. The response of CD28 Ig binding (A) decreases with increasing concentration of the RI-CD28 (6.2–412 µM). The response of CTLA-4 Ig binding (B) decreases appreciably (189 RU) only at the highest concentration (1237 µM) of RI-CD28.

 
Effect of CD28 blockade on activation of TCR-transgenic T cells specific for MBP NAc1–11

To investigate the inhibitory potential of the CD28 peptides, in vitro T cell proliferation assays were performed. MBP-specific proliferative responses of CD4+ lymphocytes from transgenic mice bearing the V{alpha}4/V{beta}8.2 TCR specific for MBP Ac1–11 were determined in vitro in the presence of varying concentrations of CD28 peptides. Fig. 4Go shows a significant decrease in the proliferative response of CD4+ LNC to MBP NAc1–11 when treated with L-CD28, EL-CD28, or RI-CD28 peptides. Maximum inhibition was observed in CD4+ LNC at 120 µM concentrations of L-CD28 (59.5%), EL-CD28 (47.6%), and RI-CD28 (45.7%) peptide. The proliferative responses of CD4+ splenocytes were also decreased but to a slightly lesser extent with the observed maximum inhibition of 50.2, 38.2, and 42% with L-CD28, EL-CD28, and RI-CD28 peptides, respectively (data not shown). In addition, the blocking effect of the synthetic CD28 peptides was assessed over a wide range of MBP NAc1–11 concentrations, reflecting widely differing TCR ligation signals. CD4+ spleen cells exhibited a significant decrease in the proliferative response when stimulated with MBP NAc1–11 at concentrations of 0.01–10 µg/ml in the presence of EL-CD28 or RI-CD28 peptides (Fig. 5Go). However, at the highest Ag concentration (100 µg/ml), this inhibitory effect was lost. These results are consistent with previous observations that CD28 costimulation can be bypassed in the presence of increased TCR ligation (2). The control RL-CD28 and D-CD28 peptides did not show inhibition (Fig. 4Go). Moreover, the hexapeptide consisting of the hydrophobic motif only did not show inhibition of T cell proliferation either. These results suggest that synthetic CD28 peptides block the CD28 signaling required for sustained T cell proliferation in vitro.



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  		FIGURE 4. Ag-specific T cell-proliferative responses of LNC from MBP-specific TCR-transgenic mice (V{alpha}4/V{beta}8.2 TCR) cultured with CD28 peptides. Single cell suspensions of CD4+ T cells isolated from the lymph nodes (5 x 104 cells/well) were stimulated with the encephalitogenic peptide of MBP, NAc1–11 (10 µg/ml), and cultured for a total of 72 h (including an 18-h pulse with [3H]thymidine) either alone (light gray column) or in the presence of 25 (black column), 75 (open column), and 100 µM (dark gray column) concentrations of the CD28 peptide analogs as shown. Data represent mean thymidine uptake and are plotted as {Delta} cpm ± SE. Results are the mean of three different experiments. Baseline proliferation of CD4+ LNC cultured in the absence of MBP NAc1-11 was 310.06 ± 42.88 cpm. Proliferative responses of CD4+ LNC treated with L-CD28, EL-CD28, and RI-CD28 at all concentrations used were significantly less than untreated and control peptide (RL-CD28 and D-CD28)-treated cells (#, p < 0.05; *, p < 0.01 by ANOVA).

 


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  		FIGURE 5. Effect of synthetic CD28 peptides over a range of TCR stimulation. Single cell suspensions of CD4+ splenic T cells (5 x 104 cells/well) were stimulated with MBP NAc1-11 (0.1–100 µg/ml) and cultured for a total of 72 h (including an 18-h pulse with [3H]thymidine) in the presence of indicated concentrations of CD28 peptide analogs as shown. Data represent mean thymidine uptake and are plotted as {delta} cpm ± SE. Baseline proliferation of CD4+ cells cultured in the absence of MBP NAc1-11 was 2845.6 cpm (light gray columns, 0.1 µg/ml), 2855 cpm (light gray columns, 0.1 µg/ml), 3102.53 cpm (black columns, 1 µg/ml), 4170 cpm (open columns, 10 µg/ml), and 4004.22 (dark gray columns, 100 µg/ml) cpm, respectively. Proliferative responses were significantly less in CD4+ spleen cells stimulated with NAc1-11 at 0.01–10 µg/ml and treated with EL-CD28 and RI-CD28 than in untreated cells. No significant difference was observed in the proliferative responses of CD28 peptide-treated and untreated CD4+ spleen cells at the highest NAc1-11 concentration (100 µg/ml).

 
Expansion of T cells following CD28 ligation results from an increase in IL-2 production (34).

To determine whether the observed reduction in proliferation is reflected in IL-2 secretion, the ELISPOT assay was performed. Fig. 6Go shows that MBP-stimulated CD4+ LNC bearing V{alpha}4/V{beta}8.2 TCR exhibited a significantly reduced frequency of IL-2-secreting cells in the presence of 75 µM EL-CD28 or 100 µM RI-CD28 peptide when compared with untreated cells.



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  		FIGURE 6. Fewer cells secrete IL-2 when stimulated in the presence of CD28 peptides. CD4+ T cells were isolated from the peripheral lymph nodes of V{alpha}4/V{beta}8.2 TCR-transgenic mice, stimulated with 10 µg/ml of MBP NAc1-11 peptide either alone or in the presence of the specified concentrations of CD28 peptides, and assayed by ELISPOT. A significant decrease in the number of NAc1-11 responders was observed following treatment with EL-CD28 and RI-CD28 at 75 and 100 µM concentrations. Results are representative of two experiments (#, p < 0.05 by one-way ANOVA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The induction of a productive immune response and maintenance of T cell homeostasis is largely determined by the balance of costimulation provided by the interacting B7:CD28/CTLA-4 molecules. In this study, we selectively blocked T cell CD28 costimulation by using a peptide mimic of its ligand-binding epitope as an antagonist for B7:CD28 interactions. Synthetic T cell costimulatory receptor peptides incorporating the MYPPPY sequence fold into a PP II helical structure, bind CD80 Ig with low affinity, and block the expansion of encephalitogenic T cells in vitro.

Previous studies targeting the members of B7:CD28/CTLA-4 pathway for immunotherapeutic modulation of T cell responses reported variable outcomes (5, 6, 35). In EAE, anti-B7-1 treatment suppressed disease induction, injection of both anti-B7-1 and anti-B7-2 or anti-B7-2 alone exacerbated the disease (36). Interestingly, treatment with CTLA-4 Ig, which blocks both B7-1 and B7-2, has been shown to either suppress or enhance clinical signs of EAE depending on the time of administration (37). These opposing results have been attributed to unintended simultaneous inhibition of down-regulatory B7:CTLA-4 interactions. Therefore, a method to selectively block B7:CD28 interaction alone, sparing the B7:CTLA-4 interaction, would be an attractive approach for achieving maximum therapeutic advantage. Perrin et al. (11) have shown that adoptive transfer of MBP-primed cells cultured in the presence of anti-CD28 Fab resulted in decreased incidence and severity of EAE. In recent years, there has been considerable interest in the development of nonimmunogenic peptide therapeutics with greater access across tissue barriers as antagonists for protein-protein interactions.

The importance of the polyproline motif in the CDR-3-like region of CD28 for binding B7 ligands is well documented by mutagenesis (16, 29, 30, 38). However, the results of a previous study (39) and our observations have shown that a synthetic peptide consisting of the MYPPPY motif alone does not constitute a minimal ligand-binding epitope. In general, a "functional epitope" defined by mutagenesis is much smaller than the "structural epitope" seen in the three-dimensional structures (40).

Using the solution structure of the extracellular domain of CTLA-4 as a template, Bajorath et al. (16) constructed a model of the extracellular domain of CD28 and predicted that the conserved 99MYPPPY104 motif may be modeled as a {beta} bulge. However, the authors also suggested that this region is likely to be more flexible in CD28 than in CTLA-4 and may possibly adopt a different conformation due to the presence of an N-linked glycosylation site at N107 (16). In this study, we show that 20-mer synthetic CD28 peptides consisting of the MYPPPY motif and flanking residues exhibit a typical CD spectrum of a PP II helix. Similar nonrepetitive proline-rich sequences implicated in binding include the PXXP motif in Src homology 3 domains of cytoskeletal proteins and the PPPGHR motif in the cytoplasmic region of CD2 (41). Proline-rich sequences in these proteins have been demonstrated to preferentially adopt a PP II helical conformation presenting an easily accessible hydrophobic surface as well as a good hydrogen-bonding site (18). The presence of proline with a bulky side chain in the solvent-exposed region precludes tight binding. However, the weaker binding by proline-rich regions offers the potential advantage of rapid modulation that favors very fast on and off rates. Such interactions are commonly observed under conditions of rapid recruitment and/or interchange of proteins such as cytoskeletal rearrangements. To our knowledge, this is the first report of a PP II helical conformation in the ligand-binding region of a TCR protein involved in signal transduction.

We evaluated the kinetics of binding of synthetic CD28 peptide to B7 ligands. Both EL-CD28 (2.34 µM) and RI-CD28 (2.53 µM) interacted with CD80 Ig with an affinity constant that is slightly lower than that of CD28 Ig (3.17 µM). Comparable binding with fast kinetics and low affinities for B7-1:CD28 interaction has been previously reported (19). In contrast, a linear peptide derived from the conserved regions of the human CTLA-4 molecule (KICKVELMYPPPYYLGIGNGA) did not bind B7-1 (39). A plausible explanation for the lack of binding by this peptide is the presence of multiple glycines distal to the polyproline motif in the CTLA-4 molecule, in accord with the CD studies of glycine/proline copolymers showing that the presence of glycine in polyproline peptides disrupts the PP II helical structure that may be important for binding (42).

Significantly, synthetic CD28 peptides competed efficiently with CD28 Ig to bind CD80. This demonstrates that the CD28 peptides form complexes with CD80 and thus represent a ligand-binding epitope. Due to their weaker affinity for binding CD80, the synthetic CD28 peptides compete weakly at high concentrations (3 mg/ml) with CTLA-4 fusion protein for binding the CD80 ligand. The use of synthetic CD28 peptides at such high concentrations may not be therapeutically achievable in vivo without toxic effects. Collectively, these results suggest that the CD28 peptides have a greater potential to selectively block B7:CD28 interactions while maintaining the higher affinity B7:CTLA-4 interactions largely intact.

Functionally, CD28 peptides act as antagonists of B7:CD28 interaction as evidenced by their ability to inhibit MBP-stimulated encephalitogenic T cells in vitro. Furthermore, treatment with CD28 peptides also resulted in a significant decrease in the frequency of IL-2-secreting cells among activated encephalitogenic T cells, substantiating the inhibitory potential of the synthetic CD28 peptides. This suggests that the competition between the synthetic CD28 peptides and the cell surface CD28 for binding the B-7 ligands may result in a quantitative reduction in CD28 receptor occupancy, thereby decreasing the probability of signaling. Previous studies have reported a similar reduction in the expansion of encephalitogenic cells following CD28 blockade by anti-CD80 Ab or CTLA-4 Ig (43, 44, 45, 46).

Aside from the ability to augment T cell proliferation, CD28 costimulation has the potential to directly enhance the survival of T cells in vitro by preventing their deletion by apoptosis (44, 47). A previous study has shown that blockade of B7:CD28 interaction decreases T cell survival in response to Ag stimulation in vitro (48, 49). Our preliminary results show that the number of apoptotic cells is significantly higher in cultures treated with EL-CD28 peptide than in untreated or control peptide-treated cells stimulated with MBP Ac1–11.

Current strategies for immunomodulation of autoreactive T cell responses include the use of mAbs to block the critical molecules involved in T cell activation (50). However, the value of these Abs as effective therapeutic agents is limited by virtue of their inherent immunogenicity. By comparison, small peptide-based therapeutics are less likely to be immunogenic, with the potential for use over longer periods. Furthermore, in contrast to large proteins, peptides have substantially lower m.w. and are more likely to cross tissue barriers into the target organ such as the CNS. Previously, Jameson et al. (13) showed that insertion of a "P-G-P" motif in the peptide analogs derived from the CDR3-like region of murine CD4 resulted in a structural mimic of CD4 that exhibited similar therapeutic effects as anti-CD4 Abs in EAE.

Data presented in this study demonstrate that a peptide mimic of the ligand-binding region of CD28 inhibits expansion of encephalitogenic CD4+ T cells in vitro. Thus, we have identified an active peptide derived from a well-characterized cell surface receptor, CD28, wherein the binding affinity of the large protein has been transferred to a short peptide mimic. Recently, our results show that a single in vivo administration of the EL-CD28 or RI-CD28 peptide prevented the induction of EAE and, more importantly, suppressed established EAE in B10.PL mice.4 Inhibition of T cell activation by synthetic CD28 peptides has been achieved in the absence of a cross-linking agent. Thus, in addition to providing a novel approach to develop new therapeutic agents, synthesis of bioactive peptides designed to mimic the surface epitopes involved in protein-protein interaction offers a powerful technique for characterization of the mechanisms involved in immune responses.


   Footnotes
 
1 This work is supported in part by National Institutes of Health Grants RO1 AI40302 (to P.T.P.K.) and RO1 AI43376 (to C.W.). Back

2 Address correspondence and reprint requests to Dr. Pravin T. P. Kaumaya, College of Medicine, Obstetrics, and Gynecology, Suite 302, Comprehensive Cancer Center, 410 West 12th Avenue, Columbus, OH 43210. E-mail address: kaumaya.1{at}osu.edu Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CDR, complementarity-determining region; PP II, polyproline type II; RU, Back

4 M. Srinivasan, I. E. Gienappp, S. S. Stuckman, C. J. Rogers, P. T. P. Kaumaya, and C. C. Whitacre. Suppression of experimental autoimmune encephalomyelitis using a retro-inverso peptide mimic of CD28. Submitted for publication. Back

Received for publication November 7, 2000. Accepted for publication April 26, 2001.


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