T-Cell Antigen Receptor Peptides Inhibit Signal Transduction within the Membrane Bilayer

Clinical Immunology Vol. 105, No. 2, November, pp. 199 –207, 2002 doi:10.1006/clim.2002.5270

T-Cell Antigen Receptor Peptides Inhibit Signal Transduction within the Membrane Bilayer Xin M. Wang,* ,1 Julianne T. Djordjevic,† ,1 Nozomu Kurosaka,* Stephen Schibeci,† Lianne Lee,* Peter Williamson,† and Nicholas Manolios* ,2 *Department of Rheumatology and †Department of Immunology, Westmead Hospital, Sydney, New South Wales 2145, Australia

Previous studies have shown that a synthetic peptide (core peptide, CP) corresponding to a 9-aminoacid region in the transmembrane domain of the ␣ subunit of the T-cell antigen receptor (TCR) can suppress T-cell function in vitro and in vivo. The aim of these experiments was to determine the cellular site and molecular mechanism of CP inhibition in T cells. The cytochrome c-sensitive TCR-expressing hybridoma (2B4) was stimulated with pigeon cytochrome c antigen, anti-CD3 crosslinking, or PMA and ionomycin, in the presence or absence of CP, and the resulting IL-2 produced was measured in a bioassay using an IL-2-dependent cell line (CTLL-2). In the presence of CP, IL-2 production was inhibited following antigeninduced stimulation. By contrast, when stimulated with cross-linking antibodies to the CD3 complex or with PMA and ionomycin, both of which activate T cells downstream of the TCR antigen recognition site, CP had no effect on IL-2 production. These experiments suggest that CP interferes with TCR function by inhibiting T-cell activation at the transmembrane/receptor level. In addition, we show that CP inhibits early TCR signal transduction events such as TCR ␨ chain phosphorylation following stimulation with either antigen or anti-CD3-crosslinking antibodies, although this is unlikely to be the mechanism leading to the reduced IL-2 production. © 2002 Elsevier Science (USA) Key Words: T-cell antigen receptor; core peptide; Tcell activation; signal transduction; phosphorylation. INTRODUCTION

T cells constitute a critical cellular component of our immune system and are concerned with immune surveillance and antigen recognition. Loss of “self-tolerance” and subsequent recognition of “self” antigens by T cells lead to a large spectrum of autoimmune diseases including diabetes, allergy, arthritis, psoriasis, 1

Both authors contributed equally to the work. To whom correspondence and reprint requests should be addressed. Fax: 0061-02-98913749. E-mail: [email protected]. edu.au. 2

and multiple sclerosis (1– 4). The pathological consequences seen in these disorders are initiated by T-cell antigen receptor (TCR) activation present on the cell surface of T cells. Inhibition of T-cell function can suppress the T-cell-mediated autoimmune diseases (5–9). Hence, an understanding of TCR structure, function, and means of inhibition have important clinical ramifications. The TCR complex is a multichain structure composed of six distinct polypeptide chains. The TCR-␣ and -␤ chains form a heterodimer and are responsible for the recognition of foreign antigens that are presented in the context of MHC molecules on the surface of antigen-presenting cells (APCs). The invariant chains of CD3 (consisting of ␥, ␦, and ␧) and the ␨ chain dimer represent two signal-transducing modules responsible for coupling ligand binding to the signaling pathways that result in T-cell activation and the elaboration of cellular immune responses (reviewed by Cantrell (10) and Kane (11)). Antigen recognition by the TCR-␣ and -␤ chains induces receptor aggregation with accessory molecules (CD2, CD4, or CD8), leading to phosphorylation of CD3 subunits. This results in activation of kinases and an increase in intracellular calcium and culminates in the production and secretion of IL-2. A region critical for TCR assembly and function resides in the transmembrane region of the TCR-␣ chain (12, 13). This region consists of a stretch of nine amino acids, two of which are hydrophilic (lysine and arginine) and separated by four hydrophobic amino acids (GLRILLLKV). Previous studies have demonstrated that the two positively charged amino acids within this region are important for the interaction of the TCR-␣ chain with CD3 molecules and subsequent T-cell signaling (12–20). Previous results from our laboratory have shown that core peptide (CP), a synthetic peptide corresponding to this sequence, can suppress the immune response in a number of animal models of T-cellmediated inflammation (21). One of the earliest events to follow T-cell receptor activation is phosphorylation of the CD3 chains on

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tyrosine residues contained within immunoreceptor tyrosine-based activation motifs (ITAMs) by Src-family tyrosine kinases such as Lck and Fyn (22–26). The CD3-␥, -␧, and -␦ chains all contain one copy of ITAM. The TCR-␨ chain however, contains three ITAMs. As there are two tyrosine phosphorylation sites per ITAM, the TCR-␨ chain contains a total of six potential tyrosine phosphorylation sites, making it one of the most prominent phosphorylation substrates in T cells. Two major tyrosine phosphorylated forms of TCR-␨ (molecular masses of 21 and 23 kDa) have subsequently been consistently identified in primary T cells and T-cell clones and in murine T-cell clones, both phosphorylated forms are induced following TCR stimulation with antigen, coinciding with Src-kinase activation of ZAP-70 (27). The presence of multiple CD3 ITAMs with different phosphorylation potentials may be required to establish a threshold level of activation, thus distinguishing between a strong and weak TCR stimulus (28). The recent finding that CD3-␥, -␦, and -⑀ ITAM phosphorylation can substitute for -␨ ITAM phosphorylation suggests the existence of a phosphorylation hierarchy leading to the production of IL-2 (29). T cells can be activated in a number of ways. The TCR-␣␤ chains can be stimulated with a specific antigen presented by an antigen-presenting cell. Alternatively, monoclonal antibodies (mAbs) can be used to cross-link the extracellular domain of CD3 subunits, thus bypassing the TCR-␣␤ antigen recognition site. When used under appropriate conditions, these mAbs can induce resting T cells to secrete lymphokines, such as IL-2 (30, 31) and interferon-␥ (IFN-␥) (32), express a number of new cell surface molecules such as CD40 ligand (33), and to proliferate (34). Finally, activation can be achieved by incubating T cells with calcium ionophores and phorbol esters, bypassing the TCR complex entirely (35). Moreover, these reagents could induce lymphokine secretion in mutant cell lines that fail to express the TCR (36), suggesting that these pharmacologic reagents mimic or activate important signals downstream of the TCR. Calcium ionophores such as ionomycin induce an increase in cytoplasmic free calcium which activates calcineurin. Activated calcineurin subsequently activates transcription factors involved in IL-2 synthesis and secretion (37). Phorbol esters such as PMA activate the serine-threonine kinase, PKC, which also results in upregulated transcription and T-cell activation (10, 38, 39). The aim of this study was to determine the molecular site of IL-2 inhibition by CP. The approach involved stimulating T cells with antigen, anti-CD3 antibodies, or PMA/calcium ionophore and measuring the effect of CP on the level of IL-2 produced. The effect of CP on TCR-␨ ITAM phosphorylation, an early TCR signaling event, was also investigated.

MATERIALS AND METHODS

Cells The 2B4.11 cell line is a murine T helper cell hybridoma obtained by fusing pigeon cytochrome c (PCC)primed lymph node T cells with BW 5147 cells (40). This cell line expresses a complete TCR on the cell surface and recognizes a fragment of PCC presented by the class II MHC molecule, I-E K (40). LK35.2 cells are a MHC I- E K genotype B lymphoma cell line and are used in these experiments to present PCC fragments to 2B4.11 cells (41). CTLL-2 cells are an IL-2 dependent cell line used to measure IL-2 produced by 2B4.11 cells following antigen presentation (42). Antibodies and Reagents Monoclonal hamster IgG directed against mouse CD3-␧ chain (anti-CD3; 2C11) was purified from hybridoma supernatant. Rabbit anti-human ␨ was a kind gift from Dr. R. Klausner (NIH). Anti-rabbit and antimouse HRP conjugates were purchased from Amersham Pharmacia Biotech (Sydney, Australia). Monoclonal anti-phosphotyrosine antibodies (4G10) were obtained from Auspep (Melbourne, Australia). PCC, PMA, and ionomycin were purchased from Sigma (Sydney, Australia). Super signal (enhanced chemiluminescence reagent) was obtained from Selby Biolab (Sydney, Australia) and protein A–Sepharose was obtained from Amersham Pharmacia Biotech. Peptides Peptides (shown in Table 1) were purchased from Auspep. Synthesis was by FMOC chemistry, and synthesized peptides were shown to be 95% pure by HPLC. CP was modified by replacing the two positively charged amino acids (lysine and arginine) with either neutral hydrophobic amino acid (glycine, G) or two negatively charged amino acids (glutamic acid, E; aspartic acid, D). The modified peptides were named Peptide C and Peptide E, respectively. CP and LH-RH were dissolved in 0.1% acetic acid. MCC, Peptide C, and Peptide E were dissolved in RPMI medium or DMSO, respectively. The stock concentrations of CP, Peptide C, Peptide E, LH-RH, and MCC were 10 mM, 10 mM, 10 mM, 10 mg/ml, and 1 mM, respectively. Antigen Presentation Assay This has been previously described by Manolios (21). Briefly, 2B4 cells (5 ⫻ 10 4), LK cells (5 ⫻ 10 4), and PCC (50 ␮M) were incubated in a 96-well microtiter plate for 24 h at 37°C/5% CO 2 in the presence of peptides or solvent controls (acetic acid or DMSO) at various con-

TCR PEPTIDES INHIBIT SIGNAL TRANSDUCTION IN MEMBRANE BILAYER

centrations. Dose titration curves were performed to determine the lowest effective dose of CP and the maximum inhibition dose. CP is effective at 10 ␮M and reaches maximum inhibition at 25–50 ␮M. Higher doses of 100 ␮M do not lead to greater inhibition. As such we have opted to use 50 ␮M. Following incubation, the supernatants were collected and stored at ⫺20°C until IL-2 analysis using a CTLL-2 bioassay. MCC at a final concentration of 5 ␮M stimulated 2B4 cells equally as well as PCC (50 ␮M). IL-2 Bioassay IL-2 activity of the supernatant was determined using the IL-2-dependent cell line CTLL-2. Supernatants, collected following T-cell activation, were serially diluted and incubated with CTLL-2 (2 ⫻ 10 4) cells for 18 h at 37°C with 5% CO 2. [ 3H]thymidine (0.5 ␮Ci/ml) was added for 6 h and CTLL cells were collected onto glass fiber filter mats using a Titertek cell harvester. Radioactivity was counted using a Liquid Scintillation Analyzer (TRI-CARB). Sample radioactivity was compared to standard curves and expressed as IU per milliliter. The amount of IL-2 present in samples was compared to the IL-2 present in the positive controls and expressed as a percentage. We also note that CTLL cells can respond to IL-4. TCR Activation with Anti-CD3 Antibodies Microtiter plates were coated with anti-CD3 antibody (10 ␮g/ml) in PBS for 24 h at 4°C. 2B4 cells (5 ⫻ 10 4/well) were added with CP (50 ␮M) or acetic acid (0.005%) for 24 h at 37°C/5% CO 2. Following incubation the supernatants were collected, stored at ⫺20°C, and analyzed for IL-2 content. To exclude the possibility that CP, added with the anti-CD3 antibody, may not have had sufficient time to penetrate cell membranes in some experiments, 2B4 cells (1 ⫻ 10 6/well) were incubated in serum-free RPMI 1640 with CP (50 ␮M) or acetic acid (0.005%) in 6-well plates for 6 or 18 h at 37°C with 5% CO 2, prior to adding the anti-CD3 antibody. Following incubation, the cells were washed three times with RPMI 1640 medium and were added to anti-CD3 antibody-coated wells for 24 h at 37°C with 5% CO 2. Following incubation, the supernatants were collected, stored at ⫺20°C, and analyzed for IL-2 content. T-Cell Activation with PMA and Ionomycin PMA (40 ng/ml) and ionomycin (5 ␮g/ml) were added to 2B4 cells (5 ⫻ 10 4) and incubated with CP (50 ␮M) or acetic acid (0.005%) in 96-well microtiter plates for 24 h at 37°C with 5% CO 2. Following incubation the

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supernatants were collected, stored at ⫺20°C, and analyzed for IL-2 content. T-Cell Activation with Antigen following Preincubation of APCs with MCC and CP This has been previously described by Anderson et al. (43). Briefly, LK cells (5 ⫻ 10 5) and MCC (5 ␮M) were incubated with CP (50 ␮M) or acetic acid (0.005%) for 18 h at 37°C with 5% CO 2. Following incubation cells were washed twice with HBSS buffer and fixed with 1% paraformaldehyde for 15 min at room temperature. LK cells (1.5 ⫻ 10 5) and 2B4 cells (1.5 ⫻ 10 5) were incubated together for 24 h at 37°C with 5% CO 2 following washing with HBSS containing glycine (100 mM) buffer. After incubation the supernatants were collected, stored at ⫺20°C, and analyzed for IL-2 content. TCR-␨ Chain Phosphorylation To detect ␨ chain phosphorylation, 2B4 cells (1 ⫻ 10 7) were preincubated with or without CP or LH-RH (control peptide) at various concentrations (0.1 or 1 mM) in 1 ml of RPMI containing 2% FBS, 2 mM glutamine, and penicillin/streptomycin (RPMI/FBS medium), for 30 min at 37°C. Following washing with HBSS buffer, 2B4 cells were incubated with MCC (100 ␮M) and LK cells (1 ⫻ 10 7) or anti-CD3 antibody in 2 ml RPMI/FCS for various times (0, 3, 7, 15, and 30 min) at 37°C. Stimulation was terminated by adding 30 ml ice-cold HBSS buffer and placing samples on ice. Cells were pelleted by centrifugation and pellets were dissolved in 1 ml of NP40 lysis buffer (50 mM Tris–HCl, pH 7.5; 40 mM NaCl; 5 mM EDTA; 1% NP-40; 1 ␮g/ml aprotinin; 1 ␮g/ml leupeptin; 1 mM sodium orthovanadate, and 1 mM PMSF) and incubated for 30 min on ice. Lysates were centrifuged for 10 min at 800g to remove nuclei and mitochondria. Supernatants were recentrifuged for 10 min at 10, 000g to remove any remaining cell debris. Supernatants were precleared with 100 ␮l of a 20% slurry of protein A–Sepharose with mixing at 4°C for 30 – 60 min. Following centrifugation at 1000g for 1 min the supernatant was incubated with 5 ␮l of rabbit anti-␨ serum for 1 h at 4°C with mixing. Immune complexes were captured by an overnight incubation at 4°C with 100 ␮l of a 20% slurry of protein A–Sepharose. Protein A–Sepharose was collected by centrifugation and washed with 1 ml of 0.5% NP-40 lysis buffer five times. A final wash was performed with 10 mM Tris– HCl, pH 7.5 (1 ml), and proteins were eluted by boiling in 20 ␮l SDS–PAGE sample buffer for 7 min. Eluted proteins were analyzed by 13% SDS–PAGE. Proteins were transferred to nitrocellulose and analyzed by Western blotting.

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TABLE 1 Peptide (Amino Acid) Sequences Name

Sequence

CP Peptide C Peptide E MCC LH-RH

G-L-R-I-L-L-L-K-V-OH G-L-G-I-L-L-L-G-V-OH G-L-D-I-L-L-L-E-V-OH K-A-N-E-R-A-D-L-I-A-Y-L-K-Q-A-T-K E-H-W-S-Y-G-L-R-P-G

Western Blotting Phosphorylated ␨ chain was detected with anti-phosphotyrosine (4G10) antibody as described by the manufacturer. Briefly, nitrocellulose blots were blocked with 3% milk in PBS (PBS/milk) and then incubated with anti-phosphotyrosine (4G10) antibody (1 ␮g/ml) in PBS/milk, overnight at 4°C. Blots were washed and then incubated with anti-mouse-HRP (1:2000 dilution) for 90 min at room temperature. Following washing, bands were detected by ECL on X-ray film. In some cases, blots were stripped using reagents from Chemicon International Inc. and reprobed with rabbit antihuman ␨ (1:1000 dilution) followed by anti-rabbit HRP (1:2000 dilution). RESULTS

LK or 2B4 cells were incubated with varying concentrations of CP (1, 10, 50, or 100 ␮M) for 3 days. No cytotoxic effects were observed as assessed by trypan blue uptake or flow cytometric analysis following propidium iodide incubation (results not shown). Cell viability was greater than 95%.

show that IL-2 production as a result of CD3 stimulation did not significantly change in the presence of CP at a concentration of 50 ␮M when compared to the positive control (Fig. 2A; P ⬎ 0.1, n ⫽ 9). To exclude the possibility that CP, added with anti-CD3 antibody, may not have had sufficient time to penetrate cell membranes and inhibit crosslinking activation, 2B4 cells were preincubated with CP and then crosslinked with anti-CD3 antibody. When CP was preincubated with 2B4 cells for 6 or 18 h prior to TCR stimulation, the IL-2 production was the same as the positive control (Fig. 2B; P ⬎ 0.1, n ⫽ 9). CP Does Not Inhibit IL-2 Production Induced by PMA/Ionomycin Activation 2B4 cells were also stimulated with PMA/ionomycin to bypass the entire TCR complex. Similarly, CP had no significant effect on the level of IL-2 produced at a concentration of 50 ␮M when T cells were activated with PMA and ionomycin (Fig. 3; P ⬎ 0.1, n ⫽ 9). IL-2 Production Induced by Antigen Is Not Inhibited by CP following Preincubation of APCs with Moth Cytochrome c and CP A 17-amino-acid peptide corresponding to the antigenic region of moth cytochrome c (MCC) contains positively charged amino acids similar to that of CP. This MCC peptide sits in the binding groove of the MHC class II molecule and stimulates specific T cells. The ability of CP to compete with the MCC peptide for

CP Inhibits IL-2 Production in Antigen-Stimulated T Cells The effect of CP on IL-2 production in antigen-activated T cells is shown in Fig. 1. CP at concentrations of 10, 50, and 100 ␮M caused a decrease in IL-2 production by 20% ⫾ 5%, 45% ⫾ 7%, and 47% ⫾ 2%, respectively (n ⫽ 9). The dose-dependent decrease in IL-2 production was statistically significant with all CP concentrations except 1 ␮M (P ⬍ 0.01, n ⫽ 9). Because similar inhibitory effects were observed with 50 and 100 ␮M CP, a CP concentration of 50 ␮M was used in all subsequent experiments. When CP was substituted with neutral peptide (Peptide C) or negatively charged peptide (Peptide E), no effect on antigen-induced IL-2 production was observed. CP Does Not Inhibit IL-2 Production Induced by Anti-CD3 Antibody Crosslinking 2B4 cells were activated with anti-CD3 antibodies to bypass the ␣␤ antigen recognition site. The results

FIG. 1. CP inhibits antigen-induced IL-2 production by T cells. 2B4 cells and LK cells were incubated with PCC for 24 h in the absence or presence of CP, Peptide C, or Peptide E at the concentrations specified. CP inhibition of IL-2 was dose dependent. Peptide C and Peptide E had no affect on IL-2 production. Levels of IL-2 produced in these experiments were approximately 700 IU/ml.

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FIG. 3. CP does not inhibit IL-2 produced by T cells stimulated with PMA and ionomycin. 2B4 cells were incubated with PMA and ionomycin in the absence or presence of CP for 24 h. CP did not inhibit PMA/ionomycin-stimulated IL-2 production. Levels of IL-2 produced in these experiments were approximately 150 IU/ml.

compete with MCC for binding to the LK cells to reduce IL-2 production (P ⬎ 0.1, n ⫽ 9). CP Inhibits TCR-␨ Chain Phosphorylation

FIG. 2. CP does not inhibit IL-2 production by T cells activated by CD3 crosslinking. (A) 2B4 cells were incubated with anti-CD3 for 24 h at 4°C, in the absence or presence of CP. CP had no effect on IL-2 production following CD3 crosslinking. (B) 2B4 cells were preincubated with CP for 6 or 18 h prior to anti-CD3 activation. IL-2 production was not significantly different compared to control. Levels of IL-2 produced in these experiments were approximately 200 IU/ml.

binding to MHC class II and thus exert its inhibitory effects at the level of antigen presentation was examined. LK cells were preincubated with MCC peptide and CP or MCC peptide alone prior to incubation with T cells, and the IL-2 produced in each case was compared. We have previously performed dose titration studies and have shown that 5 ␮M MCC peptide is equivalent to 50 ␮M PCC (data not shown). As such, for these experiments the dose of MCC used was 5 ␮M. For these experiments, MCC was used instead of PCC because of its ability to compete with CP outside of the MHC II process pathway. In Fig. 4, the results show that there was no difference in IL-2 production between the two groups, suggesting that CP does not

The ability of CP to inhibit the early TCR signal transduction event of ␨ chain phosphorylation was also investigated. Time-course experiments of ␨ chain phosphorylation following antigen-induced T-cell activation were carried out. The results show that the amount of the phosphorylated ␨ chain isoforms (p21/23) detected by the anti-phosphotyrosine antibody (4G10) was substantially elevated using antigen incubation times of 15 to 30 min (Fig. 5A). These phosphorylated isoforms were absent when no cells or LK cells only were used in

FIG. 4. CP does not compete with MCC for binding to LK cells. LK cells were incubated with MCC in the absence or presence of CP for 18 h. Following washing, cells were fixed and incubated with 2B4 cells of 24 h. There was no difference in IL-2 production in cells pretreated with MCC and CP, suggesting that there is no competition between MCC and CP. Levels of IL-2 produced in these experiments were approximately 700 IU/ml.

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the ␨ immune capture assays. These controls are essential to demonstrate that the bands running above p21/23 represent the heavy and light chains of the immune capturing antibody (recognized by the secondary antibody in the Western blot) and that the bands running at 21 and 23 kDa represent the T-cell-specific ␨ chain phosphoisoforms. Incubating T cells with CP (0.1 and 1 mM) inhibited ␨ chain phosphorylation by 50 and 98%, respectively (Fig. 5B; lanes 3 and 4). By contrast, there was no effect on inhibition when CP was substituted with the same amounts of control peptide (LH-RH; Fig. 5B; lanes 5 and 6). LH-RH was chosen as a control peptide because of its similarity to CP in length and charge distribution. Protein content in cell lysates was equal in all samples tested as determined by standard protein approximation, excluding the possibility of differences in sample loading. At least a fivefold increase in ␨ chain phosphorylation (p21/p23) was achieved by incubating T cells with anti-CD3 antibody for 15 min (Fig. 5C, top, compare lanes 1 and 2). The high level of endogenous phosphorylation in lane 1 can be attributed to the fact that twice as much ␨ chain (p18 nonphosphorylated isoform) is present. This was determined by reprobing the membrane with anti-␨ antibodies (Fig. 5C, bottom, compare lanes 1 and 2) and quantitating the ␨ band following densitometric scanning. Preincubation of T cells with CP reduced ␨ phosphorylation to the level achieved without stimulation, after correcting for protein loading (Fig. 5C, top, compare lanes 1, 2 and 3). It should be noted that the ␨ chain polyclonal antibody used in these experiments can immune capture p21/p23 and p18 but only recognizes p18 in a Western blot. To assay for ␨ chain phosphorylation the number of T cells used for stimulation was greater (1 ⫻ 10 7/ml) than that required for the antigen presentation assay (2 ⫻ 10 5/ml). To account for this discrepancy in CP/cell availability the concentration of CP in the ␨ phosphorylation experiments was increased to 1 mM (ideally should have been 2.5 mM). Although the lower concentration (1 mM) was used, it was still effective in inhibiting ␨ chain phosphorylation. Cationic peptides similar to CP, such as magainin-2 (44 – 46), have been shown to lyse cells by forming pores within the cell membrane. To demonstrate that the observed decrease in ␨ chain phosphorylation was not due to CP exhibiting cytotoxic effects on T cells, PI uptake by treated and nontreated cells was assessed by flow cytometry. Results (not shown) show that PI uptake is not enhanced by CP-treated cells. DISCUSSION

We have previously published that administration of CP can inhibit in vitro IL-2 production by antigen-

FIG. 5. Treatment of T cells with CP inhibits TCR-␨ chain phosphorylation. (A) 2B4 cells were incubated with LK cells and MCC for the times indicated (lanes 3–7). TCR-␨ chain, immune-captured from each cell lysate (lanes 3–7) and from 2B4 and LK cells alone (lanes 1 and 2, respectively), was subjected to SDS–PAGE and immunoblotting with anti-phosphotyrosine (4G10). Lane 8 is a negative control without cells necessary to establish which bands in the Western blot are specific to the immune-capturing antibody. (B) 2B4 cells which had been pretreated with or without CP or LH-RH for 30 min at 37°C as indicated were incubated with LK cells and MCC for 15 min (lanes 2– 6). Cells in lane 1 were not stimulated with MCC and lane 7 is a negative control without cells. TCR-␨ chain was immune-captured from each cell lysate and subjected to SDS–PAGE and Western blotting with anti-phosphotyrosine (4G10). (C) 2B4 cells pretreated with or without CP for 30 min at 37°C were stimulated with antiCD3 antibody for 15 min as indicated. TCR-␨ chain was immunecaptured from each cell lysate and subjected to SDS–PAGE and Westernblotting with anti-phosphotyrosine (4G10). Blots were then reprobed with anti-TCR-␨ antibodies. The position of the TCR-␨ chain isoform is indicated by the arrows. The position of each molecular weight marker in kDa and the antibody used in each blot are also indicated.

activated T cells and suppress the immune response in a number of animal models of T-cell-mediated inflammation (21). The CP sequence is derived from a charged, 9-amino-acid region within the transmembrane domain of the TCR-␣ chain which is critical for

TCR PEPTIDES INHIBIT SIGNAL TRANSDUCTION IN MEMBRANE BILAYER

TCR assembly and function (12–20). Experiments in a nonlymphoid cell system have shown that the TCR-␣ chain can assemble with CD3-␦ and CD3-␧ via transmembrane charge interactions (12, 16, 47) and that these subunit interactions represent the initial steps in the binding hierarchy of TCR subunits (48). Based on the origin of the CP sequence, a possible mechanism of immunosuppression by CP may involve the disruption of TCR complexes by direct competition with the positively charged TCR-␣ chain for binding to the negatively charged ␦ and/or ␧ chains (12–20). In support of this hypothesis, we have recently demonstrated using confocal microscopy that CP specifically colocalizes with clustered plasma membrane TCRs. In the current study, we use antigen-stimulation assays, to further define the site of CP inhibition of IL-2 production. The results show that CP inhibited IL-2 production initiated by stimulation of the ␣/␤ heterodimer with antigen/MHC II class but not following antibody crosslinking of the ␧␦/␧␥ component of the CD3 complex, which bypasses the antigen-recognizing ␣/␤ heterodimer. In addition, CP did not inhibit IL-2 production due to PMA-activated PKC and downstream signaling molecules. The TCR antigen inhibition appears to be dependent on the presence of the two positive charges, as neutral peptide (Peptide C) and negatively charged peptide (Peptide E) did not reduce IL-2 production following antigen stimulation. This is consistent with the previous findings, that Peptide C and Peptide E exerted no immunosuppressive effects in animals with T-cell-mediated adjuvant-induced arthritis (21), and recent confocal microscopy results showing that biotinylated Peptide C and Peptide E interacted poorly with cell membranes (53). We have used BiaCore instrumentation to compare the membrane penetration efficiencies of the control Peptide C and Peptide E with CP. In contrast to CP, the acidic Peptide E showed little absorption to artificial membrane liposomes (DMPC and DMPG). However, Peptide C did absorb in a moderate fashion compared to CP (10% vs 20%, respectively, results not shown). Despite this absorbance, there is no corresponding decrease in IL-2 production, making Peptide C a “reasonable” peptide control for cytokine production assays. TCR-specific tyrosine phosphorylation events occur rapidly and can be observed within seconds of TCR stimulation (49). To exclude the possibility that TCR stimulation occurred before CP had a chance to penetrate the membrane, CD3 stimulation was also initiated following preincubation of CP with cells for 6 and 18 h. By using these conditions, no inhibition of IL-2 production was observed. To rule out the possibility that CP is not exerting its effect at the site of antigen presentation to T cells, APCs were stimulated with MCC in the absence or presence of CP and then incubated with T cells. No inhibition of IL-2 production was

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detected, indicating that CP does not compete with MCC for binding to the APCs. The similar result was also obtained by flow cytometry (results not shown). The results presented suggest that the site of CP action leading to reduced IL-2 production occurs within the T-cell membrane at the site between the antigen recognition complex and the CD3 signaling module. We next examined whether CP has an effect on early TCR signaling events such as TCR-␨ chain phosphorylation. The results showed that CP inhibited TCR-␨ chain phosphorylation triggered by stimulation through either the ␣/␤ heterodimer or the CD3 complex. Although the result for antigen stimulation was expected, the latter result with CD3 stimulation was not. Although CP inhibited ␨ chain phosphorylation triggered by CD3 crosslinking, such an alteration in early signaling is apparently not sufficient to produce a decrease in IL-2 production, as demonstrated in Fig. 2. Thus inhibition of ␨ chain phosphorylation is unlikely to be the mechanism leading to reduced IL-2 production and is most likely a separate effect. In support of our results, transfecting ITAM-defective ␨ chain into a TCR-␨ ⫺/⫺ cell line to reconstitute cell surface TCR expression did not affect IL-2 production relative to cells transfected with wild-type (ITAM-intact) ␨ chain (29) suggesting that ␨ chain phosphorylation is not essential for IL-2 production. The conclusion from this finding is that there must be redundancy in the contribution of CD3 ITAM phosphorylation to IL-2 production as previously suggested (29). Although models have been postulated to explain TCR structure with respect to the arrangement of the ␣/␤ heterodimer and the CD3 complex based on the formation of a hierarchy of assembly intermediates (13, 16, 20, 48), the positioning of the ␨ chain is still not known. The addition of the ␨ dimer, however, is thought to be the final and ratelimiting step involved in TCR assembly and association of ␨ chain with the ␣/␤, and the CD3 complex is essential to obtain expression of functional TCR at the cell surface (48). Although ␨ chain ITAM phosphorylation coincides with TCR activation it does not appear to be essential for IL-2 production (29). At this stage we can only speculate about how CP is exerting its effect on IL-2 production. The mechanism of action could be due to a failure to induce a configurational change required for signaling by CD3 or steric inhibition of supramolecular complexes formed during T-cell activation in lipid rafts. This latter point remains the current focus of research. The amino acid sequence of CP bears a resemblance to a growing family of antimicrobial peptides. Common structural features of these peptides as seen in magainins, mastoparans, alamethicin, cecropin A, sarcin, and antiflammins are that they are amphipathic and in the lipophilic milieu of membranes most likely form ␣-helical conformation. This amphipathic character en-

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ables membrane permeabilization and/or perturbation to occur. The net positive charge of two or more basic amino acids could facilitate their interaction with negatively charged membrane phospholipids. These peptides have recently been the subject of a series of biophysical studies, investigating interactions between peptides and lipid surface (50 –52). These studies concluded that such peptides could either absorb onto the membrane surface or insert into the membrane as in a cluster of helical bundles. The ability to engineer different peptides and quantitatively measure the effect of CP and analogues on T-cell function by changing the physicochemical characteristics of the peptide by amino acid/charge substitutions offers a unique opportunity in understanding structure–function relationships and protein–lipid interactions. We have modified the C-terminal valine of CP by linking it to gly-tris-palmitate, -dipalmitate, and –tripalmitate and demonstrated that both the CP and its lipopeptide conjugates translocated into fibroblasts and T cells using FITC as a fluorochrome. A more dramatic biological effect was noted with lipopeptides in preventing IL-2 antigen production in vitro and the onset of arthritis in the adjuvant induced arthritis model (data unpublished). We believe that the number and type of lipid residues have a significant effect on membrane insertion of the peptide conjugates. In summary, these results support the hypothesis that the site of CP inhibition leading to reduced IL-2 production is upstream of the CD3 complex and most likely at the interface between the antigen recognition unit and the CD3 signal transducing unit. These results highlight the potential use of CP as a probe for TCR function and possibly in the future as a treatment of T-cell-mediated autoimmune diseases. ACKNOWLEDGMENT We thank Dr. V. Bender for her technical support and helpful discussions.

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