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J. Biol. Chem., Vol. 283, Issue 18, 12528-12537, May 2, 2008

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Identification of Human T Cell Receptor -recognized Epitopes/Proteins via CDR3 Peptide-based Immunobiochemical Strategy^*

From the Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Peking Union Medical College, National Key Laboratory of Medical Molecular Biology, Beijing 100005, China

Received for publication, September 27, 2007 , and in revised form, March 5, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Human T lymphocytes, bearing T cell receptor (TCR) , play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCR have been defined so far. To investigate antigenic epitopes/proteins recognized by T cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR chain (CDR3) peptide-mediated epitope/protein-binding assays. CDR3 peptides synthesized using the CDR3 region in TCR V2 chain were validated for their binding specificity to target cells or tissues. These CDR3 peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionization-tandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCR, including human mutS homolog 2 (hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to T cells but also functionally activate T cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCR. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of V2 T cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCR. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by T cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
T lymphocytes are classified structurally into two types according to different T cell receptors (TCR)³ β or . TCRβ specifically recognize antigenic peptides (epitopes) presented by MHC I/II molecules. Although many peptide ligands have been identified for TCRβ (1), only a few ligands have been identified for TCR so far (2, 3).

Human T cells account for 5% of CD3⁺ T cells in the peripheral blood, but constitute a major T cell subset in other anatomic locations, such as the intestine. In the peripheral blood of healthy individuals, TCR V2 chain pairing with one particular V9 chain is expressed on 50-90% of the T cells, whereas intestinal intraepithelial T cells frequently express the V1 gene, which can associate with different V elements (4). The antigen-binding site of TCR is formed primarily from three complementarity-determining regions (CDRs) contributed by each V and V domain. CDR1 and CDR2 fragments are encoded by germ line V genes, whereas the CDR3 is formed by somatic rearrangement of V(D) and J fragments. Sequence diversity in antigen receptors is not evenly distributed among all six CDRs but is highly concentrated in one or two CDR3. It had been proposed that the principal antigen specificity of an immunoglobulin or TCR is derived from its most diverse CDR3 fragments (5).

In addition to TCR diversity, structures of the TCR-CD3 complex, and tissue distribution, the pattern of antigenic recognition is another key difference between T cells and conventional βT cells. TCRT cells have two important features in their antigenic recognition. First, TCR is structurally similar to the B cell receptor (BCR) on B lymphocytes, binding to its antigens or epitopes directly to trigger a fast response against foreign pathogens or self-malignant cells (6). Second, TCR and Toll-like receptors (TLRs) are functionally alike, because these receptors recognize conserved molecules (7).

Although T cells only account for a small proportion in the human T lymphocyte pool, they play important roles in both anti-tumor and anti-microbe responses in innate immunity (8). Human T cells recognize stress-induced or malignancy/infection-related antigens. Such recognition is mediated through a non-MHC restricted manner in most cases. Among the known antigens for T cells, the most frequently identified ones are non-peptide antigens, such as pyrophosphoantigen (naturally occurring and synthetic antigens) recognized by T cells bearing V92 heterodimer TCR chain (9-11). In addition, classical MHC and MHC-like molecules can directly serve as ligands for T cells (12). MHC class I chain-related gene A (MICA) and B (MICB) are recognized by V1 T cells (13, 14). UL-16 binding protein (ULBP) is a novel ligand for NKG2D receptor in humans (15). We found that RAET1E2, a soluble isoform of the UL16-binding protein RAET1E produced by tumor cells, inhibits NKG2D-mediated NK cytotoxicity (16). Because of the sequence and structure similarity of the extra-cellular domains between MICA and ULBPs, ULBPs are also considered as antigens for V1 T cells (17). Heat shock proteins (HSP) are highly conserved among prokaryotes and eukaryotes and are known to be involved in various stress conditions. HSP had been implicated in T cell-mediated anti-tumor response (18, 19). Moreover, other protein antigens, such as ectopically expressed mitochondrial ATPase, have also been identified as ligands for TCR (20).

However, a crucial issue regarding T cells is the paucity of the identified ligands for these cells. To date, only a few human TCR-recognized protein antigens have been reported. To better understand T cell function, we have developed a new technical strategy to identify human TCR-recognized epitopes/proteins by combining immunological and biochemical methods using the CDR3 peptide of T cell receptor 2 chain (CDR3)-mediated epitope/protein-binding assays (CEPAs). This CDR3 peptide-based immunobiochemical strategy was based on a hypothesis that the primary sequence of CDR3, especially CDR3, because of its similarity to CDR3β of TCRβ and V_H CDR3 of BCR in gene composition, could serve as the key determinant of specificity in antigen binding by the TCR. We have confirmed this hypothesis using in vitro binding assays. Synthesized TCR V2 CDR3 peptides derived from tumor-infiltrating lymphocytes (TILs) in ovarian epithelial carcinoma (OEC) could specifically bind tumor cell lines and tissues, suggesting the determinant role of CDR3 in antigen binding. Moreover, CDR3 peptide-mediated binding specificity was blocked by preincubation with the same peptide, which decreased the cytotoxicity of T cells to OEC cells in vitro, further indicating such binding is specific (21). Based on our previous findings, we then used synthesized CDR3 peptides as specific probes to identify putative TCR-recognized antigenic epitopes in a peptide library and TCR-recognized proteins in an affinity chromatography system. As shown in supplemental Fig. 1, CDR3 peptide-based immunobiochemical strategy is technically composed of four steps as follows: selecting probes for epitopes/proteins, screening epitopes/proteins, identifying epitopes/proteins sequences, and validating epitopes/proteins functions. Based on the specific binding of CDR3 peptide to its target, we developed a set of CEPAs, which contains eight assays running through all the four steps of our strategy, including the following: 1) CDR3 peptide-mediated surface plasmon resonance (SPR) to determine interaction between CDR3 peptides and target proteins; 2) CDR3 peptide-mediated immunofluorescence assays to detect specific binding of CDR3 peptides to target cells; 3) CDR3 peptide-mediated enzyme immunoassays to detect specific binding of CDR3 peptides to target proteins and tissues; 4) competitive test to confirm CDR3 peptide sharing antigenic binding specificity with TCR; 5) panning putative epitope peptides in a 12-mer random peptide phage-displayed library with immobilized CDR3 peptides; 6) screening antigenic proteins in affinity chromatography with CDR3 peptide as affinity molecule; 7) phage-ELISA to select single CDR3 peptide-binding phage clone; and 8) CDR3 peptide-mediated Western blotting to detect CDR3 peptide-binding proteins.

With this strategy, we have identified seven tumor-related epitopes and two HBV infection-related epitopes, as well as two self-proteins, including HSP60 and human mutS homolog 2 (hMSH2). HSP60 has been identified as a ligand for T cells (18, 19), whereas hMSH2 is a DNA mismatch repair protein in the cell nucleolus (22). We show that hMSH2 is expressed on tumor cell membrane and recognized by V2 T cells, suggesting that hMSH2 may be a novel ligand for TCR. Taken together, our findings provide a novel strategy to identify epitopes/proteins for TCR.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Peptide Synthesis—The following four V2 CDR3 peptides were synthesized, including three (OT1, OT2, and OT3) corresponding to the sequences derived from TIL in OEC and one CDR3 peptide HP1, whose sequence was derived from a peripheral T cell clone from an HBV-infected patient: OT1, CACDSHGPSRLMMEGGLLGTDKLIFGKG; OT2, CARKDLPINNWGIPRIDKLIFGKG; OT3, CDFPSHTFHSTGGHTTDKLIFGKG; and HP1, CACDHLPLGDTRVHDKLIFGKG. Meanwhile, a structurally very similar peptide HP1m was synthesized with mutations of the first four amino acids and was employed as the control for HP1: HP1m, TQEHHLPLGDTRVHDKLIFGKG. The conformational preference of the CDR3 peptides OT3 and HP1 was analyzed by molecular dynamics. The results suggested that both of them displayed loop-like conformations in water (supplemental Fig. 2 shows the C- root mean square deviation of the CDR3 peptides). The modeled structure of OT3 and HP1 is shown in Fig. 1A and Fig. 2A, respectively. Seven tumor-related epitope peptides, named as EP1 to EP7, and two HBV infection-related epitopes, EP8 and EP9, were also synthesized (Table 1). All peptides were biotinylated by adding a biotin tag at the N terminus of each peptide sequence for detection in the following assays. Peptides were synthesized by the Academy of Military Medical Sciences, China. The purity of each peptide was 85% in high performance liquid chromatography analysis.

SPR Assay—SPR experiment was performed using an IAsys biosensor (Affinity Sensors). The surface of CMD chip was activated by 200 mM N-ethyl-N'-dimethylaminopropylcarbodiimide and 50 mM N-hydroxysuccinimide. 10 ng of HSP60 was coupled to the activated CMD chip. After immobilization, the surfaces were blocked with 1 mM ethanolamine (pH 8.5) and then washed with 1 mM formic acid to remove noncoupled proteins. OT3 peptide from low to high concentrations (0.25 to 2 mM) consecutively and HP1/HP1m peptides (0.5 mM) were employed for the binding assay at room temperature. BIAevaluation version 3.1 (Biacore AB) was used for data analysis.

FCM and Confocal Microscopy Assays—Immunofluorescence assays were applied to assess CDR3 peptide/OT3 graft immunoglobulin (OT3-Ig), whose V_H CDR3 region of a human autoantibody (IgG) recognizing thyroglobulin was replaced with OT3 peptide sequence (21), epitope peptide-mediated binding activity to target cells (tumor cells/T cells), and the expression of hMSH2 on tumor cells. For FCM assay, cells were incubated with biotinylated CDR3/epitope peptides, OT3-Ig (mouse IgG), and rabbit anti-hMSH2 polyclonal antibody (Santa Cruz Biotechnology). FITC-conjugated streptavidin (Pierce) or FITC-conjugated goat anti-mouse/rabbit IgG antibody (Pierce) was then added and incubated for 30 min at 4 °C. Wide type immunoglobulin (WT-Ig) and rabbit IgG (Zhongshan, China) were used as controls for OT3-Ig and rabbit anti-hMSH2 polyclonal antibody. The cells were analyzed on a FACSort flow cytometer (BD Biosciences). For confocal microscopy, cells were fixed on slides by 2% cold paraformaldehyde and in turn incubated with biotinylated synthesized HP1 peptide and with FITC-conjugated streptavidin. Cells incubated with biotinylated synthesized HP1m peptide instead of the HP1 peptide were used as controls. Slides were examined with a confocal laser microscope (LSM 510; Carl Zeiss).

Immunohistochemistry and ELISA—Immunohistochemistry assay was used to evaluate CDR3 peptide-mediated binding activity to target tissues. Briefly, formalin-fixed paraffin-embedded sections of HBV-infected liver tissues or normal tissues were deparaffinized and then boiled by microwave for antigen retrieval. After quenching with hydrogen peroxide, the sections were blocked with 5% goat serum. Biotinylated synthesized HP1 peptide (5 µg) was then added to the slides. The sections were subsequently incubated with horseradish peroxidase (HRP)-conjugated streptavidin (Zhongshan, China). Binding was visualized using diaminobenzidine (Sigma) as the substrate and observed under a light microscope. In CDR3-ELISA, 96-well plates were coated with proteins extracted from SKOV3 cells (10 µg/well) in 0.1 M NaHCO₃ (pH 9.6), or formalin-fixed SKOV3 cells (4 x 10⁵/well). After blocking with 3% bovine serum albumin solution, the plates were incubated with biotinylated CDR3 peptides or OT3-Ig/WT-Ig. After reaction with HRP-conjugated streptavidin (Pierce) or HRP-conjugated goat anti-mouse IgG antibody (Pierce) and substrate (Sigma), the plates were then read on a microplate reader (Labsystem) at 450 nm.

MTT Colorimetric Test and CDR3 Peptide Competitive Test—This test was used for evaluation of T cell cytotoxicity to tumor lines. Briefly, SKOV3 cells as target cells were seeded onto 96-well plates (1 x 10⁴/well). For competitive testing, target cells were preincubated with CDR3 peptides (0.1 mg/ml) or anti-hMSH2 polyclonal antibody (0.2 mg/ml) at 37 °C for 2 h, and equivalent rabbit IgGs (Zhongshan, China) were used as controls for anti-hMSH2 polyclonal antibody. Then T cells as effector cells were incubated with target cells for another 8 h (the T cells were preincubated with epitope peptides for 2 h in stimulating assay). MTT solution (5 mg/ml) was added to wells (15 µl/well) and incubated at 37 °C for 4 additional hours. The reaction was stopped by the addition of 100 µl of lysis buffer (10% SDS) to dissolve the tetrazolium crystals. The plate was examined in a Multiskan Microplate Reader (Thermo Lab-systems, Finland) and the percentage of specific lysis was calculated by using Equation 1,

(Eq.1)

T_A = absorbance of target cells for control; E_A = absorbance of effector cells for control, and (E + T)_A = absorbance of effector cells cultured with target cells. The absorbance was determined at 570 and 630 nm.

Panning of CDR3 Peptide-binding Phage Clones in a 12-mer Random Peptide Phage-displayed Library—A 12-mer random peptide phage-displayed library (New England Biolabs) was screened by CDR3 peptides as follows: a 96-well plate was coated with 100 µl/well coating buffer (containing 10 µg CDR3 peptide) for 2 h at 37 °C and then blocked with PBS containing 3% bovine serum albumin overnight at 4 °C. The primary library solution was added to the wells (10 µl per well containing 10¹¹ colony-forming units) and shaken gently at room temperature for 1 h. After thoroughly washing with PBS, 0.1% Tween 20, the CDR3 peptide-binding phages were eluted by acidic buffer (0.2 M glycine-HCl (pH 2.2)) and neutralized by 1 M Tris-HCl (pH 9.1) immediately. In some cases, a high concentration of CDR3 peptide (200 µg/ml) was used for competitive elution as well. The concentration of Tween 20 in washing buffer was increased to 0.3 and 0.5% in the following two rounds of biopanning and was kept at 0.5% thereafter.

Affinity Chromatography—Affinity chromatography was used to enrich CDR3 peptide-binding proteins with the following procedures. CDR3 peptide was coupled to a 1-ml HiTrap N-hydroxysuccinimide-activated HP column (Amersham Biosciences) (5 mg) through its C-terminal hydroxyl group in coupling buffer (0.2 M NaHCO₃, 0.5 M NaCl (pH 8.3)). The excess active groups uncoupled to CDR3 peptide were deactivated with solution (0.5 M ethanolamine, 0.5 M NaCl (pH 8.3)). CDR3 peptide-coupled column was then thoroughly equilibrated by binding buffer (20 mM PBS (pH 7.0)). After the proteins were applied to the column, the absorbance value of the outflow at 280 nm was detected, and elution buffer (1% formic acid) was changed until the A₂₈₀ returned to the base line. The fractions containing eluted proteins were collected and neutralized quickly with 1 M Tris-HCl (pH 9.1). throughout the process, the flow rate was kept under 0.2 ml per min.

Identification for CDR3 Peptide-binding Phage Clones by Phage-ELISA, PCR, and Sequencing—After five rounds of panning, individual plaque was picked randomly, and positive phage clones were selected by phage-ELISA using HRP-anti-M13 phage antibody (Amersham Biosciences). Briefly, wells of ELISA plate were coated with CDR3 peptides and blocked as in the panning procedures. Single colony phage was added into CDR3 peptide-coated wells and incubated at 37 °C for 2 h. After fully washing, HRP-anti-M13phage antibody was added and incubated at 37 °C for 1 h. The positive phage clones in ELISA were then amplified, and epitopes containing parts were obtained by PCR and sequenced with the ABI automatic sequencer 377 (PerkinElmer Life Sciences) using the -96 sequencing primer, 5'-CC CTC ATA GTT AGC GTA ACG-3' (supplied in the kit).

Identification of CDR3 Peptide-binding Proteins by SDS-PAGE and CDR3 Peptide-mediated Western Blotting—The enriched proteins through affinity chromatography were separated by SDS-PAGE. After proteins were electrically transferred to a nitrocellulose membrane, the blot membrane was placed in blocking buffer. The biotinylated CDR3 peptide (10 µg/ml) was used as the primary antibody, and HRP-streptavidin was employed as the secondary antibody for usual Western blotting. Chemiluminescent HRP substrate (Pierce) was added, and the blot was exposed to a x-ray film for an appropriate duration.

Data Base Search and Protein Identification—The in-gel trypsin digestion and MS analysis of a certain SDS-PAGE protein band was carried out at Beijing Proteome Research Center, China. LC ESI-MS/MS analyses were performed using an LTQ system (Thermo Finnigan). Tryptic peptides mixtures were first loaded onto a capillary column (15 cm length, 75 µm inner diameters). The elution from reverse phase liquid chromatography column was directed on line to be detected by MS. All the MS/MS spectra were searched using SEQUEST algorithm-based Bioworks version 3.2 (Thermo Finnigan, San Jose, CA) against the International Protein Index human data base 3.19, which had 60,397 entries (23). Proteins with at least two unique "identity" score peptides were considered as being unambiguously identified, and the single peptide-matched proteins were renounced.

Epitope Peptide-immobilized Expanding Assay and [³H]TdR Incorporation Assay—For expanding assay, PBMC were cultured in 24-well plates with immobilized epitope peptides (10 µg/well), using blank wells and anti-pan-TCR-monoclonal antibody (1 µg/well)-coated wells as negative and positive controls, respectively. Cells were then cultured in RPMI 1640 medium (Invitrogen) with 10% FCS and IL-2 (200 units/ml). The percentage of T cells was measured 2 weeks later by FCM. In [³H]TdR incorporation assay, T cells (purity >90%) were first cultured without IL-2 for 48 h. The resting T cells were then seeded onto 96-well plates with 1 x 10⁵ cells per well. After 60 h of stimulation with epitope peptides or IL-2 plus epitope peptides, [³H]TdR was added into wells, and the cells were cultured for an additional 8 h. The proliferation of T cells was measured by counts/min value.

Reverse Transcription-PCR and Sequence Analysis—Total RNA was isolated from EP1- and EP8-expanded T cells with TRIzol reagent (Promega). cDNA was synthesized using oligo(dT) (Promega) and Moloney murine leukemia virus reverse transcriptase (Promega) in the reverse transcription reaction. The V regions of 9 and 2 chains were amplified by PCR using specific primers as follows: V2(+), 5'-GCCATTGAGTTGGTGCCTGAACAC-3';V2(-), 5'-CGGATGGTTTGGTATGAGGCTGAC-3';V9(+), 5'-GCAGGTCACCTAGAGCAACCTC-3'; and V9(-), 5'-GGCTTGGGGGAAACATCTGCATC-3'. Amplified cDNA was cloned into the pGEM-T easy vector (Promega) and sequenced with the ABI automatic sequencer 377.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Specific Binding Activity of Synthesized CDR3 Peptides to OEC Cell Line SKOV3—To work as probes, synthesized CDR3 peptides must bind specifically to tumor cells. Although we previously reported the specific binding activities (21), several binding experiments were repeated to confirm the binding. Fig. 1B showed that the peptide OT3 bound to tumor cell lines but not to normal PBMC. OT3-mediated ELISA was used for evaluation of the interactions between OT3 and tumor cells. Both SKOV3 cells (Fig. 1C) and SKOV3 total protein extracts (Fig. 1D) bound OT3 peptide in a dose-dependent manner. OT3 competed with TCR on T cells expanded from PBMC (Fig. 1E), resulting in a reduced cytotoxicity of human T cells to SKOV3 cells in vitro (Fig. 1F). These data suggest that OT3 peptide and TCR bind the same ligands on SKOV3 cells. Similar results have been obtained from peptides OT1- and OT2-mediated binding and competitive assays (data not shown). The interaction between the OT3 graft immunoglobulin (OT3-Ig) and tumor cell lines/protein extracts were analyzed by FCM and ELISA, respectively. Similar to OT3 peptide, the OT3-Ig bound to SKOV3 and HeLa, whereas the WT-Ig did not bind to any cell lines (Fig. 1G). OT3-Ig bound to SKOV3 total protein extracts in ELISA, and the WT-Ig did not (Fig. 1H), suggesting specific binding.

CDR3 Peptide HP1 Specifically Binds to HBV-infected Tissues/Cells—To further investigate CDR3 peptide binding specificities, we analyzed the binding of HP1 peptide to HBV-infected liver tissues and cell line 2.2.15 containing the intact dimers of the HBV genome, respectively. HP1 peptide specifically bound 2.2.15 cells but not normal cells (PBMC) and other types of tumor cell lines, such as SKOV3 and HepG2 (Fig. 2B). In immunohistochemistry assay, HP1 peptide specifically bound hepatocarcinoma tissue (Fig. 2C). Strong fluorescent staining was detected on HBV-infected 2.2.15 cells after incubating with HP1 peptide in confocal microscopy assay (Fig. 2D). Consistent with the above results, ELISA shows the specific interaction of HP1 peptide and HBV-infected 2.2.15 total protein extract (Fig. 2E). Taken together, our data suggest that the binding of synthesized HP1 peptides to targets, either tissue, cell, or protein extract, is specific.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Synthesized CDR3 peptide OT3 specifically bound tumor cell line SKOV3. A, representative structure of OT3 peptide in molecular dynamics simulation. C- trace was shown with residues colored according to their types (blue for acidic amino acids, red for basic, yellow for polar, and gray for nonpolar residues). B, flow cytometry analysis of surface staining of tumor cell lines by OT3 peptide. Normal PBMC was used as control. Cells were stained with biotinylated (bio)-OT3 peptide and FITC-conjugated streptavidin. C and D, ELISA of OT3 peptide binding to SKOV3 cell and its protein extracts. SKOV3 cells (4 x 105/well) or protein extracts (10 µg/well) have been immobilized on ELISA plate. The bio-OT3 peptide was added with various concentrations, and HRP-conjugated streptavidin was added subsequently. Each assay was run in duplicate. *, p < 0.05, A value of SKOV3 cell-coated wells detected with bio-OT3 (1 µg/well), when compared with that of mock wells; **, p < 0.01, A value of SKOV3 cell coated wells detected with bio-OT3 peptide (10 µg/well), when compared with that of mock wells; A value of SKOV3 protein extracts coated wells detected with bio-OT3 peptide (1 or 10 µg/well) compared with that of mock wells. E and F, OT3 peptide competitive binds SKOV3 in vitro. MTT assay evaluating cytotoxicity inhibition of T cells against SKOV3 after OT3 peptide preincubation. PBMC-derived T cells with the purity more than 90% in FCM analysis were used as effector cells in MTT assay (E). OT3 peptide (10 µg/well) was incubated with SKOV3 cells for 2 h before adding T cells. Results are representative of three independent experiments (F). The A value of OT3 peptide added wells was compared with that of wells without OT3 (*, p < 0.05, E:T ratio = 10:1; **, p < 0.01, E:T ratio = 20:1, and E:T = 40:1). E:T stands for the ratio of effector cells to target cells. G, flow cytometry analysis of surface staining by OT3-graft Ig with tumor cells SKOV3 and HeLa (blue). WT-Ig was used as control (red). Results are representative of three independent experiments. H, ELISA of OT3-graft Ig to SKOV3 total protein extract. The proteins extracts have been coated on an ELISA plate (1 µg protein/well). The OT3-Ig and WT-Ig were added, respectively, to the plate in the same concentration (5 µg/ml). Each assay was run in duplicate.

BLAST—BLAST search was performed to identify human proteins containing motifs in the sequences of the CDR3 peptide-bound phage displaying peptides. The BLAST results of the nine sequences that we chose as candidate epitopes are listed in supplemental Table 3. No exactly matched protein sequences were returned from BLAST analysis, which was based on primary sequence searching, and the best matching manner had a maximum of 7 uninterrupted amino acids matching all 12 amino acids. In addition, most proteins in BLAST results were from prokaryotes or insects and hypothetical proteins, consistent with a role of TCR in innate immune recognition. The BLAST results of other CDR3 peptide-bound motifs were similar (data not shown).

View larger version (43K): [in this window] [in a new window]   FIGURE 2. HBV infection-related CDR3 peptide HP1 specifically bound HBV-infected tissues/cells/cell extracts, and HBV infection-related epitope peptides stimulatedT cells in vitro. A, representative structure of HP1 peptide in the molecular dynamics simulation. C- trace was shown with residues colored according to their type (the same as in Fig. 1A). B, flow cytometry analysis of surface staining by HP1 peptide to PBMC, tumor cell lines (SKOV3 and HepG2), and HBV-infected cells 2.2.15 (red). For cells with HP1m peptide, a structurally very similar peptide to HP1 with mutations of the first four amino acids, staining served as negative controls (green). Blue line shows the staining with PBS. C, immunohistochemistry staining of hepatocarcinoma tissues. The same tissues were stained with biotinylated HP1 peptide, HP1m peptide, and PBS as shown from left to right. Staining was visualized using diaminobenzidine as the substrate (brown, x200). D, confocal microscopy analysis of the staining of HP1 peptide on HBV-infected cells 2.2.15. Binding of biotinylated HP1 peptide was analyzed by confocal microscopy after staining with FITC-conjugated streptavidin. As a control, control peptide HP1m and PBS instead of HP1 peptide was used. E, HP1 peptide specifically bound 2.2.15 protein extracts in ELISA. **, p < 0.01, A value of 2.2.15 cell protein extract-coated wells detected with HP1 peptide, when compared with all controls, including blank, HepG2 protein extracts, and HP1m peptide. F, immobilized EP8 and EP9 could induce expansion of T cells from PBMC. We used panel 1, RPMI 1640 medium mock as negative control; panel 2, immobilized 1 µg of anti-pan-TCR monoclonal antibody as positive control; panels 3 and 4, immobilized 10 µg of epitope peptides EP8, EP9 respectively. G, EP8 and EP9 could induce proliferation of resting T cells at 10 µg/ml in [3H]TdR incorporation assay.

Identification of OT3-specific Tumor Proteins—We screened the total cell lysate of SKOV3 tumor cells directly using OT3-mediated affinity chromatography. The 1-ml HiTrap N-hydroxysuccinimide-activated HP column coupled with 4.5 mg of OT3 with a coupling ratio of 90% (data not shown). After washing with elution buffer, the elution peak was collected (Fig. 4A). The protein fractions were pooled and concentrated. SDS-PAGE showed that only two protein bands with molecular mass of 45 and 60 kDa, respectively, can be obviously observed (Fig. 4B). Western blotting analysis indicates that only the 60-kDa protein was OT3-specific, and its amount was enriched when compared with SKOV3 total proteins (Fig. 4C). The specific band in the SDS-polyacrylamide gel was analyzed by LC ESI-MS/MS. A total of 15 proteins with at least two unique peptides was identified for the 60-kDa protein under the appropriate cutoff parameters for SEQUEST search, among which pyruvate kinase 3 isoform 1, DNA mismatch repair protein (hMSH2), and HSP60 demonstrated highly matching unique peptides count and higher cover percentage (supplemental Table 4).

Validation of HSP60 and hMSH2—Stressful conditions up-regulate the expression of HSP60, which has been considered as a target antigen involved in the activation of T cells (18, 19). We examined the binding of CDR3 peptide OT3 to HSP60 using SPR. OT3 peptide indeed bound HSP60 in a dose-dependent manner (supplemental Fig. 3). We chose hMSH2 as a potential new ligand for TCR for further characterization. In FCM, 21% of HepG2 and 43% of SKOV3 tumor cell lines were positive for surface staining with antibody against hMSH2, whereas less than 7% of normal T cells expressed hMSH2 (Fig. 4D). In cytotoxicity blocking assay, cytotoxicity of V2 T cells to SKOV3 cells was blocked by anti-hMSH2 antibody, whereas cytolytic activity of V1 T cells could not be inhibited (Fig. 4, E and F), suggesting that hMSH2 may be a ligand for V2 T cells. In ongoing investigations, we are cloning and expressing recombinant hMSH2 protein for further functional tests. Preliminary data revealed that hMSH2 cloned from SKOV3 cDNA was mutated (data not shown), suggesting that the mutant hMSH2 might be displayed on tumor cell surface to be recognized by immune cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we demonstrate a novel strategy to identify epitopes/proteins recognized by T cells by combining immunological and biochemical methods. With this strategy, we identified nine T cell-reactive epitopes and two antigenic proteins, HSP60 and hMSH2. Another putative antigenic protein (pyruvate kinase 3 isoform 1) showed possible sequence characteristics as a ligand for TCR according to MS analysis, although the validation proceedings continue. Therefore, our method provides a novel and specific high throughput system to identify epitopes/proteins recognized by T cells.

This strategy includes four independent but closely related technical steps, including selecting probe for epitopes/proteins, screening epitopes/proteins, identifying epitope/protein sequences, and validating epitope/protein functions. The binding specificity of synthetic CDR3 peptides to target molecules forms a principal base for a set of CEPAs.

In selecting probes for T cell-recognized epitopes/proteins, we used four assays to verify the specificity of these peptides, including CDR3 peptide-mediated SPR, enzyme immunoassay, immunofluorescence assays, and peptide competition tests. Biotin-streptavidin affinity system makes an amplification signal, which includes enzyme immunoassays as well as immunofluorescence assays. These results were consistent with the nonbiotinylated CDR3 peptide in the SPR assay, indicating the CDR3 peptides were specific and could be used as selecting probes. Moreover, data from the CDR3 peptide competitive test further confirmed the specificity of CDR3 peptide for their targets. Although CDR3 peptide showed good binding activities to targets, there were some shortcomings. A linear peptide with less than 30 amino acid residues would have some unspecific binding. To circumvent this, we constructed and expressed a CDR3-graft antibody, whose CDR3 sequences in heavy chains were replaced by CDR3. The CDR3-graft antibody showed the similar binding specificity with CDR3 peptides. In summary, the tested CDR3 peptides showed specific binding activities to target proteins, cells, and tissues and can serve as probes for screening epitopes/proteins.

CDR3 peptides were employed in two different ways: panning a 12-mer random peptide phage-displayed library and binding by affinity chromatography. One issue is whether we can obtain possible epitope peptides in the 12-peptide library. It is reported that T22, an identified protein recognized by murine T cells, formed a TCR-recognized epitope 15-18 amino acid residues in length (24). Therefore, the 12-mer random peptide phage-displayed library might cover most parts of the TCR-recognized epitopes, suggesting that the strategy is feasible. We used four CDR3 peptide probes in this study. As a result, we found that CDR3 peptide had sufficient affinity to its bound proteins in the CDR3 peptide-mediated affinity chromatography, which was confirmed by the enriched collection of eluted proteins from SKOV3 cell total proteins with an elution peak. Therefore, our data suggest that CDR3 peptidemediated affinity chromatography is useful for isolation of ligand for TCR. Because the principle of this technique is based on interaction between the ligand-binding side (such as CDR3) and the receptor-binding side (such as epitope), the peptides from the ligand-binding side in other immune receptors, such as NKG2D, TLR, and BCR, might also be tested for their ligand proteins in a similar manner.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Tumor-related epitope peptides bound and stimulated T cells in vitro. A, flow cytometry analysis of T cells surface staining by biotinylated epitope peptide EP7. The same staining of J.RT3-T3.5 cells was performed as control group. B, expansion of T cells from PBMC by immobilized epitope peptides. Panel 1, RPMI 1640 medium was added as negative control; panel 2, 1 µg of anti-pan-TCR monoclonal antibody was immobilized as positive control; panels 3-8, 10 µg of epitope peptides EP1-6 were immobilized, respectively. The figure shows phycoerythrin (PE)-anti-TCRβ and FITC-anti-TCR double staining after culturing for 2 weeks. C and D, epitope peptides EP3, EP4, and EP5 triggered the proliferation of resting T cells in [3H]TdR incorporation assay. The counts/min value showed the activation status. E, MTT assay to evaluate the change of cytolytic activity of the T cells against SKOV3 on the binding of EP6. T cells expanded from PBMC as mentioned above were used as effector cells, and SKOV3 were target cells. Different doses of EP6 were incubated with T cells for 2 h before adding to target cells. Results are representative of three independent experiments.

In validating epitopes/proteins functions, we tested the binding of putative epitopes to T cells through a series of experiments. These CDR3-bound epitope peptides were able to bind T cells and could trigger their proliferation and cytotoxicity to target cells in vitro. The finding of HSP60 as a ligand for TCR with our strategy is sufficient and powerful evidence for the validity of the CDR3 peptide-based immunobiochemical strategy. HSPs were first discovered in cellular response to hyperthermia stress (25). As molecular chaperones, HSPs were first known to play critical roles in protein folding (26). Moreover, it was found that stressful conditions up-regulate the expression of HSPs, and HSPs are involved in immune responses to various pathogens and tumors (27, 28). HSPs have been identified as target antigens of tumor cells for T cells (18, 19). The T cells of oral cancer patients were able to lyse tumor cells of the same origin via recognition of HSP60 on the surface of oral tumor cells (29). The cell surface expression of HSP70 on heat-stressed tumor cells but not on unstressed tumor cells increases their susceptibility to lysis by V92 T cell clones derived from autologous blood lymphocytes, and both the anti-V2 monoclonal antibody and anti-HSP70 monoclonal antibody inhibit the responses (30). Our SPR analysis confirmed again the binding of synthesized CDR3 peptide to HSP60 (supplemental Fig. 3). Besides, hMSH2 was identified.

View larger version (42K): [in this window] [in a new window]   FIGURE 4. The enrichment, detection of OT3-bound proteins, and functional validation of hMSH2. A, chromatogram of OT3 peptide-bound proteins of total protein extracts of SKOV3 cells in affinity chromatography. The 1st vertical line at 0 ml pointed the time that sample was added. After protein loading, most proteins unable to bind OT3 were washed away in the first throughput peak. The smaller figure at the top right corner showed the absorbance of the whole throughput peak at 280 nm with most uncombined proteins. The elution peak appears after changing to elution buffer at the 2nd vertical line; the arrow indicates the elution peak we pooled. B, 10% SDS-PAGE analysis of pooled fractions of affinity chromatography. Lane 1, low molecular weight protein markers. Lane 2, total protein extracts from SKOV3 cells. Lane 3, concentrated fractions of affinity chromatography. The arrows marked the main protein bands. C, detection of OT3-binding proteins by Western blotting. Western blotting using bio-OT3 and HRP-streptavidin was done to reconfirm which protein band in the fractions bind OT3. Lanes 2 and 3 are the same as B. The arrows indicate the OT3-bound protein bands in Western blotting. D, surface staining of hMSH2by flow cytometry analysis of tumor cells and normal cells (T cells). Rabbit IgG instead of anti-hMSH2 antibody was employed as control. E and F, the cytotoxicity ofT cells against SKOV3 was blocked by anti-hMSH2-antibody in MTT assay. PBMC-derived V2T cells with the purity of 91% and TIL-derived V1T cells with the purity of 88% in FCM analysis were used as effector cells in MTT assay, respectively (E). Anti-hMSH2 polyclonal antibody (0.2 mg/ml, 5 µl) was incubated with SKOV3 cells for 2 h before adding T cells. SKOV3 cells with rabbit IgG blocking were used as control. The cytolytic ratio was measured under an E to T ratio of 5:1 (F).

	FOOTNOTES

 
^* This work was supported by Grants 2001CB510009, 2004CB518706, and 2007CB512405 from the National Program for Key Basic Research Projects; Grants 2006AA02Z480 and 2006AA02A245 from the National Program for Biomedical Hightech, the Ministry of Science and Technology of China; and Grant 30490244 from the National Natural Science Foundation of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1-5 and Figs. 1, 2 and 3.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, National Key Laboratory of Medical Molecular Biology, 5 Dong Dan San Tiao, Beijing 100005, China. Tel.: 86-10-65136981; Fax: 86-10-65105909; E-mail: heweiimu{at}public.bta.net.cn.

³ The abbreviations used are: TCR, T cell receptor; BCR, B cell receptor; CDR, complementarity-determining region; CDR3, 3 in T cell receptor chain; CEPA, CDR3 peptide-mediated epitope/protein-binding assay; FCM, flow cytometry; FCS, fetal calf serum; HBV, hepatitis B virus; hMSH2, human mutS homolog 2; HSP, heat shock protein; MHC, major histocompatibility complex; MICA/B, MHC class I chain-related A/B; OEC, ovarian epithelial carcinoma; PBMC, peripheral blood mononuclear cell; SPR, surface plasmon resonance; TIL, tumor-infiltrating lymphocyte; TLR, Toll-like receptor; ULBP, UL-16 binding protein; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; WT, wild type; LC/ESI/MS/MS, liquid chromatography/electrospray ionization-tandem mass spectrometry; TdR, thymidine; IL, interleukin.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Dr. Keng Shen for providing tumor cell line SKOV3 and Dr. Yemei Fan for help on peptide synthesis.

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