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Potent Cytolytic Response by a CD8⁺ CTL Clone to Multiple Peptides from the Same Protein in Association with an Allogeneic Class I MHC Molecule¹

Shigeki Kageyama^2,*, Theodore J. Tsomides^3,*, Naomi Fukusen^4,*, Ioannis A. Papayannopoulos^5,*, Herman N. Eisen^* and Yuri Sykulev^6,

^* Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CTL clone 2C recognizes the allogeneic class I MHC molecule L^d in association with peptides derived from -ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface L^d than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with L^d as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.

	Introduction

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Natural ligands for the Ag-specific receptor (TCR) on CD8⁺ CTL are complexes of short peptide fragments bound to MHC class I molecules. These peptides are normally generated by proteosomes from cytosolic proteins in the target cells (1) and are transferred into the lumen of the endoplasmic reticulum (ER)⁷ by transporter molecules. In the ER, peptides bind nascent class I MHC molecules to form peptide-MHC (pMHC) complexes that are translocated to the cell surface for survey by CTL (2).

Clone 2C was originally derived from an H-2^b mouse immunized with H-2^d cells expressing K^d, L^d, and D^d class I MHC proteins (3). 2C CTL specifically responds to L^d in association with the naturally processed peptide LSPFPFDL (p2Ca), isolated from spleen and other tissues (4), or the longer natural peptide VAITRIEQLSPFPFDL (p2Cb), isolated from the same source and containing the entire sequence of p2Ca (5). Both peptides are derived from a ubiquitous intracellular protein, oxoglutarate dehydrogenase (OGDH) (5, 6). A synthetic p2Cb peptide was digested in vitro with cellular extracts containing proteosomes and was found to produce active fragments, suggesting that it might be a natural precursor of smaller active peptides (5).

The synthetic peptide QLSPFPFDL (QL9), extending by one amino acid from the N terminus of p2Ca in the murine OGDH sequence, was 100-fold more potent in sensitizing L^d+ target cells for specific lysis by 2C CTL than naturally occurring p2Ca (7). Because this peptide has not been isolated to date from tissue extracts (5, 8), it is unclear whether it is naturally processed and presented on target cells as an epitope for 2C CTL or behaves as a heteroclitic ligand. To address this issue, we tested p2Ca, p2Cb, and QL9 using three experimental approaches: peptide binding to L^d on live L^d+ cells, in vitro digestion of p2Cb by purified 20S proteosomes, and the endogenous expression of minigenes encoding these peptides in target cells. We show that QL9 can be produced in vitro from the longer natural precursor p2Cb by 20S proteosomes and that cells transfected with the QL9 minigene become susceptible to specific lysis by 2C CTL. We also found that the N-terminal glutamine residue of QL9 is rapidly converted to pyroglutaminyl in the low pH trifluoroacetic acid (TFA) solution, the medium generally used to extract peptides from tissues. This conversion precludes the verification of QL9 by Edman degradation, which may explain our inability to identify this peptide in tissue extracts.

In addition to QL9, several longer peptides sharing a common C-terminal sequence with the QL9 end were cleaved from p2Cb by 20S proteosomes. Synthetic analogs of these peptides bind L^d to form peptide-L^d complexes recognizable by 2C CTL. All these data indicate that the strong allogeneic response of 2C CTL to L^d+ target cells is mediated by numerous peptides presented on target cells in the context of L^d. Although most of these peptides are derived from OGDH, the presence of other L^d-binding peptides from different sources that might serve as ligands for 2C CTL cannot be excluded. Recognition of multiple ligands by the same TCR could help to account for the exceptional vigor of some allogeneic T cell responses.

	Materials and Methods

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Cells

The CD8⁺ CTL 2C from the original clone was maintained as described previously (3). T2-L^d is a human cell line T2 that lacks the TAP transporter genes and was transfected with the L^d gene (9). C1R, a mutant human cell line that does not express HLA-A2, was derived from the B lymphoblastoid cell line (10). C1R cells transfected with the gene for L^d are called C1R-L^d. T2-L^d and C1R-L^d (gifts from P. Cresswell, New Haven, CT) were maintained in K medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME) in the presence of 250 and 400 µg/ml of the antibiotic G418, respectively.

Peptides

Peptides were synthesized using conventional tBoc chemistry (Biopolymers Laboratory, Massachusetts Institute of Technology, Cambridge, MA), and many of them were purified by HPLC. Peptide concentrations were determined by quantitative amino acid analyses and/or bicinchoninic acid assay.

Peptide radiolabeling

The monoiodinated radioactive form of the mouse CMV peptide (¹²⁵I₁-pMCMV) was prepared as previously described (11). Briefly, Na¹²⁷I and Na¹²⁵I were mixed to give a 4- to 5-fold molar excess of total iodide of predetermined specific activity over peptide. The reaction was conducted in the presence of Iodobeads (Pierce, Rockford, IL) for 30 min at pH 7.0. The mono- and di-iodinated peptide derivatives and unlabeled peptide were separated by HPLC. Fractions containing the monoiodinated peptide were lyophilized and resuspended in H₂O. The specific activities of the radiolabeled peptides were determined by counting aliquots in a gamma counter (Packard, Downers Grove, IL).

Cytotoxicity assay

⁵¹Cr-labeled target cells (2 x 10⁴ cells/well) were incubated in triplicate with peptide in K medium for 30–60 min (37°C) in 96-well round-bottom microtiter plates before addition of 2C CTL at a CTL:target cell ratio of 5:1. After 4 h at 37°C in a CO₂ incubator, plates were centrifuged (200 x g, 5 min), and 100-µl aliquots from each supernatant were counted in a gamma counter. Percent specific lysis was determined as: 100 x (⁵¹Cr release in the experimental supernatant - spontaneous release)/(total release in detergent - spontaneous release). Spontaneous release controls contained a peptide plus ⁵¹Cr-labeled target cells but no 2C CTL. Total release controls contained ⁵¹Cr-labeled target cells in the presence of 10% detergent (Nonidet P-40). In the absence of peptide, the target cell lysis by CTL was <1% for C1R-L^d cells and did not exceed 10% for T2-L^d cells.

Competition assay to measure peptide binding

Peptide binding to L^d on live cells was measured as previously described (12). In brief, T2-L^d cells were suspended at 10⁷ cells/ml in either K medium or PBS supplemented with 0.1% BSA containing protease inhibitors aprotinin (2 µg/ml), PMSF (100 µg/ml), EDTA (5 mM), and iodoacetamide (20 µg/ml). Cells (2.5 x 10⁶) were incubated in microfuge tubes with a fixed concentration of HPLC-purified monoiodinated pMCMV (¹²⁵I₁-pMCMV) and various concentrations of unlabeled peptides in a total volume of 500 µl. After 2 h at 37°C, the cells were rapidly pelleted, washed twice with chilled K medium, and transferred to tubes containing 400 µl of oil (84% silicon oil, density 1.050; 16% paraffin oil, density 0.838, v/v) to separate cell-bound and unbound ¹²⁵I-peptide by centrifugation. Radioactivity in cell pellets and supernatants was measured in a gamma counter. Nonspecific binding of ¹²⁵I₁-pMCMV, measured in the presence of a 500-fold molar excess of unlabeled pMCMV, was subtracted to determine the amount of radioactivity specifically bound to the cells.

Construction of episomal vectors with minigene DNA

Episomal plasmids were constructed using the expression vector p8901 as previously described (13, 14). In brief, two oligonucleotide fragments with sequences that overlap the desired peptide were annealed, extended with Klenow DNA polymerase, and amplified by PCR. The PCR product was cloned into the BamHI-NotI site of the p8901 vector, and the identity of the resulting plasmid was confirmed by direct sequencing.

Minigene transfection

C1R-L^d and T2-L^d cells were transfected with p8901 plasmids encoding p2Ca or p2Cb or their variants as well as with a plasmid containing the adenovirus E3/19-kDa protein ER translocation signal sequence RYMILGLLALAAVCSA(A) followed by the QL9 sequence as previously described (13, 14, 15). The transfected cells are referred to as C1R-L^d(p2Ca), C1R-L^d(p2Cb), and C1R-L^d(sig-QL9), respectively (see Table I).

Peptides were isolated from minigene-transfected C1R-L^d cells as previously described (8). In brief, 10⁹ cells were washed with K medium and immediately frozen in liquid nitrogen for storage at -70°C until subjected to acid extraction. Thawed cells were combined with 10 ml of 0.7% TFA and immediately homogenized with 50 strokes in a Dounce homogenizer (Kontes, Vineland, NJ). After 30 min on ice, the homogenate was centrifuged (31,000 x g) for 60 min at 4°C, and the supernatant was subjected to ultrafiltration (2,600 x g, 4°C) using a Centricon 10 membrane (M_r cut-off, 10 kDa; Amicon, Beverly, MA). Filtrates were freeze dried and dissolved in 0.1% TFA for reversed-phase HPLC, using a C₁₈ column (218TP104; Vydac, Hesperia, CA) and a gradient (0.067%/min) of solvent B in solvent A, where solvent A is 0.1% TFA and solvent B is acetonitrile containing 0.085% TFA. Fractions (1.0 ml) collected at 1-min intervals were dried by Speed-Vac (Savant, Farmindale, NY), dissolved in water, and assayed for their ability to sensitize T2-L^d cells for specific lysis by 2C CTL.

Processing of p2Cb by 20S proteosomes

20S proteosomes were purified as previously described (1). Before digestion the synthetic peptide p2Cb was HPLC purified to homogeneity. The purified peptide (0.35 µg/ml) was incubated with exhaustively dialyzed 20S proteosomes (0.32 µg/ml) in a final volume of 15 µl of 13 mM Tris-HCl (pH 8.0) containing 5 mM DTT. After 2 h at 37°C, the digestion was stopped by adding 285 µl of 0.2% TFA. The reaction mixture was then subjected to ultrafiltration using a Centricon 10 membrane (Amicon) to separate peptides from high molecular mass material as described above. An aliquot of the isolated peptides was examined by mass spectrometry to determine the molecular masses of the peptide fragments.

Mass spectrometric analysis

Samples were analyzed by matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry (16) on a linear Vestec VT2000 instrument (Vestec/PE Biosystems, Houston, TX), operating at 25-kV accelerating voltage (17). To each sample the MALDI matrix, -cyano-4-hydroxycinnamic acid (Aldrich, Metuchen, NJ), was added as a 10 mg/ml solution in 60% water-40% acetonitrile. Spectra were calibrated with a mixture of peptides of known sequence in the appropriate mass range (external calibration). All mass spectral data were acquired and processed using a data system developed at the Massachusetts Institute of Technology mass spectrometry facility.

	Results

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Expression of p2Cb in L^d+ target cells renders the latter susceptible to TCR-mediated lysis by 2C CTL

Since p2Cb is thought to be a longer natural precursor of p2Ca (5), we investigated processing and presentation of p2Ca and p2Cb in live cells using the minigene expression system. To reduce the confounding effect of peptides from endogenous mouse OGDH, we chose the human cell line C1R-L^d as a recipient target cell. It has been previously shown that the synthetic human analog VAITRIEHVSPFPFDL of p2Cb was >10 times less potent in the cytotoxicity assay, while the p2Ca human analog VSPFPFDL can be presented by L^d to 2C CTL with similar efficacy (our unpublished observations). Nevertheless, neither untransfected C1R-L^d cells nor the cells transfected with a minigene coding for human p2Cb were lysed to a measurable extent by 2C CTL (data not shown). In contrast, transfection of C1R-L^d cells with murine p2Cb rendered these cells susceptible to specific lysis (Table I and Fig. 1A). Lysis of C1R-L^d(p2Cb) cells by 2C CTL was TCR mediated, since the clonotypic anti-TCR Ab 1B2, in a concentration-dependent manner, blocked the cytolysis (Fig. 1B). When the signal peptide was introduced upstream of p2Cb to promote peptide translocation into the ER (14), the specific lysis of C1R-L^d(signal-p2Cb) cells was not enhanced. Extension of p2Cb from the C-terminal end by 6 aa residues matching the murine OGDH sequence also led to less efficient lysis of the target cells, indicating less efficient processing and presentation of this longer peptide (Table I). C1R-L^d cells transfected with the plasmid containing p2Ca were not, however, lysed by 2C CTL.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Specific lysis of C1R-Ld cells transfected with minigenes coding for naturally occurring OGDH-derived peptides. A, 51Cr-labeled C1R-Ld transfectants were incubated with 2C CTL at the indicated E:T cell ratio for 4 h at 37°C. The specific lysis was based on released 51Cr in the culture supernatant. Data of a representative experiment are shown. The transfectants were C1R-Ld (p2Cb; •) and C1R-Ld (p2Ca; ). Untransfected C1R-Ld () were used as a control. Mean values and error bars (SD) are based on triplicate determinations at every E:T cell ratio. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (determined from comparison of the specific lysis of C1R-Ld (p2Cb) and C1R-Ld (p2Ca), by unpaired t test). B, The clonotypic anti-TCR Ab (1B2) blocks lysis of C1R-Ld(p2Cb) by 2C CTL. 51Cr-labeled C1R-Ld(p2Cb) cells were incubated with 2C CTL in the presence of the indicated concentrations of 1B2 Ab for 4 h at 37°C. The E:T cell ratio was 25:1. The dotted line indicates specific lysis in the absence of the Ab.

To confirm that p2Cb was expressed and processed in the C1R-L^d(p2Cb) cells, we extracted peptides from these cells with 0.1% TFA, separated them by HPLC, and measured the cytolytic activity of each fraction. As shown in Fig. 2, peptides in one broad peak at a retention time of 56–58 min were effective in eliciting cytotoxicity. Retention times of synthetic peptides separated under the same conditions were p2Ca and QL9 at 52 min, pyroQL9 (see below) at 56 min, and p2Cb at 58 min. These data indicate that the active peptide fraction isolated from C1R-L^d(p2Cb) most likely contains p2Cb and/or pyroQL9, but not p2Ca or QL9.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. HPLC fractionation of peptides derived from C1R-Ld transfected with p2Cb minigene, CIR-Ld(p2Cb). Low molecular mass fraction (<10 kDa) isolated from homogenized C1R-Ld(p2Cb) cells was fractionated by reversed-phase chromatography. Fractions collected at 1-min intervals were tested in the cytotoxicity assay using T2-Ld as target (see Materials and Methods for details).

The activities of synthetic OGDH peptides in CTL assays and the apparent binding constants for the reaction of these peptides with recombinant L^d protein have been previously determined (5, 7, 12, 18, 19, 20, 21). Although the affinities of p2Ca, p2Cb, and QL9 measured here with live L^d+ cells were somewhat lower, QL9 still bound more strongly to cell surface L^d than did p2Ca or p2Cb peptides (see Fig. 3A and Table II). Also, the concentration of QL9 in the extracellular medium required for half-maximal lysis (SD₅₀) of T2-L^d target cells was 50- to 100- and 1000-fold lower than the concentrations required for p2Ca and p2Cb, respectively (Fig. 3B and Table II), consistent with the higher affinity of 2C TCR for QL9-L^d than for p2Ca-L^d (7).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Presentation of OGDH peptides to 2C CTL. A, Peptide binding to cell surface Ld. A total of 2.5 x 106 T2-Ld cells was combined with 125I1-pMCMV (19.6 nM) and the unlabeled, noniodinated test peptide at various concentrations in 500 µl of RPMI 1640 containing 0.1% BSA and a mixture of protease inhibitors. After 2 h at 37°C, cell-bound and free 125I1-MCMV were separated to measure cell-bound radioactivity. The amount of bound reference peptide (cpm) corrected for nonspecific binding is plotted as a function of the unlabeled peptide concentration. B, Percent specific lysis of 51Cr-labeled T2-Ld cells in the presence of various concentrations of the indicated peptides (for peptide structures, see Table II). The results of a representative experiment are shown. Arrows indicate the peptide concentration required for half-maximal lysis (SD50).

To determine which amino acid residues stabilize the QL9-L^d complex, we systematically replaced with alanine the amino acid residues in every position of QL9 and measured the binding of the mono-substituted QL9 derivatives to L^d on T2-L^d cells. As indicated by the binding constant values in Table II, positions P7 and P9 are critical for QL9 binding to L^d. These data are consistent with the results of a similar analysis of the p2Ca-L^d reaction (22) and with x-ray crystallographic analysis of the L^d protein (23). They indicate that QL9, like p2Ca, uses an alternative L^d binding motif (22, 24), depicted in bold in Table II.

PyroQL9 binds more strongly to L^d and exhibits higher activity in the CTL assay than QL9

The reversible cyclization of N-terminal glutamine in QL9 to pyrrolidone carboxylate is not only catalyzed by glutamine cyclase (QC) (25), but also occurs nonenzymatically, especially at low pH (26). To prepare pyroQL9 we incubated freshly dissolved QL9 in 0.1% TFA followed by HPLC separation. QL9 and pyroQL9 were eluted at 52 and 56 min, respectively, and their identities in the eluted fractions were confirmed by amino acid analysis and Edman degradation (data not shown). As is evident from Fig. 3 and Table II, pyroQL9 was more active than QL9 in both binding to L^d and cytotoxicity assays. Consistent with its higher activity in the cytolytic assay, pyroQL9 bound 3-fold more strongly to cell surface L^d (Table I and Fig. 1).

QL9 and longer OGDH peptides with a conserved C terminus are generated in vitro from p2Cb by purified 20S proteosomes

Because p2Cb and pyroQL9 were not resolved by HPLC under the standard conditions used, we analyzed the products generated from incubating purified p2Cb with 20S proteosomes by means of mass spectrometry. From their molecular masses, several peptide fragments matching the C-terminal sequence of p2Cb could be identified (Fig. 4); these molecular masses correspond to TRIEQLSPFPFDL (TL13), RIEQLSPFPFDL (RL12), IEQLSPFPFDL (IL11), EQLSPFPFDL (EL10), and pyroQL9 (QL9 minus NH₃; Fig. 4). Interestingly, we could not detect a peptide with molecular mass corresponding to p2Ca. The results indicate that proteolytic cleavage of p2Cb yields several fragments including QL9, which had been converted to pyro-QL9 upon exposure to TFA during its isolation.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. MALDI-time-of-flight mass spectrum analysis of the mixture of peptides produced upon digestion of p2Cb with purified 20S proteosomes. Several peptides were detected, the molecular masses of which can be assigned to contiguous segments of the p2Cb sequence, as discussed in the text.

To demonstrate that endogenously generated QL9 can be presented effectively on the cell surface in association with L^d, we transfected a minigene-coding signal-QL9 peptide into C1R-L^d+ and found that 2C CTL lysed the sig-QL9 transfectants (Fig. 5). Because synthetic pyroQL9 and QL9 are both very active in cytotoxicity assays (see above), the low level of specific lysis observed in these experiments indicates that only a very small amount of QL9 was available at the cell surface.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. 2C CTL specifically lyse Ld+ target cells transfected with a minigene encoding sig-QL9. 2C CTL and 51Cr-labeled C1R-Ld cells transfected with the sig-QL9 minigene were incubated at various E:T cell ratios for 4 h at 37°C. The experiment shown is representative of three independent titrations. **, p < 0.01; ***, p < 0.001 (determined from comparison of the specific lysis of transfected and untransfected target cells, by unpaired t test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Assuming that the transfected DNA is expressed equally well within various transfectants, we note that the p2Cb minigene downstream of the signal sequence resulted in much weaker sensitization of the target cells than the minigene without the signal sequence (Table I). Although p2Cb has been shown to be readily transported from the cytosol to the ER (30), it might be too long to be effectively "trimmed" in the ER to produce an optimal peptide epitope (31). It is possible that p2Cb could be retrotranslocated from the ER to the cytosol to be shortened and then recycled back to the ER (31). Such recycling would result in a lower yield of optimal length peptide and could explain the inefficient lysis of C1R-L^d(sig-p2Cb) target cells.

Almost complete lack of sensitization of the target cells carrying the p2Ca minigene was perhaps surprising, since this is a naturally processed peptide, and its synthetic analog readily renders L^d+ targets susceptible to specific lysis by 2C CTL (8). An explanation for this apparent disparity could be that peptides of an optimal length are produced in the ER rather than in the cytosol (32, 33). Optimal length peptides lack the flanking residues that are thought to be essential for their association with cytosolic chaperones that facilitate TAP-dependent peptide translocation into the lumen of the ER (33). The above considerations may also explain our failure to detect presentation by transfected cells carrying the minigene-encoded QL9 without the signal sequence (data not shown).

That the presumed expression of p2Ca in the ER of transfected C1R-L^d cells did not render the cells susceptible to lysis by 2C CTL (see Table I) is at odds with experiments demonstrating that optimal peptides expressed in the ER are usually effectively presented on the cell surface (14, 15). A low copy number of this minigene in the transfected cells may also account for the lack of detectable peptide presentation. In addition, p2Ca forms very short-lived complexes with L^d protein (half-life is 5 min; I. Vturina, Y. Sukulev, and H. N. Eisen, unpublished observation), and consequently, the epitope density (i.e., number of cognate pMHC complexes per cell) required to elicit cytotoxicity would not be achieved despite a continuous flux of p2Ca-L^d complexes to the cell surface. These results also suggest that p2Ca may not be responsible alone for the strong allogeneic response of 2C CTL against L^d+ targets cells (such as P815 cells) under physiological conditions.

Why has QL9 not been identified in tissue extracts as a natural peptide? We have shown here that the N-terminal glutamine of this peptide is rapidly converted to pyroglutamine in TFA, which is commonly used to extract peptides from tissues. This correlates with the finding that one of the peptide fragments extracted with TFA from the digest of p2Cb produced by purified 20S proteosomes has a molecular mass corresponding to pyroQL9 but not to QL9. Since p2Cb and pyroQL9 were not resolved by HPLC under the standard conditions used (see above), naturally produced QL9 in tissue extracts would not necessarily have been detected chromatographically.

The conversion of N-terminal glutamine to pyroglutamine is catalyzed by QC, which is present in various tissues, including spleen (25). Whether QC converts the N-terminal glutamine of QL9 to its cyclized form under physiological conditions is not clear.

Besides the fragment with a molecular mass of pyroQL9 found in the 20S proteosome digest, there were other fragments corresponding to OGDH peptides of various lengths, all sharing with p2Cb the same C-terminal end: TRIEQLSPFPFDL (TL13), RIEQLSPFPFDL (RL12), IEQLSPFPFDL (IL11), and EQLSPFPFDL (EL10; see Fig. 4). That these peptides might also play a role in the recognition of L^d+ target cells by 2C CTL is consistent with our findings demonstrating that synthetic analogs of the various OGDH peptides, including p2Cb, bind soluble L^d protein to form pMHC complexes that are recognized by 2C TCR on the surface of live 2C cells (Ref. 21, and Y. Sykulev, A. Brunmark and H. N. Eisen, unpublished observations) and that such peptides have considerable activity in cytotoxicity assays. In support of this, C1R-L^d cells sensitized with highly purified p2Cb induced a calcium flux in 2C CTL that was detectable within about 1 min; this time is too short to be accounted for by processing of p2Cb into a shorter active peptide fragment that could be responsible for the observed response (5).

How are all of these multiple length peptides accommodated in the L^d-binding groove to produce biologically active peptide-MHC complexes? One explanation lies in the critical role of an alternative L^d-binding motif in the C-terminal sequence shared by all active OGDH peptides (22, 24). We have shown that amino acids at P9 and P7 are essential for the binding of QL9 to L^d (Table II). A similar conclusion has been reached by others who found that homologous amino acid residues (P8 and P6) are responsible for the binding of p2Ca to L^d (22, 24). It is remarkable that even the C-terminal tetrapeptide PFDL of p2Ca, but not the N-terminal fragment LSPF, is still able to interact with L^d, albeit weakly, and that the soluble PFDL-L^d complex binds to 2C TCR with measurable affinity (Ref. 34, and Y. Sykulev, A. Brunmark, and H. N. Eisen, unpublished observations). It seems that L^d-bound OGDH peptides use the same register in the binding groove, with the peptide’s C terminus anchored in the F pocket of the L^d molecule (23).

Based on the crystal structure of the L^d protein (23, 35), Speir et al. (23) built a model of the QL9-L^d complex. This model confirmed that the P9 and P7 amino acid residues of the peptide are essential for stability of this peptide-MHC complex. The modeled structure of QL9-L^d also explains why different OGDH peptides sharing a common C terminus form L^d-peptide complexes that all bind 2C TCR with relatively high affinities ranging from 10⁵ to 10⁷ M^-1 (Ref. 21, and Y. Sykulev, A. Brunmark, and H. N. Eisen, unpublished observations). The presence of bulky amino acids Trp⁷³, Trp⁹⁷, and Tyr⁹⁹ in a prominent ridge on the floor of the L^d molecule binding groove forces bound peptides to bulge out toward the solvent. This peptide bulging increases the proximity of the peptide’s C terminus to the 2C TCR surface, resulting in increased electrostatic interactions between the negatively charged penultimate aspartic acid of the peptide and the positively charged residues of the TCR -chain (23). Because negatively charged Asp in the penultimate position is present in all OGDH peptides sharing C-terminal sequence, these peptides display the same epitope to the 2C TCR.

In summary, the data discussed above indicate that QL9 and several longer OGDH-derived peptides might be naturally processed from p2Cb and presented on L^d+ target cells. Although complexes of these peptides with the L^d protein bind the 2C TCR with relatively high affinity, they are short-lived. Even the most stable of these complexes, QL9-L^d, has a half-life of about 20 min (20). We have previously shown that fewer than 10 QL9-L^d complexes/target cell can render these cells susceptible to specific lysis by 2C CTL (20). However, the magnitude of the observed lysis induced by a few cognate pMHC complexes is far lower than that of the lysis of the L^d+ mouse mastocytoma cell line P815, which is commonly used as an allogeneic target for 2C CTL (3, 8). To date, only p2Ca was isolated from cell surface L^d molecules on P815 cells (8). It is possible that the other OGDH peptides discussed here are also presented on the cell surface, but at much lower densities, which would make it difficult to prove their identities by direct isolation. Based on these considerations, we submit that QL9 and other OGDH peptides acting in concert with p2Ca and p2Cb on the surface of the L^d+ target cells elevate the total density of 2C-recognized epitopes and thus account for the exceptionally vigorous lysis of the target cells by T cells expressing the 2C TCR.

	Acknowledgments

 
We thank Dr. Ike Eisenlohr for valuable discussions of this manuscript, Prof. Klause Biemann for help with the interpretation of the mass spectrometry data, Mimi Rasmussen and Carol McKinley for excellent technical support, and Richard F. Cook and colleagues at the Massachusetts Institute of Technology Biopolymers Laboratory for peptide synthesis and amino acid analyses.

	Footnotes

 
¹ This work was supported by National Institutes of Health Research Grants AI44477 and CA60686 (to H.N.E.) and AI3254 (to Y.S.) and The W. W. Smith Charitable Trust Award (to Y.S.).

² Current address: Fuji Photo Film Co. Ltd., Asaka Research Laboratories, Asaka, Saitama, Japan 351-8585.

³ Current address: Department of Medicine, Maine Medical Center, Portland, ME 04102.

⁴ Current address: Department of Biochemistry and Nutrition, National Institute of Public Health, Tokyo, Japan 108-8638.

⁵ Present address: AstraZeneca R&D Boston, Worcester, MA 01605.

⁶ Address correspondence and reprint requests to Dr. Yuri Sykulev, Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107.

⁷ Abbreviations used in this paper: ER, endoplasmic reticulum; pMHC, peptide-MHC; OGDH, oxoglutarate dehydrogenase; TFA, trifluoroacetic acid; MALDI, matrix-assisted laser desorption-ionization; QC, glutamine cyclase.

Received for publication September 15, 2000. Accepted for publication December 18, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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