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KelsoOutWest – 2006
Abandoned Post Office at the ghost town of Kelso, California. The adjacent Union Pacific whistlestop, in danger of being destroyed by the railroad, has been renovated and now serves as the Visitor's Center for the Mojave National Preserve.
3021CaliforniaGhost townKelso DepotMojave National PreservePost officeUnion Pacificabandonedderelictmojaveskytrees. road
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CTL clone 2C recognizes the allogeneic class I MHC molecule Ld in association with peptides derived from α-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 Ld 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 Ld 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.
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-2b mouse immunized with H-2d cells expressing Kd, Ld, and Dd class I MHC proteins (3). 2C CTL specifically responds to Ld 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 Ld+ 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 Ld on live Ld+ 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 Ld to form peptide-Ld complexes recognizable by 2C CTL. All these data indicate that the strong allogeneic response of 2C CTL to Ld+ target cells is mediated by numerous peptides presented on target cells in the context of Ld. Although most of these peptides are derived from OGDH, the presence of other Ld-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
The CD8+ CTL 2C from the original clone was maintained as described previously (3). T2-Ld is a human cell line T2 that lacks the TAP transporter genes and was transfected with the Ld 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 Ld are called C1R-Ld. T2-Ld and C1R-Ld (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 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 × 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 × g, 5 min), and 100-μl aliquots from each supernatant were counted in a gamma counter. Percent specific lysis was determined as: 100 × (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-Ld cells and did not exceed 10% for T2-Ld cells.
Competition assay to measure peptide binding
Peptide binding to Ld on live cells was measured as previously described (12). In brief, T2-Ld 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 × 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-Ld and T2-Ld 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-Ld(p2Ca), C1R-Ld(p2Cb), and C1R-Ld(sig-QL9), respectively (see Table I⇓).
Acid extraction and HPLC fractionation of low molecular mass material from minigene-transfected C1R-Ld cells
Peptides were isolated from minigene-transfected C1R-Ld 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 × g) for 60 min at 4°C, and the supernatant was subjected to ultrafiltration (2,600 × g, 4°C) using a Centricon 10 membrane (Mr 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-Ld 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, α-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.
Expression of p2Cb in Ld+ 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-Ld 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 Ld to 2C CTL with similar efficacy (our unpublished observations). Nevertheless, neither untransfected C1R-Ld 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-Ld cells with murine p2Cb rendered these cells susceptible to specific lysis (Table I⇑ and Fig. 1⇓A). Lysis of C1R-Ld(p2Cb) cells by 2C CTL was TCR mediated, since the clonotypic anti-TCR Ab 1B2, in a concentration-dependent manner, blocked the cytolysis (Fig. 1⇓B). When the signal peptide was introduced upstream of p2Cb to promote peptide translocation into the ER (14), the specific lysis of C1R-Ld(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-Ld cells transfected with the plasmid containing p2Ca were not, however, lysed by 2C CTL.
A low molecular mass fraction extracted from C1R-Ld(p2Cb) cells contains peptides recognized by 2C CTL
To confirm that p2Cb was expressed and processed in the C1R-Ld(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-Ld(p2Cb) most likely contains p2Cb and/or pyroQL9, but not p2Ca or QL9.
QL9 binds to cell surface Ld with high affinity and is the most potent OGDH peptide by cytotoxicity
The activities of synthetic OGDH peptides in CTL assays and the apparent binding constants for the reaction of these peptides with recombinant Ld protein have been previously determined (5, 7, 12, 18, 19, 20, 21). Although the affinities of p2Ca, p2Cb, and QL9 measured here with live Ld+ cells were somewhat lower, QL9 still bound more strongly to cell surface Ld than did p2Ca or p2Cb peptides (see Fig. 3⇓A and Table II⇓). Also, the concentration of QL9 in the extracellular medium required for half-maximal lysis (SD50) of T2-Ld target cells was ∼50- to 100- and 1000-fold lower than the concentrations required for p2Ca and p2Cb, respectively (Fig. 3⇓B and Table II⇓), consistent with the higher affinity of 2C TCR for QL9-Ld than for p2Ca-Ld (7).
Alanine scan of QL9 reveals alternative Ld-binding motif
To determine which amino acid residues stabilize the QL9-Ld 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 Ld on T2-Ld cells. As indicated by the binding constant values in Table II⇑, positions P7 and P9 are critical for QL9 binding to Ld. These data are consistent with the results of a similar analysis of the p2Ca-Ld reaction (22) and with x-ray crystallographic analysis of the Ld protein (23). They indicate that QL9, like p2Ca, uses an alternative Ld binding motif (22, 24), depicted in bold in Table II⇑.
PyroQL9 binds more strongly to Ld 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 Ld and cytotoxicity assays. Consistent with its higher activity in the cytolytic assay, pyroQL9 bound 3-fold more strongly to cell surface Ld (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.
Expression of QL9 in the endoplasmic reticulum of Ld+ target cells leads to specific lysis of these cells by 2C CTL
To demonstrate that endogenously generated QL9 can be presented effectively on the cell surface in association with Ld, we transfected a minigene-coding signal-QL9 peptide into C1R-Ld+ 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.
Although the MHC moiety of an allo-MHC-peptide complex plays a critical role in allogeneic T cell responses (27), the MHC-bound peptide can also determine the specificity of a T cell-mediated alloreaction (8, 28, 29). The octapeptide p2Ca and the 16-mer p2Cb were the first naturally occurring peptides found to be critical for recognition of allogeneic MHC class I molecules by CTL (5, 8). Since p2Cb contains the entire sequence of p2Ca, it was reasonable to ask whether it is the natural precursor of p2Ca (5). To address this, we found that expression of p2Cb in Ld+ target cells renders the latter susceptible to specific lysis by 2C CTL. However, HPLC separation of peptides extracted from these target cells did not provide evidence for the presence of p2Ca. Instead, the retention time of the active CTL assay fractions eluted by HPLC indicated that these fractions contained p2Cb and/or pyroQL9. Because both peptides have very similar retention times, we simulated p2Cb processing in vitro with 20S proteosomes and analyzed the fragmented peptide products by mass spectrometry. We found several fragments with various molecular masses (Fig. 4⇑), one of which (m/z 1046) matches the molecular mass of pyroQL9. Further evidence suggesting that QL9 is naturally processed derives from the finding that expression of this peptide in the ER results in specific lysis of transfected cells by 2C CTL (Fig. 5⇑). All of these results indicate that QL9 could be naturally processed and contributes to the allogeneic response of 2C CTL against Ld+ target cells. In accord with this conclusion, we found that QL9 is not only the most potent OGDH peptide in the cytolytic assay, but that it also binds more strongly to Ld on live cells than the other OGDH-derived peptides (Table II⇑).
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-Ld(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 Ld+ 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-Ld 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 Ld 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-Ld 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 Ld+ 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 Ld+ target cells by 2C CTL is consistent with our findings demonstrating that synthetic analogs of the various OGDH peptides, including p2Cb, bind soluble Ld 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-Ld 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 Ld-binding groove to produce biologically active peptide-MHC complexes? One explanation lies in the critical role of an alternative Ld-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 Ld (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 Ld (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 Ld, albeit weakly, and that the soluble PFDL-Ld complex binds to 2C TCR with measurable affinity (Ref. 34, and Y. Sykulev, A. Brunmark, and H. N. Eisen, unpublished observations). It seems that Ld-bound OGDH peptides use the same register in the binding groove, with the peptide's C terminus anchored in the F pocket of the Ld molecule (23).
Based on the crystal structure of the Ld protein (23, 35), Speir et al. (23) built a model of the QL9-Ld 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-Ld also explains why different OGDH peptides sharing a common C terminus form Ld-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 Ld 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 β-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 Ld+ target cells. Although complexes of these peptides with the Ld protein bind the 2C TCR with relatively high affinity, they are short-lived. Even the most stable of these complexes, QL9-Ld, has a half-life of about 20 min (20). We have previously shown that fewer than 10 QL9-Ld 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 Ld+ 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 Ld 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 Ld+ 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.
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.
↵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. E-mail address: ysykulev{at}lac.jci.tju.edu
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 September 15, 2000.
- Accepted December 18, 2000.
- Copyright © 2001 by The American Association of Immunologists


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I've loved star shapes for as long as I can remember. If you go check my blog out, you'll see a couple of different star projects (mostly jewelry and hair pieces). I also hoard (and yes, I mean HOARD) star shapes – plastic glow in the dark stars, wooden stars, glass stars, metal stars – you name it, I most likely will have it, and if not, I'll be compelled to get it. Same thing happened when I saw these stars – they're so pretty and with dimensional details to boot. I wondered what I was going to make with them, but it seemed apt to make these really easy-to-assemble DIY Christmas gift earrings – looks festive enough to wear throughout the holiday season, no?
Star Dangle Earrings DIY Christmas Gift Supplies:
- Darice Tin Stars
- 1/8″ Metal Puncher
- Darice Gold Spray Paint
- Darice Semi-Precious Stone Beads
- Head Pins
- Round Nose Pliers
- Wire Cutter
- Earring Hooks
- Jump Rings
- DIY Christmas Gift Free Printable Earring Cards < click to print
Step 1. Get your stars and pick out what size you want to use. For the pair I have made, I used two of the second-to-the-smallest ones.
Step 2. Use your metal puncher to punch the top hole (one of the star tips) and a hole directly opposite that top hole as well. Alternatively, you can drill these holes with a really small bit, but it's much easier to use the metal puncher.
Step 3. Paint your stars. I spray painted mine, but you can also use Rub n Buff or nail polish. Make sure to spray the backside first before spraying the front.
Step 4. Now it's time to assemble the bead dangle – just thread it through the head pin, bend at the top, cut off the excess wire and loop to seal it. Easy peasy!
Step 5. Attach your bead dangle onto the bottom hole with a jump ring…
Step 6. And attach an earring hook on the top hole.
Step 7. Repeat steps 4-6 for the other DIY Christmas gift earring and you are done!
Step 8. Optional – If you're going to give these out as stocking stuffers, a small DIY Christmas gift for friends or whatever, you may want to download these earring tags I've made. They come in grey, red and navy blue and they're about 3″ x 4.5″ in size. You can also use these tags as mini Christmas cards! Download them here.
Are you ready for Christmas? I'm not yet – but I'm getting there! I hope you'll love this easy DIY Christmas gift craft, and don't forget to check out my blog for similar tutorials and printables! 🙂
Have fun making these and giving them out to friends!


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The syllables creep through her teeth. Maybe their character painted the flower, or maybe their character is the flower. Joni Chancer, teacher-consultant of the South Coast Writing Project Californiahas paid a lot of attention to the type of questions she wants her upper elementary students to consider as they re-examine their writing, reflecting on pieces they may make part of their portfolios.
Write a review of an imaginary production of the play we have just finished studying in class.
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The situation left girls feeling overwhelmed, according to Waff, and their "voices faded into the background, overpowered by more aggressive male voices. I made a small frame out of a piece of paper and placed it down on one of her drawings — a sketch she had made of a visit with her grandmother.
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The Writing Assignment Framework and Overview represent the effort of National Writing Project teachers to create tools for themselves and their colleagues that might substantially strengthen their writing assignments.Download


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New Yorker
Runs Rough
1995 Chrysler New Yorker motor runs rough and misfires at a
I put this car in the shop to have it repaired and after 1100.00 dollars, the problem still is not fixed. I will be seeking reimbursement from this shop, do the idiots not knowing what they were doing. This is what the car does, it starts and seems to run fine, until you give it a little gas around 12000-15000 RPM's the car seems to misfire and carry on, if you give it a little more gas the motor runs great. I have checked the TPS, IAC, fuel injectors I got 15.6 or better on all of them. New Spark plug wires, plugs, coil pack, I am running out of idea's on what might be the problem. Garage ran diagno on car and they were still unable to find case of problem, but still require that I pay the bill. Fuel pressure is fine, just don't know at this point. Car is getting worse, smells like fuel, flooding out when running. What would cause this problem. Have taken it to 2 more mechinec's and they can't figure it out still.
Do you
have the same problem?
Friday, April 2nd, 2010 AT 7:26 PM
3 Replies
Hi receiver25. Welcome to the forum. You have an unusual problem, YOU are running out of ideas, and the mechanics are idiots? I don't know of anyone who works for free including you. Well, ... Maybe me! Doctors get paid for doing diagnostics even when they don't know what's wrong. Why are mechanics held to a higher standard?
You sound like you know more about cars than the average person, but the things you mentioned shouldn't be related to the symptom of fuel smell and flooding. The only thing the IAC, (automatic idle speed motor in Chrysler terminolgy) does is adjust idle speed and provides the idle flare-up at engine startup. He ain't the problem. The TPS will cause a hesitation but not rough running. Do you mean the injectors measured 15.6 ohms? They shouldn't be the problem either unless one is stuck open. That would be REALLY weird. Injector trouble is real common on GM products but it's almost unheard of on Chryslers.
How did you check the TPS and AIS motor? Do you have a scanner? If not, do you know someone who has one you could borrow? The fuel supply system is not monitored by the Engine Computer but all of the engine sensors are. The scanner will let you read that data while the engine is running. If you're in danger of spending a lot more money on mechanics, you might be better off buying a scanner, but you will want more than a simple code reader.
Sounds like you replaced everything in the ignition system so spark should not be the problem. If the computer was not firing one coil, two cylinders would be dead all the time. Doesn't sound like that's the case either. There actually is one thing that can cause intermittent spark at specific engine speeds but it almost always results from previous service. That is the crankshaft position sensor does not have the proper air gap. I never actually replaced one on an Intrepid / New Yorker / LHS, but I've seen this happen on the minivans when the paper spacer is not installed to set the proper gap. What you might try is using an inductive pickup timing light on each plug wire, one at a time, and hold the throttle open until the misfire occurs. See if all three coils are firing. If one of them cuts out, check the crankshaft position sensor.
Since the problem is rpm-specific, I don't think fuel delivery is the issue either. A problem with volume will show up mostly during coasting from highway speeds. GM has a ton of trouble with pressure regulators leaking fuel into the vacuum hose causing a flooding condition. I've never heard of that on a Chrysler either, but to check, pull the vacuum hose off the regulator and look for any sign of wetness.
Now for the harder stuff. The MAP sensor has the biggest say in how much fuel is commanded by the computer. The acceptable range of signal voltage is.5 to 4.5 volts. The voltage can be wrong, but as long as it falls within that range, no diagnostic fault code will be set. This is where watching live data with a scanner comes in handy. These sensors caused a pile of trouble in the late '80s - early '90s, but by the time these LH cars showed up, the problem was solved. A fast and easy test is to replace it, but rather than buying a new one, search for one in a salvage yard. They are pretty reliable now so your chances of finding a bad one are real small. One clue that suggests the sensor is the problem is if the rough running occurs at different engine speeds dependent on engine load. In other words, the misfire might not be occurring at a specific engine speed; it might be occurring at a specific intake manifold vacuum. That would also mean the misfire occurs more during driving than just sitting still in park or neutral.
Another thing to look for is a vacuum leak in the intake manifold gaskets. While the engine is still cool, spray water wround the gaskets and watch for a change in speed. A leak will usually introduce extra air into one or two cylinders which can cause a misfire, then the oxygen sensor picks up the unburned oxygen in the exhaust and tells the Engine Computer to enrichen the mixture for all of the cylinders. No matter how much fuel is added, that unburned oxygen will always be detected so more fuel will always be requested. O2 sensors don't respond to fuel, just oxygen. The clue here is to view the short and long-term fuel trim numbers on the scanner. High positive numbers means the computer is adding fuel, ... Lots of it, in response to seeing too much oxygen in the exhaust. High negative numbers means there is too much unburned fuel in the exhaust and the computer sees it and is trying to do something about it by subtracting fuel from the calculated value.
Finally, here's a weird one that's real hard to find but also not real common. An exhaust leak before the catalytic converter can cause a rich exhaust smell. Between the pulses of exhaust gas, the momentum creates tiny pulses of vacuum. Those vacuum pulses can draw in outside air. The oxygen sensor picks up that oxygen and tells the computer the mixture is too lean so it adds more fuel to every cylinder. The clue here is the problem will not occur for the first few minutes after cold startup. The O2 sensors have to reach 600 degrees before they start to work. Until that happens, they won't report any extra unburned oxygen that might be present. The computer will determine fuel delivery based solely on pre-programmed values without modifying the amount as a result of the O2 sensor readings.
If it appears you have a single cylinder misfire, and you can hold engine speed long enough in the range where it occurs, you can use an infrared thermometer on the exhaust manifolds to figure out which cylinder is causing the problem. Once you determine that, you can switch injectors and spark plug with those from different cylinders to see if a different cylinder develops the problem. It will also be helpful to try to observe if you have low power at higher engine speeds when driving up a hill. When raising engine speed in a garage, a misfire that is still there can sometimes appear to be gone because the power pulses smooth out at higher speeds. What seems like smooth running at high speeds might not be so smooth under load.
As a last resort, consider using an oscilloscope to view the waveforms coming from the crankshaft position sensor and the camshaft position sensor. I am not friendly with automotive scopes. I use one from my many years as a tv repairman because that's what I'm familiar with. Also, you might have to seek out an engine performance specialist. I know there is a shop in Jolliet, IL that specializes in these kinds of problems. Their main customers are other shops. One of the owners teaches very high level classes for Carquest. Some of the things I suggested are things they have found and analyzed in the past. They even developed a strategy for determining if a misfire is being caused by lack of spark, lack of fuel, or lack of compression by just looking at the waveform coming from the oxygen sensor. I'll never be that smart!
Was this
Sunday, July 4th, 2010 AT 2:46 AM
Car seems to be getting worse the more it is driven, garage thought that it was the exhaust so removed it and car still runs like crap, not a plugs exhaust. Removed timing belt cover, checked timing, one tooth off, adjusted. Put back together still no improvement. Replaced coil, no changed. Injectors replaced 2 due to sticking. Replaced caburator gaskets due to leaks, all vacuum like replaced, and still this car will not run right. I say it must have something to do with the car getting to much fuel, due to the fact, It smell like it is getting to much fuel and flooding out. I have to put the car in Park or Neutral and push the gas pedal to the floor to get it to clear the engine, then back into drive. Car use to get up to about 45 mph, and then start bucking and acting up. Now it does it from start up. Maybe injectors are dumping to much fuel, maybe fuel pump is going bad, even though pressure reading is good. This car is really a mechinacs nightmare. Running out of ideas at this point. Guess it might be best to start looking for another car. Will check air gap on Cam sensor and CPS tomorrow to make sure they are still there. What is the Start pressure suppose to be, 45, and running. Even changed ECM and no change. Any other ideas would be helpful. Thanks
Was this
Monday, July 5th, 2010 AT 6:44 PM
Did you check any of the things I mentioned previously? In particular, have you tried a new MAP sensor? Has anyone read the fuel trim numbers to determine if the computer is requesting additional fuel or if it sees the excessive fuel and is trying to subtract some? Have you checked for an exhaust leak?
A fuel pump can't cause too much fuel, only not enough if the pressure or volume are low. If the pressure tried to increase, the extra fuel would just go through the pressure regulator back into the tank. Pop the vacuum hose off the regulator and be sure there is no raw fuel in it. Has anyone measured fuel pressure to see if it is too high? Remove the return hose from the fuel rail and run it into a container. If the engine runs better, the return line is kinked or blocked. If no fuel comes out while the engine is running, the regulator is blocked. Be sure the vacuum hose going to the regulator doesn't have any leaks and is not blocked. Fuel pressure should change when a hand-operated vacuum pump is used to apply vacum to the regulator. As vacuum increases, pressure should drop. That will tell you if incorrect fuel pressure is the problem.
Has anyone used an infrared heat gun to measure exhaust temperature? If one cylinder is significantly different than the rest, that cylinder has a problem. Look at the injector and spark plug. Try switching these parts with those from a different cylinder to see if the problem moves to that cylinder. If all cylinders are about the same temperature, look for something they all have in common such as sensors, fuel supply, and the computer.
Another possibility is a broken flexplate around the crankshaft bolts. Check spark timing. If it is severely advanced, the flexplate may have turned a little. Sometimes that will also cause a banging noise while the engine is running.
Was this
Tuesday, July 6th, 2010 AT 12:28 PM
to post a reply.
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Gold has grown throughout Africa the world with offices , partner offices ranging from west to east, with its commercial hub in Johannesburg . ACM Gold Sandton City Office Tower, Johannesburg, Sandton, Forex Trading is located at 12, 158 5th Street, 2196 South Africa. Directory of building contractors builders construction companies in. No Fees on Deposits." We hope to achieve resolution by next week " Robyn Francis said Friday in an e- mail. Spreads from 0 pips. By: acmgold Subscribe. For example, J- HE W I S D O M OF V A L U E. PressReader - Sowetan: Zinhle' s bae gets R60 000.
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Archived Better Off Deadbeat: People Get Into Debt, Learn the Law, Become Credit Terrorists (blacklistednews.com)
submitted ago by indelible
[–] Empire_of_the_mind 0 points 5 points 5 points (+5|-0) ago
this guy is a hero
[–] doctorconimac 0 points 1 points 1 points (+1|-0) ago (edited ago)
So what's the website the author keeps writing about??


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Hi ladies. I've found out today that I'm having my second boy 😊 we decided prior to finding out the sex that we would keep it between us.
Anyway, our 2 year old is named Jude which we love and he has his dad's first name as a middle name.
We like names that aren't too common or trendy, but that are heard of. When we were having Jude, Eli was the only real contender, so it is an option but it's maybe a bit more common than I'd like.
Also, the name can't begin or end in a 'c/ck/k' sound and our last name is 3 syllables. I kind of like Reid and Roman but my husband doesn't really. It's so much harder this time!
So, does anyone have any suggestions that fit the (many) criteria? 💙 thanks ladies 💙
I think the name Eli sounds absolutely amazing with you. Jude is pretty trendy so I don't think that Eli will seem any many more popular than Jude. And they're both gorgeous names that flow well together, similar vibe for sure manly but soft.
Others ideas:
Thanks ladies ❤
I'm in Australia and Jude isn't in the top 100 and hasn't been in at least the last 5 or so years so it's not very common here. I'd LOVE something like that, it's just so hard!
Thanks @jellens I feel we may end up sticking with Eli I'm just not sure, feels like he's getting our second name because we couldn't come up with anything haha! Maybe I'm being ridiculous.
thanks for your suggestions, my husband really doesn't like the idea of using a name then shortening it all the time eg. I wanted Judah and we could nn our Jude, Jude but he was so against it 😣 ps. did you used to go by 'kiwi_lass' or similar on this board a couple of years ago? If so, I remember you from when I was using this app while pregnant with Jude 😊
oh really, so interesting! See Jude's not even in the top 100 in Aust, Eli I think is sitting around number 47, so not SUPER overly used but it still has been a factor in my mind. Anyway, we are thinking of going with Eli. As @HF23 said we probably won't think of Eli being our second choice name once he's here (at least I hope not!) and we both really do like the name. It does go well with our surname too 😊😁😍


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2018. All Rights.


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Big Picture - November/December 2017 - 31
design to collaborate and create prototype products. This past
summer, the program produced diverse stick figure family
decals, a dollhouse, and what Allen calls "soft sculptures - aka
throw pillows," she laughs. "But shaped ones!"
The soft sculpture project was spearheaded by Richard
Stone, a Jefferson University student with an interest in
sewing. Stone discovered Fresh Artists' "Silly City" collection
of hand-drawn architecture, primarily drawn by third- through
sixth-graders, depicting "architectural gems from their own
community," explains Allen, such as Independence Hall, City
Hall, the art museum, The Betsy Ross house, and the Liberty
Bell. Stone took one look at the collection and knew this
artwork was perfect for creating fun and unique throw pillows.
He transformed the designs into soft sculpture form utilizing
the shop's Sawgrass Virtuoso VJ 628, printing onto Fisher
Textiles 8330 Hercules fabric, and cutting with a Mutoh Kona
1400. From there, Stone stuffed and sewed the 25 architectural
pillows sure to liven up any living room or office space.
Who says design is only for adults (or that fun is only for
kids)? Through Fresh Artists, children who may not otherwise
get the opportunity to flex their creative muscles are encouraged to let that creativity flow. And who knows: Today's Fresh
Artists students just may be tomorrow's art directors, large-format
design specialists, shop owners, and studio executives.


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text: I'm not too sure which type of router I should buy. I'm clueless about routers, actually. I'm currently using a desktop that is directly wired to my modem. I'm looking for a router that I can attach to my modem(I think that's how it goes?) and in turn, the router will be wired to my desktop and be wireless to my laptop. (I plan to purchase a Macbook as my laptop, do I need a special router?) Also, I heard something about the Airport Extreme. Not sure if that's the only router I can use? Again, I'm clueless to routers.


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Sarah Rayl's grandmother died seven years ago. That was the last time the Scecina senior saw any of her biological family.
People at Scecina see Sarah as a happy student who doesn't seem to have many cares. Most don't know that she spent 11 years in foster homes, starting when she was just 2 months old. Or that her biological parents gave up custody of her and six siblings because they couldn't care them.
Sarah lived in 11 different homes and attended many different schools in Fort Wayne.
For overcoming these challenges, she was honored ... Read More
By Beth Murphy, Director of Marketing Communications
On my first trip to the March for Life with Scecina's Crusaders for Life, I spent four straight days with 48 teenagers, including 22 hours on a bus (not including the bus time in Washington, D.C., traffic).
Not only did I survive, I had a great experience! ...Read More
By Beth Murphy, Director of Marketing Communications
There were more than a few tears as 15 Spanish students said goodbye to Scecina one recent morning.
The students hailed from Andalusia in the southern Spain, several came from the capital city of Seville and others live in smaller towns. They came as part of the Short-Term Program of Foreign Links Around the Globe (FLAG), an international student exchange program.
The tears that flowed on departure day were real. There were long embraces ... Read More
All schools and parishes under the guidance of the Roman Catholic Archdiocese of Indianapolis admit students of any race, color, national and ethnic origin to all the rights, privileges, programs, and activities generally accorded or made available to students at the school. The schools do not discriminate on the basis of race, color, national and ethnic origin in administration of educational policies, admissions policies, scholarship and loan programs, and athletic and other school-administered programs.
©2018 Scecina Memorial High School All Rights Reserved


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This is a poll intended to see how big a percentage of the current XTM users would be willing to go through a game-restart in order for the Mod to aquire some additional content.
Additional content can be (notice the 'can', meaning the below doesnt indicate everything is WiP atm):
-Custom Universe
-Race specific and/or more custom turrets
-More custom docking options (internal/external)
-Race specific SY's/ED's/Fabs
-Different weapon types instead of A/B/G types.
-Different Shields
-A Clean mod without the excess bagage from previous builds.
With the current benefit of foresight to the 2.0 patch from Egosoft, any comments on this in regards to pre-releases from XTM meanwhile or just the opposite, would also be appriciated.