Bakker et al. 10.1073/pnas.0709717105. |
Fig. 5. Establishment of an optimal MHC concentration for ELISA analysis. HLA-A1 complexes containing a control HLA-A1 ligand pA1 or the UV-sensitive ligand p*A1 were exposed to 366 nm of UV for 0 or 60 min, and serial dilutions were analyzed by ELISA. Vertical line indicates the MHC concentration used for subsequent analyses. MHC concentrations as established for the other MHC gene products were also in the 10-20 nM range.
Fig. 6. Tetramer titration on T cell clones. CD8+ T cell clones specific for HLA-A1 CMV-pp65 and HLA-A3 EBV-EBNA-3a were stained with the indicated amounts of HLA-A1 CMV-pp65 (Top) and HLA-A3 EBV-EBNA-3a (Bottom) tetramers. "Exchanged" indicates MHC tetramers generated via peptide exchange, whereas "Traditional' indicates MHC tetramers generated via classical MHC refolding with the specific peptide. Images designated "Relevant Clone" and "Control" represent staining with the matched MHC tetramer and cross-sample controls, respectively. Numbers indicate MFI of CD8+ cells.
Fig. 7. Dose-response curves of a competitive binding FP assay after 30-min UV irradiation and 45-h incubation using HLA-A3 p*A3 and the indicated peptides. IC50 values were determined from three independent experiments. A representative experiment is shown. mP indicates millipolarization.
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Labeling of Peptides.
Fluorescent labeling of peptides was performed as follows: 6 mg of peptide (50 mg/ml in DMSO) was diluted in 1.5 ml 50 mM Hepes buffer and low amounts of N,N-dimethylformamide (DMF; Biosolve) were added whenever precipitates formed. 2.5 mg of TMR-5-maleimide (Tebu-Bio) from a 50 mg/ml DMF stock solution was added, and the reaction mixture was incubated at room temperature for 36 h on an orbital shaker. Five microliters of the reaction was analyzed by LC-MS, and the reaction was terminated by the addition of DTT to a final concentration of 5 mM. Before purification, 1.5 ml of 10% trifluoroacetic acid (TFA; Biosolve)/5 mM DTT/50 mM Hepes was added. Labeled peptides were purified by reverse-phase HPLC (1525EF; Waters) with H2O/0.05%TFA as solvent A and acetonitrile/0.05% TFA as solvent B at a flow rate of 18 ml/min on a 10-μm 30 × 250-mm dc18 Atlantis column (Waters). A linear gradient of 10-95% solvent B over 14 min was used for a first elution. The fractions were then analyzed by LC-MS, and fractions containing fluorescently labeled peptide were further purified by using a similar protocol with a linear gradient of 25-80% solvent B over 19 min for elution. Fractions were then lyophilized, dissolved in DMSO, and stored at -20°C.Peptide Elution.
HLA-A2 Flp*A2 complexes were either left untreated or exposed to UV light for 60 min. For examination of the effect of UV treatment on the total pool of MHC-associated plus released fluorescent ligand, peptides were extracted with 10 μl of 10% TFA (Biosolve) for 60 min at room temperature. The material was then analyzed by reverse-phase chromatography with H2O/0.05%TFA as solvent A and acetonitrile/0.05% TFA as solvent B at a flow rate of 1 ml/min on a 300 ×3.9 Delta Pak 15μ C18 column (Waters). A linear gradient of 10-60% solvent B over 60 min was used for elution. To analyze the composition of material remaining MHC-associated after UV treatment, reactions were separated by gel-filtration chromatography after UV exposure and material eluting with the retention time of pMHC complexes was collected. Subsequently, samples were adjusted to contain an equal amount of fluorescent signal and concentrated to 100 μl. Ten microliters of 10% TFA was added, and samples were incubated for 60 min at room temperature. Nine hundred microliters of H2O was added, and samples were analyzed by reverse-phase chromatography as described above.Fluorescence Polarization Assay.
For all fluorescence polarization binding assays peptides were diluted in bovine γ-globulin in PBS (0.5 mg/ml; Sigma). The HLA-A3-binding peptide KVPCALINK was labeled with TMR-5-maleimide (Anaspec) and purified by reverse-phase HPLC as described above. This peptide (pTAMRA) was used as a tracer for fluorescence polarization (FP) experiments. Labeled peptide was standardized against a fluorescence intensity curve by using free TMR-5-maleimide as a standard.FP measurements were performed on a PerkinElmer Wallac EnVision 2101 Multilabel Reader. Samples were measured by using a 531-nm excitation filter, 579-nm S-channel emission filter, a 579-nm P-channel emission filter, and a Bodipy-TMR FP Dual mirror (all filters and mirrors obtained from PerkinElmer). FP values are given as mP (millipolarization) and calculated by using the following formula: polarization (mP) = 1,000 × (S-GP)/(S+GP), where S and P are the fluorescence intensities measured in the S (parallel to polarization plane) and P (perpendicular to polarization plane) directions and G is the grating factor. The G factor is a correction factor for instrument (filters, mirror)-dependent variations in sensitivity for measurements in the S and P directions and is determined according to the instrument manufacturer's instructions by measuring a 1 nM sample of free TAMRA. The G factor is then calculated using the following formula: G = (S/P) × (1-L/1,000)/(1+L/1,000), where L is the theoretical or literature polarization value for the fluorophore, here TAMRA (L = 50 mP). Typically, G factors of 1-1.1 were obtained.
Epitope Screening.
To determine the binding capacity of the predicted ligands for HLA-A3, percentage inhibition of binding of tracer peptide was determined at 5 μM in an FP competition assay with conditional p*A3. For this purpose, an FP assay was modified for application with UV-mediated peptide exchange for HLA-A3. In a 384 well black nonbinding surface assay plate (Corning) each well was loaded with 10 μl of a 2.5× peptide solution (12.5 μM), 5 μl of a pTAMRA solution (5 nM) and 10 μl of a 2.5× HLA-A3 p*A3 solution (1.25 μM). For all preparations 0.25 mg/ml bovine γ-globulin in PBS was used as a buffer. The plate was spun for 1 min at 1,000 × g at room temperature. To start UV-mediated peptide exchange, the plate was placed 10 cm under a 365-nm UV lamp (2 × 15 W blacklight blue tubes, LxWxH 505 × 140 × 117 mm; Uvitec) located in a cold room (4°C). After 30 min irradiation, the plate was sealed with thermowell sealing tape (Corning) and incubated at room temperature for 45 h, when periodic readings showed no further increase in polarization, indicating the establishment of equilibrium. Controls included peptide free samples (1% DMSO) for 0% inhibition of tracer binding and 200 μM ILRGSVAHK (A3-Flu epitope) samples for 100% inhibition of tracer binding. All data points were determined in triplicate.Competition Assay.
For peptides displaying >63% inhibition at 5 μM IC50 values were determined by generating dose-response curves of serial peptide dilutions covering a range of 50 μM to 50 nM using the UV-mediated FP competition assay described above. The binding affinity (IC50 value) of each competitor peptide was defined as the concentration that inhibits 50% binding of pTAMRA tracer peptide. Data were analyzed by using GraphPad Prism software (GraphPad). Fifty percent inhibitory concentrations were determined in at least three independent experiments for each peptide. Peptide concentrations used for determination of IC50 values were calculated by assuming maximum synthetic yield (2 μmol). For 13 peptides containing tyrosine or tryptophan residues, concentrations were verified by measuring absorption at 280 nm (for calculations of extinction coefficients see: www.expasy.org/tools/protparam.html). Peptide concentrations as determined by absorption at 280 nm were in between 0.47 and 1.05 of the predicted values.