This study was approved by the Institutional Review Boards of the Massachusetts General Hospital and the Nuffield Department of Medicine; all subjects gave written informed consent. All subjects were anti-HCV–positive as measured by enzyme immunoassay. HCV viral loads were measured by the Roche Amplicor Monitor assay (Roche, Branchburg, New Jersey, USA) (detection limit 300 copies/ml of plasma). Subjects 01-40, 98A, OXS, OXM, OXD and O1E spontaneously resolved HCV viremia, while subject 99D was treated with IFN/ribavirin therapy and subsequently resolved HCV viremia; these subjects had persistent documented undetectable plasma viral loads (<300 copies/ml of plasma). Subject OXK had received unsuccessful combination therapy 6 years previously, but all other subjects with chronic infection were untreated. HLA typing was performed by standard serological and molecular techniques (13
). The HLA-DR alleles of the subjects included in this study were as follows: 99D: DRB1*0401; 01-40: DRB1*0401, DRB1*07; 98A: DRB1*0401, DRB1*10; OXS: DRB1*0404, DRB1*07; OXM: DRB1*0401, DRB1*0701; OXD: DRB1*04, DRB1*07; O1E: DRB1*0401, DRB1*11; 99-24: DRB1*0401, DRB1*15; OXB: DRB1*1501, DRB1*0401; OXK: DRB1*0404, DRB1*1101; O2P: DRB1*0401, DRB1*07.
Expression of DR/CLIP precursors.
The constructs for the expression of DR/CLIP complexes were based on those previously described for DR2 (DRA, DRB1*1501), which carried Fos and Jun dimerization domains (14
). For the β chain constructs (DRB1*0101, DRB1*0401, DRB1*1501, and DRB5*0101), the covalently linked myelin basic protein (MPP)peptide was replaced with the CLIP peptide (PVSKMRMATPLLMQA, single–amino acid code). The linker carried a thrombin-cleavage site, as reported (16
). For the DRα chain construct, a BirA biotinylation site (GLNDIFEAQKIEWHE) was attached via a six–amino acid linker (GGSGGS) to the 3′ end of the Fos dimerization domain (17
). The four different DRβ chain constructs were cotransfected with the DRα chain construct into CHO cells, and clones were screened for secretion of DR molecules in an ELISA in which mAb L243 (American Type Culture Collection, Manassas, Virginia, USA) was used for capture and a polyclonal DR antiserum for detection (15
). Clones with the highest expression levels were expanded for inoculation into bioreactors. For large-scale protein production, the ACUSYST-miniMAX instrument (Cellex Biosciences Inc., Minneapolis, Minnesota, USA) was used, which allows control of temperature, pH, and media feed rate. Transfectants were grown in hollow fiber bioreactors with an internal surface area of 1.1 m2
, and supernatants from the extracellular space were harvested by the instrument at a predetermined flow rate into a collection bottle placed at 4°C. These cultures were maintained for approximately 3 months, and supernatants were frozen at weekly intervals. DR molecules were purified by affinity chromatography with mAb L243 (American Type Culture Collection) as previously described (14
Processing of DR molecules: biotinylation, thrombin cleavage, and peptide exchange.
Purified DR molecules were biotinylated using a 1:20 molar ratio of BirA to DR in a buffer containing 100 μM biotin, 10 mM ATP, 10 mM magnesium acetate, 50 mM Bicine (Sigma-Aldrich, St. Louis, Missouri, USA), and 1× protease inhibitor cocktail (Sigma-Aldrich) at pH 8.0. The final HLA-DR concentration was adjusted to 2.5 mg/ml with 10 mM Tris, pH 8.0, and the reaction was incubated for 2 hours at 30°C. The protein was then extensively dialyzed against PBS to remove free biotin. Biotinylation was confirmed by electrophoresis on native polyacrylamide gels (6%), and dimers, trimers, and tetramers of DR molecules could be visualized at different molar ratios of streptavidin and DR.
Prior to the peptide-exchange reaction, the linker was cleaved with thrombin for 2 hours to allow release of the CLIP peptide. Twenty units of thrombin (Novagen, Madison, Wisconsin, USA) were used per milligram for DRB1*0101 and DRB1*0401 molecules, and 40 units per milligram for DRB5*0101 and DRB1*1501. Thrombin cleavage was confirmed by SDS-PAGE based on a shift in the molecular weight of the DRβ chain. Thrombin-cleaved complexes are designated as *DR/CLIP. Peptide exchange was performed using dinitrophenol-labeled (DNP-labeled) peptides for affinity purification of defined DR/peptide complexes. The DNP group was attached to the N-terminus of peptides via an aminohexanoic acid linker during synthesis (New England Peptide Inc., Fitchburg, Massachusetts, USA); all peptides were HPLC-purified and analyzed by mass spectrometry.
The sequences of the HCV peptides used for construction of MHC class II tetramers were as follows: HCV 1248: GYKVLVLNPSVAATL; HCV 1579: SGENLPYLVAYQATVCARA; HCV 1770: SGIQYLAGLSTLPGNPAIASL. Control tetramers were generated with peptides from annexin II and gp100 that had been eluted from DR4 molecules of a melanoma cell line (annexin II, residues 208–223: DVPKWISIMTERSVPH; gp100, residues 44–59: WNRQLYPEWTEAQRLD) (18
). Peptide-exchange reactions were carried out with 3.3 μM *DR/CLIP and 50 μM of the respective DNP-labeled peptide in a buffer containing 50 mM sodium citrate, pH 5.2, 1% octylglucoside, 100 mM NaCl, and 1× protease inhibitor cocktail (Sigma-Aldrich). The reactions were incubated overnight at 30°C and then concentrated by ultrafiltration. The *DR/CLIP concentration and reaction temperature were chosen based on preliminary experiments designed to minimize aggregation of empty DR molecules created by CLIP dissociation. Aggregation was substantially lower at a *DR/CLIP concentration of 3.3 μM compared with 8.25 μM, and at a reaction temperature of 30°C compared with 37°C.
DR molecules were separated from unbound peptide with a Superose 12 HPLC gel filtration column (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA) using PBS at a flow rate of 0.8 ml/min. The peak representing DR molecules was collected and injected onto an anti-DNP HPLC affinity column. The anti-DNP affinity column was generated by covalent cross-linking of 10 mg of anti–DNP-1 antibody (BIOTREND Chemikalien GmbH, Cologne, Germany) to a 4.6 mm × 50 mm protein G column on POROS 20 XL media (Applied Biosystems, Foster City, California, USA). DR molecules with bound DNP-labeled peptide were eluted from the column using 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), pH 11.5, and eluates were neutralized by addition of 1 M phosphate, pH 6.0. Biotinylated, peptide-loaded DR molecules were concentrated by ultrafiltration (Centricon 2-ml concentrator; Millipore Corp., Bedford, Massachusetts, USA), and the buffer was simultaneously changed to PBS. Biotinylated DR/peptide complexes were frozen in small aliquots at –80°C and multimerized with labeled streptavidin prior to use in staining reactions.
Kinetics of CLIP dissociation and peptide association.
CLIP dissociation and HA peptide association were examined under conditions relevant to preparative loading of *DR/CLIP complexes. To study the kinetics of CLIP dissociation, 8.33 μM of *DR/CLIP was incubated overnight at 37°C with 100 nM Alexa-488–labeled CLIP peptide in 50 mM sodium citrate, pH 5.2, 1% octylglucoside, 100 mM NaCl, and 1× protease inhibitor cocktail (Sigma-Aldrich). A molar excess of the high-affinity influenza HA (residues 306–318) peptide (100 μM) was then added, and the reactions were incubated for different periods of time at 37°C and then frozen until analysis. Complexes of DR and Alexa-488–labeled peptide were separated from unbound peptide using a Bio-Silect SEC 125 gel filtration column (Bio-Rad Laboratories Inc., Hercules, California, USA) run at a flow rate of 1 ml/min with PBS. The Alexa-488–labeled peptide was detected using an HPLC fluorescence detector with excitation and emission wavelengths set at 495 and 519 nm, respectively (Varian ProStar 363; Varian Analytical Instruments, Walnut Creek, California, USA). Peptide association was examined using 3.3 μM *DR/CLIP and 50 μM Alexa-488–labeled influenza HA peptide, under the same conditions as described above. The amount of bound peptide was determined based on the surface area of the peak that represented the DR/peptide complex. Half-life for dissociation was calculated by fitting of data to an equation describing an exponential decay. Half-life for association was calculated by fitting of data to an equation describing exponential rise. The following labeled peptides were used in these experiments: CLIP (residues 87–101) Alexa-488–CGGGPVSKMRMATPLLMQA; influenza HA (residues 306–318) Alexa-488–CGGGPKYVKQNTLKLAT.
Competition assays were performed to identify HCV peptides suitable for tetramer production. HCV peptides were used as competitors for binding of a biotinylated influenza HA (residues 306–318) peptide to thrombin-cleaved DR4/CLIP complexes. Competitor peptides were tested at concentrations ranging from 30 nM to 10 μM in the presence of 30 nM labeled HA peptide and 1.7 μM *DR/CLIP. Reactions were incubated for 20 hours at 37°C in 50 mM sodium citrate, pH 5.2, 1% octylglucoside, 100 mM NaCl, 100 μg/ml of BSA, and 1× protease inhibitor cocktail. One hundred nanograms of DR was loaded onto a 96-well plate (Wallac Oy, Turku, Finland) coated with 200 ng/well of mAb L243, and DR-bound peptide was detected with europium-labeled streptavidin. Fluorescence was quantitated using a DELFIA 1234 fluorometer (Wallac Oy).
Establishment of T cell lines specific for the influenza HA peptide.
PBMCs from normal donors with the appropriate DR subtype were plated at 2 × 105
cells per well in 96-well U-bottom plates in the presence of influenza HA (residues 306–318) peptide (0.2–1 μM) and RPMI 1640, 10% human serum, 2 mM L
-glutamine, 10 mM HEPES, and 100 μg/ml penicillin/streptomycin. Recombinant IL-2 was added after 3 days to a final concentration of 5 U/ml (Roche Diagnostics, Indianapolis,Indiana, USA), and half of the media was changed every 3 days. Cell lines were maintained by restimulation with autologous irradiated mononuclear cells and HA peptide at 10- to 14-day intervals as described previously (19
Establishment of HCV-specific CD4 T cell lines.
HCV-specific CD4 T cell lines were generated as previously described (7
) by a single round of stimulation with a recombinant HCV antigen. Briefly, 5 × 106
PBMCs were stimulated with 1 μg/ml of recombinant C200 antigen (encoding NS3 and NS4 proteins) in media supplemented with 50 U/ml of recombinant IL-2. After 10–14 days of culture, T cell lines were stained with tetramers and analyzed for Vβ usage.
Intracellular cytokine staining.
Intracellular cytokine staining of PBMCs ex vivo and in short-term stimulated cell lines was performed as previously described (7
MHC class II tetramer staining of PBMCs.
For tetramer formation, biotinylated DR/peptide complexes were incubated with R-phycoerythrin–labeled streptavidin for at least 1 hour on ice, at a 4:1 molar ratio of DR to streptavidin and a final DR concentration of 0.2 mg/ml. Cells (fresh PBMCs, cryopreserved PBMCs, or cells from a short-term stimulated line) were stained in 100 μl R10 medium (RPMI, 10% FCS, 10 mM HEPES, 2 mM L-glutamine, and 50 U/ml penicillin/streptomycin) with 2 μg of phycoerythrin-conjugated (PE-conjugated) MHC class II tetramer for 2 hours at room temperature. APC-conjugated anti-CD4, peridinin chlorophyll protein–conjugated (PerCP-conjugated) anti-CD14, PerCP-conjugated anti-CD19, and either FITC-conjugated CD27 or FITC-conjugated CD45RA mAb’s were added for the last 20 minutes of incubation. For CCR7 analysis, cells were stained with unconjugated anti–human CCR7 antibody (Becton Dickinson–Immunocytometry Products, San Jose, California, USA) during the last 20 minutes of tetramer incubation, then washed twice and stained with a FITC-conjugated secondary anti-mouse IgM antibody. The cells were incubated for 20 minutes and washed twice, and then anti-CD4, -CD14, and -CD19 were added and the cells incubated for an additional 20 minutes. Cells were washed twice and then incubated with anti-PE MicroBeads (Miltenyi Biotec Inc., Auburn, California, USA) for 20 minutes at 4°C. The cells were washed once, and 90% of the cells were applied to MS separator columns (Miltenyi Biotec Inc.) according to the manufacturer’s instructions. The other 10% of the tetramer-stained cells were reserved for FACS analysis; the total number of cells in this pre-enrichment sample was multiplied by 10 to determine the input number of cells for each sample. Two to three million PBMCs were used for each tetramer stain. The PE-positive cells were then eluted from the columns, stained with Via-Probe (Becton Dickinson–Immunocytometry Products), and analyzed by flow cytometry using CellQuest software (Becton Dickinson–Immunocytometry Products). Cells were gated on CD4+, CD14–, CD19–, and Via-Probe–negative cells. Multiple control tetramers with irrelevant peptides were used in most experiments to demonstrate specificity of binding by the relevant tetramers. The frequency of tetramer-positive cells was determined by division of the number of CD4+/tetramer+ cells after enrichment by the total number of CD4 T cells as calculated by FACS analysis of the pre-enrichment sample. For Vβ analysis, cells were labeled with FITC-conjugated antibodies (Immunotech, Westbrook, Maine, USA) following magnetic enrichment and washed after a 20-minute incubation at room temperature.