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Viral peptide-specific CTLs were generated as described (20, 30). Briefly, peripheral blood mononuclear cells were separated from whole blood and restimulated with autologous phytohemagglutinin lymphoblasts. Cells were cultured in RPMI 164C (Gibco) with 10% fetal calf serum (R10) and antibiotics for 1 week, and subsequently with R10 and 10% Lymphocult T (Biotest) added. Viral peptide-specific lines were generated and maintained from the bulk culture by use of peptide-pulsed irradiated autologous B-lymphoblastoid cells weekly as feeders. The HLA-A2-Pol-specific clone 20 was cloned by LDA techniques.
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24
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10144236754
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note
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For staining, approximately 200,000 CTLs were incubated at 4°C for 1 hour with saturating concentrations of CyChrome-conjugated antibody to CD8α (clone RPA-T8, Pharmingen) and phycoerythrin-labeled HLA-A2-peptide tetramers at a concentration of HLA-A2 of approximately 0.5 mg/ml total in PBS plus 2% fetal calf serum plus 5 mM sodium azide. After washing, the cells were fixed in PBS plus 2% formaldehyde. We then analyzed the cells on a FAC-Scan (BDIS), using CELL Quest software.
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25
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10144225184
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note
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Peptide-specific lines were assayed 4 to 5 days after restimulation. Standard 4-hour chromium-51 release assays were performed with autologous chromium-51-labeled B-lymphoblastoid cells as targets. Background chromium release was less than 20%. Percent lys's was calculated from the formula 100 × (E - M)/(T - M), where E is the experimental release, M the release in the presence of R10 media alone, and T the release in the presence of 5% Triton X-100 detergent.
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Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His: I, Ile; K, Lys; L1 Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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β assignment of clone 20; R. Boone for assistance with the flow cytometer; and L. Ngyuen for technical assistance. We also thank P. Schatz for the BirA enzyme and expression plasmid, and D. Garboczi and D. Wiley for the gift of the HLA-A2 expression plasmid. J.D.A. received funding from an American Cancer Society postdoctoral fellowship, and P.A.H.M. is a Medical Research Council Clinician Scientist. M.G.M. was a fellow of the Lucille P. Markey Charitable Trust, D.H.B. was a Marshall Scholar, and P.J.R.G. is a Medical Research Council training fellow. We also acknowledge NIH for support of M.M.D., and the Medical Research Council and the Wellcome Trust for support of A.J.M. and J.I.B.
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