Simultaneous Ex Vivo Expansion of Cytomegalovirus and Epstein-Barr Virus–Specific Cytotoxic T Lymphocytes Using B-Lymphoblastoid Cell Lines Expressing.

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Simultaneous Ex Vivo Expansion of Cytomegalovirus and Epstein-Barr Virus–Specific Cytotoxic T Lymphocytes Using B-Lymphoblastoid Cell Lines Expressing Cytomegalovirus pp65 by Qi Sun, Karen E. Pollok, Robert L. Burton, Li Jun Dai, William Britt, David J. Emanuel, and Kenneth G. Lucas Blood Volume 94(9):3242-3250 November 1, 1999 ©1999 by American Society of Hematology

Immunoblot analysis for the expression of CMV pp65 and EBV EBNA2. Immunoblot analysis for the expression of CMV pp65 and EBV EBNA2. Lanes 1 through 6, probed against CMV pp65; lanes 7 through 9, probed against both EBV EBNA2 and CMV pp65. Qi Sun et al. Blood 1999;94:3242-3250 ©1999 by American Society of Hematology

Immunoperoxidase staining using a pp65-specific monoclonal antibody with (a) SF transduced with MSCV; (b) SF transduced with MSCVpp65; (c) BLCL transduced with MSCV; and (d) BLCL transduced with MSCVpp65. Immunoperoxidase staining using a pp65-specific monoclonal antibody with (a) SF transduced with MSCV; (b) SF transduced with MSCVpp65; (c) BLCL transduced with MSCV; and (d) BLCL transduced with MSCVpp65. Qi Sun et al. Blood 1999;94:3242-3250 ©1999 by American Society of Hematology

CTL cultures at 21 days after ex vivo stimulation with autologous BLCLpp65 were tested for specific cytotoxicity against EBV-transformed BLCL, CMV-infected SF, and MSCVpp65-transduced cells in chromium release assay. CTL cultures at 21 days after ex vivo stimulation with autologous BLCLpp65 were tested for specific cytotoxicity against EBV-transformed BLCL, CMV-infected SF, and MSCVpp65-transduced cells in chromium release assay. (A through D) CTL activity from 4 CMV seropositive/EBV seropositive individuals. (E through F) CTL activity from 2 CMV seronegative/EBV seropositive individuals. (E:T ratio, 12.5:1). Qi Sun et al. Blood 1999;94:3242-3250 ©1999 by American Society of Hematology

Cytotoxicity against CMV-infected cells by BLCLpp65-primed CTL cultures was mediated via pp65. Cytotoxicity against CMV-infected cells by BLCLpp65-primed CTL cultures was mediated via pp65. CTL cultures lysed autologous fibroblasts infected with a recombinant vaccinia virus encoding CMV pp65, but not the one encoding CMV pp150. (E:T ratio, 12.5:1)‏ Qi Sun et al. Blood 1999;94:3242-3250 ©1999 by American Society of Hematology

Cytotoxicity against EBV-transformed BLCL and MSCVpp65-transduced SF was mediated by CD8+ CTL. Bulk, CD8-enriched, or CD4-enriched CTL culture from donor ST were tested at different E:T ratios in chromium release assay. Cytotoxicity against EBV-transformed BLCL and MSCVpp65-transduced SF was mediated by CD8+ CTL. Bulk, CD8-enriched, or CD4-enriched CTL culture from donor ST were tested at different E:T ratios in chromium release assay. CD8-enriched CTL (B) retained the cytotoxicity displayed by the bulk CTL (A), while the CD4-enriched CTL (C) did not. Qi Sun et al. Blood 1999;94:3242-3250 ©1999 by American Society of Hematology

CMV pp65-specific cytotoxicity was HLA class I-restricted. CMV pp65-specific cytotoxicity was HLA class I-restricted. Bulk and CD8-enriched CTL from donor ST showed cytotoxicity against HLA class I partially matched allogeneic targets (AD), which shared HLA A2 and B44 with donor ST. There was no specific lysis of the complete allogeneic targets (SQ). (E:T ratio, 12.5:1)‏ Qi Sun et al. Blood 1999;94:3242-3250 ©1999 by American Society of Hematology