Abstracts/Experimental Hematology 28 (2000) 31–131 57

avoided since it is immunogenic and may be infectious. The AP-DCs had similar morphology, immunophenotype, and functionalactivity as FCS-DCs Mature AP-DCs (23105) were pulsed withpp65495–503 and b2 microglobulin, and added to 23106 CD81 cellsor 23106 CD14 depleted PBLs. Cultures were restimulated (R/S)on day 7 & 14 with peptide-pulsed monocytes at a ratio of 1 mono-cyte to 3 CTLs, and cytotoxicity to peptide-pulsed autologousPHA-blasts and CMV infected fibroblasts was measured. Peptidespecific cells were quantitated by pp65 peptide495–503/tetramers.Specific lines were produced in 17 of 19 (89%) cultures derivedfrom seropositive donors. Higher specific lysis was observed forcultures derived from CD81 cells. In contrast, using the same in-duction protocol for seronegative donors, we found that 4 R/Swere necessary to obtain specific lysis and we observed successonly when cultures were initiated with both CD41 & CD81 cells.CTL induction for seronegative donors is more difficult presum-ably due to the absence of primed/memory T-cells. Our data sug-gests that CMV-specific CTL induction from seronegative donorsis not consistent using the protocol developed for seropositive do-nors. We only generated CMV specific cultures when we did notuse purified CD81 cells as responders indicating that CD41 helpis crucial for CTL induction from CMV seronegative donors.

83 Sunday, July 9, 2000 (10:30–12:30)Session I-1: Acute Leukemia: Basic Research

SURVIVAL GENE TRANSFER INTO LEUKEMIA SPECIFIC DONOR LYMPHOCYTES FOR CHEMOPROTECTION POST ALLOGENEIC MARROW TRANSPLANTATIONSpencer, H.T.*, Shaw, D.*, Henslee-Downey, J., Gatlin, J.*, Garcia, J.V.*, van Rhee, F.South Carolina Cancer Center, Columbia, SC & University of Texas Southwestern Medical Center, Dallas, TX

ALL is the least responsive of all leukemias to immunotherapywith DLI after allogeneic stem cell transplantation. The absence ofthe GVL effect is probably due to 1) poor immunogenicity of ALLcells and 2) a high proliferative capacity of the leukemia, resultingin the need for frequent chemotherapy to control the leukemicclone which has the deleterious effect of eradicating any expandingALL-specific cytotoxic T-lymphocytes (CTLs). We have shownthat the immunogenicity of ALL cells can be increased by upregu-lating essential (co)stimulatory molecules (B7.1&2, CD40, ICAM-1,LFA-3) using CD40 ligand. CD40 ligated ALL cells are highly ef-ficient in inducing allogeneic T-cells to proliferate compared to un-treated ALL cells. CD40ligated ALL cells can also be used to gener-ate ALL-specific CTLs from the donor. The CTLs are protected fromthe toxic effects of methotrexate (MTX) or trimetrexate (TMTX)chemotherapy by introducing a drug resistant variant of dihydro-folate reductase (L22YDHFR). Gene modified ALL-specific CTLswill allow for the administration of antifolate chemotherapy whichwill control the leukemia and reduce the leukemia burden while theALL-specific CTLs will be protected from the chemotherapy, expand,and eradicate the leukemia. Transduction efficiency of T-cells wasmeasured by flow cytometry after gene modification with a repli-cation incompetent lentivirus-based vector encoding Green Fluores-cent Protein & L22YDHFR. After TMTX selection the transduc-tion efficiency was 50% compared to 13% of T-cells not exposedto TMTX. We are currently investigating if L22YDHFR transduc-tion significantly alters the function of antigen specific CTLs.

84 Monday, July 10, 2000 (16:00–17:00)Poster Session II: Immunomodulation

EBV-SPECIFIC T-CELL LINES FOR ADOPTIVE TRANSFER TO PMRD ALLOGRAFT RECIPIENTSJohnson, K.J.*, Mostafa, B.T.*, Szmania, S.M.*, Godder, K.*, Chiang, K.Y.*, Henslee-Downey, P.J., van Rhee, F.Division of Bone Marrow Transplantation, Palmetto Richland Memorial Hospital, Columbia, SC

We have developed an efficient method for generating Epstein-Barr virus (EBV)-specific T-cell lines using clinical-grade reagentsfor prophylactic use against donor derived lymphoproliferativedisease (LPD) in patients receiving an allograft from a partiallymatched related donor (PMRD). In a cohort of 249 consecutive pa-tients who received a PMRD-BMT, 13 developed EBV-LPD. Theprobability of developing EBV-LPD was 14% at two years. Con-ventional therapies were unsuccessful. Donor PBMCs were trans-formed with EBV virus and utilized to stimulate donor PBLs in thepresence of low dose IL-2. In a period of 5 weeks, we were able toculture 60–70 3 106 cells, which is sufficient for adoptive immu-notherapy. The EBV-specific T-cells showed great efficacy &specificity eradicating donor EBV-infected cells in-vitro. We testedour EBV-specific T-cell lines in cytolytic assays and found EBVtarget cell killing to consistently be in the range of 65–95%, whilekilling of patient PHA-transformed blasts was negligible. As acontrol, we utilized K562 cells, and found killing to be in the rangeof 15–25% at an E:T ratio of 50:1. The immunophenotype waspredominately CD31/81, CD45RO1 (memory), HLA-DR1,CD62L- (activation), CD49d1 (ability to cross endothelium), andCD57- (terminal differentiation). Intracellular cytokine stainingshowed a Th1 profile with TNF-a and IFN-g positivity in theCD81 population. We will utilize these EBV-specific T-cellsprophylactically for adoptive transfer in PMRD recipients.

85 Sunday, July 9, 2000 (18:30–19:30)Poster Session I: Gene Targeting-Gene Therapy

GENERATING CYTOTOXIC T LYMPHOCYTES AGAINST CERVICAL CARCINOMA WITH LENTIVIRUS TRANSDUCED ANTIGEN PRESENTING CELLSSpencer HT*, Galloway A*, Pirisi-Creek L*, Evans J*, Garcia VJ*, Henslee-Downey J, & van Rhee FSouth Carolina Cancer Center, Columbia SC & University of Southwestern Medical Center, Dallas TX

Human Papillomavirus (HPV), the cause of cervical cancer, in-fects and immortalizes keratinocytes. Since the infected cellspresent viral antigens but lack co-stimulatory molecules for induc-ing immune responses, alternative antigen presenting cells (APCs)are needed. The 98aa HPV-E7 oncoprotein represents an attractiveimmunotherapy target, as it is overexpressed and vital for immor-talization. Recent publications report abrogated transforming abil-ity and destabilization of E7 following C58G and C91G mutationsin ‘CXXC’ zinc finger motifs at positions 58–61 and 91–94. Wehave made 7 mutant E7 proteins including C94V mutation. Thisdisrupts the zinc finger motifs, but preserves the T cell receptorregion of the immunogenic 86–94 nonamer and increases its HLA-0201 binding 14-fold, based on anchor position affinities. The ex-pression and stability of the wild type and mutant E7 (m-E7) pro-teins is being determined in transfected homozygous HLA-A0201