The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in EBV-positive Hodgkin disease and is a potential target for cytotoxic T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is low, and so far the generation of LMP1-specific CTLs has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DCs) for CTL stimulation, and we reasoned that an inactive, nontoxic LMP1 mutant (⌬LMP1) could be expressed in DCs and would enable the activation and expansion of polyclonal LMP1-specific CTLs. Recombinant adenoviral vectors expressing LMP1 or ⌬LMP1 were tested for their ability to transduce DCs. LMP1 expression was toxic within 48 hours whereas high levels of ⌬LMP1 expression were achieved with minimal toxicity. ⌬LMP1-expressing DCs were able to reactivate and expand LMP1-specific CTLs from 3 healthy EBV-seropositive donors. LMP1-specific T cells were detected by interferon-␥ (IFN-␥) enzyme-linked immunospot assay (ELISPOT) assays using the HLA-A2-restricted LMP1 peptide, YLQQN-WWTL (YLQ). YLQ-specific T cells were undetectable (less than 0.001%) in donor peripheral blood mononuclear cells (PBMCs); however, after stimulation the frequency increased to 0.5% to 3.8%. Lysis of autologous target cells by CTLs was dependent on the level of LMP1 expression. In contrast, the frequency of YLQ-specific CTLs in EBV-specific CTLs reactivated and expanded using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. IntroductionImmunotherapy with cytotoxic T cells (CTLs) is increasingly used to treat malignancies and viral infections. 1,2 For example, polyclonal Epstein-Barr virus (EBV)-specific CTLs have been used for the prevention and treatment of posttransplantation EBV-associated lymphoma (PTLD). 3 EBV-specific CTLs persisted long-term, reconstituted immunity against EBV, and produced antiviral and antilymphoma effects. We have also used EBV-specific CTLs to treat 13 patients with EBV-positive Hodgkin disease and, while the results have been promising, no patient with bulky disease has been cured. 1,4 One explanation for this failure is that current methods of EBV-specific CTL generation produce CTL lines that are dominated by clones reactive to EBV proteins not expressed in the malignant Reed-Sternberg cells (H-RS cells) of EBV-positive Hodgkin disease. 5 Only a limited number of EBV-derived antigens (EBNA1, BARF0, LMP1, and LMP2) are present in H-RS cells, 6,7 and the immunodominant response of CTL lines is against EBNAs 3A, B, and C, which are not expressed by the tumor cells. Of the EBV proteins expressed in H-RS cells, only LMP2 and LMP1 are potential targets for CD8 ϩ T cells, because EBNA 1 is mainly presented on major histocompatibility complex (MHC) class II molecules 8 and the level of BARF0 expression might be too low for CTL recognition. 9 Our group and others have recently reported the generation of LMP2-specific CTLs 10-12 ; however, for future clinical protocols it is desirable to generate CTLs against more than one tumor-as...