Generating CTL against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive Immunotherapy of EBV-associated malignancies Running title: Generation of LMP1-specific CTL Scientific Section Heading: Immunotherapy

The Epstein-Barr virus (EBV) encoded LMP1 protein is expressed in EBV-positive Hodgkin’s disease and is a potential target for cytotoxic T-lymphocytes (CTL) therapy. However, the LMP1-specific CTL frequency is low and so far the generation of LMP1specific CTL has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DC) for CTL stimulation and we reasoned that an inactive, non-toxic LMP1 mutant (∆LMP1) could be expressed in DC and would enable the activation and expansion of polyclonal LMP1-specific CTL. Recombinant adenoviral vectors expressing LMP1 or ∆LMP1 were tested for their ability to transduce DC. LMP1 expression was toxic within 48 hours whereas high levels of ∆LMP1 expression were achieved with minimal toxicity. ∆LMP1 expressing DC were able to reactivate and expand LMP1-specific CTL from three healthy EBV-seropositive donors. LMP1-specific T cells were detected by IFN-γ ELISPOT assays using the HLA-A2 restricted LMP1 peptide, YLQQNWWTL (YLQ). YLQ-specific T cells were undetectable (<0.001%) in donor PBMC, however after stimulation the frequency increased to 0.5-3.8%. Lysis of autologous target cells by CTL was dependent on the level of LMP1 expression. In contrast the frequency of YLQ-specific CTL in EBV-specific CTL reactivated and expanded using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. Thus ∆LMP1 expression in DC is non-toxic and enables the generation of LMP1-specific CTL for future adoptive immunotherapy protocols for patients with LMP1-positive malignancies such as EBV-positive Hodgkin’s disease. Targeting LMP1 in these malignancies may improve the efficacy of current adoptive immunotherapy approaches. For personal use only. on November 16, 2017. by guest www.bloodjournal.org From