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Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with KSHV infection. Key to the survival and proliferation of PEL is the canonical NF-kB pathway that becomes constitutively activated following overexpression of the viral oncoprotein ks-vFLIP. This arises from its capacity to form a complex with the modulatory subunit of the IKK kinase, IKKγ (or NEMO) resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis; both of which are important drivers of tumourigenesis. Using a combination of cell based and biophysical assays together with structural techniques, we show that the observed resistance to cell death is largely independent of autophagy or major death receptor signalling pathways and demonstrate that direct targeting of the ks-vFLIP-IKKγ interaction both in cells and in vitro can be achieved using IKKγ mimetic peptides. Our results further reveal that these peptides not only induce cell killing, but potently sensitise PEL to the pro-apoptotic agents tumour necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.\ud IMPORTANCE: KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation where it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-κB pathway that requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKKγ. Whilst this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis are additional contributors. We demonstrate that the interaction between ks-vFLIP and IKKγ is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKKγ complex can be disrupted using peptides leading to direct killing and the sensitisation of PEL cells to pro-apoptotic agents. Our studies thus provide a framework for future therapeutic interventions.

Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10(6) transducing units/ml can be harvested from the final producer clones, which can be increased to 10(8) TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.

Expression of the co-stimulatory receptor 4-1BB is induced by T cell receptor recognition of antigen, while 4-1BB ligand is highly expressed on activated antigen presenting cells. 4-1BB signalling is particularly important for survival of activated and memory CD8+ T cells. We wished to test whether co-expression of antigen and 4-1BBL by dendritic cells (DC) would be an effective vaccine strategy. We therefore constructed lentiviral vectors (LV) co-expressing 4-1BBL and influenza nucleoprotein (NP). Following subcutaneous immunisation of mice, which targets DC, we found superior CD8+ T cell responses against NP and protection from influenza when 4-1BBL was expressed. However, functionally superior CD8+ T cell responses were obtained when two LV were co-injected, one expressing 4-1BBL, the other expressing NP. This surprising result suggested that 4-1BBL is more effective when expressed in trans, acting on adjacent DC. We therefore investigated the effect of LV expression of 4-1BBL in mouse DC cultures and observed induced maturation of bystander, untransduced cells. Maturation was blocked by anti-4-1BBL antibody, required cell-cell contact and did not require the cytoplasmic signalling domain of 4-1BBL. Greater maturation of untransduced cells could be explained by LV expression of 4-1BBL causing down-regulation of 4-1BB. These data suggest that co-expression of 4-1BBL and antigen by vaccine vectors that target DC may not be an optimal strategy. However, 4-1BBL LV immunisation activates significant numbers of bystander DC in the draining lymph nodes. Transactivation by 4-1BBL/4-1BB interaction following DC/DC contact may therefore play a role in the immune response to infection or vaccination.