Endogenous IL-2 production by natural killer cells maintains cytotoxic and proliferative capacity following retroviral-mediated gene transfer

Interleukin (IL)-2 therapy given at tolerable doses is insufficient to induce maximum activation of natural killer (NK) cells. We recently demonstrated that NK cells expanded in vivo can be maximally activated by short-term ex vivo incubation with 1000 U/mL IL-2. However, IL-2 withdrawal, which would occur with reinfusion, may lead to a rapid loss of cell viability and function. We hypothesized that retroviral transduction could provide an endogenous source of IL-2 to maintain NK function as measured by proliferation and cytotoxicity. Enriched NK cells were transduced with supernatants containing an MFG-based retrovirus designed to express murine IL-2 cDNA. Several supernatant transduction strategies were evaluated. NK cells were initially cultured in 1000 U/mL of huIL-2 for 7-8 days, harvested, and replated prior to transduction (4 hours at 37°C); this proved insufficient to sustain NK proliferation or maintain cytotoxicity after exogenous human IL-2 (huIL-2) withdrawal. An alternative transduction procedure using phosphate-depleted medium, centrifugation, and transduction for 16 hours at 32°C was then evaluated. NK cells transduced under these conditions maintained significant NK proliferation in the absence of exogenous IL-2 compared with sham-transduced controls. Two consecutive daily transductions resulted in less proliferation, suggesting that several exposures to retroviral supernatant may inhibit subsequent NK proliferation. Cytotoxicity of the transduced NK cells against K562 and Raji was maintained under these conditions without exogenous IL-2. Sham-transduced NK cells produced 8.3±2.6 U/mL of murine IL-2 (muIL-2) by ELISA (background) after 7 days without exogenous IL-2. In contrast, 109±23 U/mL mulL-2 was produced by NK cells transduced with supernatant from the MFG/mulL-2 producer line. These experiments demonstrate that NK cells can be successfully transduced with retroviruses and induced to express sufficient IL-2 to maintain their proliferative and cytotoxic functions. Transduction of IL-2 genes into NK cells may offer advantages over exogenous IL-2 administration in maintaining maximum function for use in antitumor immunotherapy.