IL-12 Reprograms CAR-Expressing Natural Killer T cells to Long-Lived TH1-Polarized Cells with Potent Antitumor Activity

Human natural killer T cells (NKTs) are innate-like T lymphocytes that are increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintain a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observed that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates de novo programming of memory NKTs in CD62L negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 showed enhanced antitumor activity in tumor models, persisted long-term in vivo and conserved the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic and unappreciated plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics. Human peripheral blood mononuclear cells isolated from healthy donors were obtained from a commercial vendor and NKT cells were isolated. In the first experiment, human NKT cells were transduced with either GFP or GFP plus IL12 p40 and p35 subunits. These were maintained in culture and expanded with IL-2 prior to RNA isolation and library preparation. In the second experiment, human NKT cells were transduced with either GFP plus IL12 p40 and p35 subunits, or with IL12 p40 and p35 subunits plus a GD2-directed CAR construct. Both were injected into NSG mice engrafted with tumors from the neuroblastoma cell line CHLA-255. Tumor growth was monitored and mice were sacrificed at times according to institutional guidelines and at the termination of the experiment. Peripheral blood collected from the heart, spleen, and liver were strained and washed. The presence of NKTs and CAR constructs was verified by flow cytometry prior to RNA isolation and library preparation.