Abstract 3512: Expanded human regulatory iNKT cells exhibit direct cytotoxicity against hematolymphoid tumor targets

Abstract

Abstract CD1d-restricted invariant natural killer T (iNKT) cells are rare but potent innate regulatory cells capable of immune modulation via robust production of Th1/Th2 cytokines, as well as tumor immunosurveillance via direct cytotoxicity. Protocols to expand iNKT cells and augment their cytotoxicity would broaden their application in allogeneic transplantation and adjuvant anti-tumor immunotherapy. We have optimized a protocol for ex vivo expansion of highly purified human CD3+CD4negVα24+ iNKT cells from human peripheral blood mononuclear cells (PBMC). PBMCs were stimulated with an iNKT-specific glycolipid (α-GalCer), recombinant IL-2 and IL-7 and sorted to > 98% purity at day 7. Sorted iNKT cells were further expanded in the presence of irradiated allogeneic PBMCs, Vα24-specific TCR stimulation, and low-dose IL-2 and IL-7 for 21 days. This results in roughly 10^3-fold expansion, with peak yields of at day 21-28 of expansion (range 1.5 x 10^5 - 10.6 x 10^6 iNKT cells from 1 x 10^8 - 3.5 x 10^8 starting PBMC). At 28 days, these iNKT cells secrete high levels of IL-13, TNF-α, GM-CSF, and IL-4, moderate IL-2 and IFN-γ in anti-CD2/CD3/CD28 bead-stimulated Luminex supernatant assay. Day 28 expanded iNKT cells are dose-dependent suppressors of sorted autologous CD3+CD8+ effector T cells in 72-hr mixed leukocyte reaction (MLR). Anti-CD2/CD3/CD28 activation of these iNKT cells induced expression of high levels of key cytolytic effector molecules, including granzyme B. We therefore measured cytotoxicity of activated day 28 iNKT cells against various hematolymphoid tumor targets following co-incubation of iNKT cells versus control effector populations with firefly luciferase-transduced RS4:11 and Nalm6 (B-ALL), U937 (monocytic), and K562 (CML) targets. iNKT cell effectors (E) demonstrated dose-dependent cytotoxicity against B-lymphoid tumor targets (T) (e.g.: Nalm6: 37.7% at E:T 2:1, 17.7% at E:T 1:1, 7.7% at E:T 0.5:1), with no significant cytotoxicity against myeloid targets (e.g.: K562: 11.8% at E:T 2:1, 6.7 at E:T 1:1, 1.8 at E:T 0.5:1). Our results indicate that human iNKT cells expressing high levels of Th2 and regulatory cytokines can be potently expanded ex vivo and manifest cytotoxicity against B-ALL targets, supporting their potential application in tailored immunotherapy in the pre- and post-transplant setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3512. doi:1538-7445.AM2012-3512