Abstract 4624: Natural killer cell licensing contributes to clearance of lung cancer
Authors: Lei Shi , Li Kang , Arefanian Saeed , Krupnick Alexander S. 
Published: July 2017

a) Natural Killer (NK) cell Inhibitory receptors function in a dichotomous role. Their engagement by MHC Class I upon encountering a target results in the inhibition of cytotoxicity. During NK development, however, their interaction with MHC Class I mediates the process of licensing or education, leading to an increase in responsiveness and cytotoxicity. For this reason the role of inhibitory receptors in controlling the clearance of MHC Class I+ solid tumors such as lung cancer is poorly defined. Since NK cells comprise the primary cytotoxic lymphocyte responsible for clearance of lung cancer, we set out to determine whether the licensing status of NK cells contributes to lung cancer susceptibility.

b) Growth of MHC Class I+ murine lung cancer was evaluated in MHC Class I- mice, with unlicensed NK cells in direct comparison to MHC Class I+ mice with licensed NK cells. IFN-γ production was evaluated after plate bound antibody stimulation through the NKp46 and NKG2D pathways both flow cytometrically and by quantitative RT-PCR. Bone marrow transplantation was performed by injection of 10x106 unfractionated bone marrow cells after 500cGy irradiation and NK and CD4+ T cell depletion of recipient host.

c) Injection of Lewis Lung Carcinoma (LLC) revealed a significant decrease in pulmonary tumor burden in mice with licensed NK cells (230.4±21.6 vs. 442.7±53.8 mg) that disappeared after NK depletion (421.4±36.8 vs. 421.9±66.3 mg). Freshly isolated licensed NK cells lysed LLC more robustly than unlicensed NK cells (28.7±.16% vs. 4.9±.2% specific lysis at 100:1 ratio respectively). Lung cancer expresses stress ligands recognized by NKG2D and NKp46 activating receptors. Licensed NK cells produced significantly more IFN-γ than their unlicensed counterparts after plate-bound stimulation with anti-NKp46 and anti-NKG2D (17.9±2.8% vs. 5.4±.8% respectively for dual NKp46 and NKG2D stimulation). No differences were evident between licensed and unlicensed NK cells in ribosomal mass, making it unlikely that translational defects were responsible for altered cytokine production. Licensing status also did not affect stability of IFN-γ but did affect mRNA levels. This suggests transcriptional control of licensing-mediated NK activity. IFN-γ production could be significantly increased in unlicensed NK cells by reintroduction of MHC Class I through non-myeloablative bone marrow transplantation from MHC Class I-sufficient congenic donors. Blockade of H2Kb-Ly49c (MHC Class I-inhibitory receptor) interaction during NK development in C57BL/6 mice using a non-depleting anti-Ly49c antibody (clone 4LO) decreased IFN-γ production in NK cells.

d) The role of NK cell licensing and tumor-specific cytotoxicity is highly controversial. We now demonstrate that licensed NK cells mediate a superior lung cancer-specific immune response in a murine model. Our data suggests that altering the licensing status of NK cells may be an avenue of lung cancer-specific immunotherapy.

Citation Format: Shi Lei, Kang Li, Saeed Arefanian, Alexander S. Krupnick. Natural killer cell licensing contributes to clearance of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2017-4624