Abstract 777: Antitumor role of early infiltrating interferon-gamma producing NKT cells in murine malignant mesothelioma

Introduction: NKT cells can provide adjuvant activity against cancer by producing large amounts of IFN-gamma which activate other immune cells, and orchestrate protective anti-tumor immunity. Recently, induction of the NKT cell-dependent anti-tumor immune response using its ligand alpha-galactosylceramide has been attempted in several tumor types. However, the role of NKT cells in the tumor microenvironment has not yet been fully addressed. Our aim is to elucidate the role of NKT cells in the thoracic cavity by using a murine malignant mesothelioma model. Methods: Half a million of AB12 murine malignant mesothelioma cells were injected into the right pleural cavity of female Balb/c mice for tumor development. The control mice were injected with the same amount of PBS. In this model, thoracic cavity was filled with tumors and pleural effusion by day 14. On days 0 (before tumor cell injection), 3, 6, 10 and 14 after tumor cell injection, mice were sacrificed and the pleural effusion and tumors were collected. Lymphocytes were isolated by performing a Ficoll gradient centrifugation. Cell phenotype was identified by cell surface marker staining and cytokine expression was analyzed by intracellular cytokine staining using flow cytometry. Cytokine concentrations in the pleural effusion were measured by ELISA. Resluts: The absolute number of CD3 positive cells in the pleural cavity did not increase between day 0 (4.7±0.9 x105), day 3 (5.5±0.7 x105) and day 6 (7.1x±1.6 x105) but did start to increase from day 10 (1.2±0.2 x106) after tumor cell injection. In the control mice, the absolute number of CD3 positive cells was not different throughout the experiment. Although the absolute number of CD8 positive cells and CD4 positive cells were not different between day 0 (CD8: 1.5±0.2 x105, CD4: 2.8±0.2 x105, respectively), day 3 (CD8: 1.8±0.2 x105, CD4: 2.6±0.2 x105, respectively) and day 6 (CD8: 1.9±0.4 x105, CD4: 4.3±0.2 x105, respectively) after tumor cell injection, the absolute number of NKT cells (CD3 and DX5 double positive cells) increased dramatically at relatively earlier phase from 1.6±0.1 x104 on day 0, to 6.4±0.3 x104 on day 3 and 12.1±1.0 x104 on day 14. Intracellular IFN-gamma staining of NKT and CD8 T cells showed that the ratio of IFN-gamma positive NKT to whole NKT cells started increasing from day 3 (day0: 7.0±1.3%, day3: 31±3%, respectively) and peaked on day 6 (59±11%) whereas the ratio of IFN-gamma positive to whole CD8 T cells started increasing from day 6 (day0: 12.1±3.5%, day6: 36±6.5%) and peaked on day 10 (39±3.4%). Conclusion: These results indicate that the NKT cells are recruited to the pleural cavity at a relatively early phase of tumor formation and could potentially affect the subsequent CD8 T cell response by the production of IFN-gamma. Further experiments are on going to see the role of NKT cells and to test the impact of chemotherapy on these cells in this model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2011-777