1. Interferon-γ (IFNγ), a cytokine secreted by effector lymphocytes upon activation, is known to cause upregulation of MHC class I in tumor cells, which has been associated with natural killer (NK) cell inhibition. NK cells are very heterogeneous with respect to receptor expression and their function depends on an interplay between these inhibitory and activating signals. In order to optimize NK cell therapy, it is essential to identify the NK cells expressing the optimal combination of receptors for a given target. In this study we evaluated tumor cell lines from the Pediatric Preclinical Testing Program to determine the impact of IFNγ on their expression of NK cell activating and inhibitory ligands, death receptors, and adhesion molecules using mass cytometry, and their correlation with tumor lysis. We found that IFNγ treatment can significantly increase ICAM- 1 and/or HLA-ABC levels together or independently. When HLA-ABC increases without concomitant ICAM-1 upregulation, this is associated with decreased lysis. Conversely, when ICAM-1 upregulation exceeds HLA- ABC upregulation this is associated with increased conjugate formation and better NK cell cytotoxic activity. This increase in cytotoxicity and conjugate formation is lost after blocking ICAM-1. Time-lapse imaging of the NK cell interaction with tumor cells shows that NK cells require less time to form stable contacts with IFNγ treated tumor cells when compared to untreated tumor cells. This suggests that IFNγ induced upregulation of ICAM-1 enhances NK cell cytotoxic activity by reducing the time required for NK cells to form stable contacts with their target. Methods Human NK cells were purified from anonymized blood bank buffy coats using the RossetteSep Human NK cell Enrichment Cocktail. NK cells were expanded for 21 days using a protocol developed at our laboratory in which K562 with membrane bound-IL21 are used as feeder cells (1). Expanded NK cells were used for all experiments. Tumor cell lines were obtained from the Pediatric Preclinical Testing Program (PPTP) in vitro panel. Conclusions References 1.Denman, C.J., et al., PLoS One, 2012. 7(1): p. e30264. 2.Moore, M., W.J. White, and M.R. Potter, Int J Cancer, 1980. 25(5): p. 565-72. 3.de Fries, R.U. and S.H. Golub, J Immunol, 1988. 140(10): p. 3686-93. 4.Wang, R., et al., J Leukoc Biol, 2012. 91(2): p. 299-309. 5.Helander, T.S. and T. Timonen, 1998. 230: p. 89-99. IFNγ has variable effects on expression of MHC Class I and ICAM-1 in pediatric cancers which alter sensitivity to NK cell mediated lysis Arianexys Aquino-López 1, Vladimir Senyukov 1, Gabrielle Romain 2, Navin Varadarajan 2, and Dean A. Lee 1 1 Department of Pediatric Research, University of Texas MD Anderson Cancer Center Houston, TX 2 Department of Chemical & Biomolecular Engineering, University of Houston, TX Results A) Abstract This presentation is supported by the National Cancer Institute through the U54 CA096297/CA096300: UPR/MDACC Partnership for Excellence in Cancer Research Training Program. For more information, please contact Arianexys Aquino-López at aaquino@mdanderson.org Introduction NK cell function depends on a complex interplay between inhibitory and activating signals. Although NK cell therapy is a promising treatment for cancer, a better understanding of the NK-ligands present on target cells and how they can be affected by the microenvironment can help optimize the use of NK cells as a therapy. Interferon-γ (IFNγ) is secreted by effector lymphocytes upon activation. The NK cell expansion protocol developed at our laboratory produces expanded NK cells with 30 times higher levels of IFNγ secretion than fresh NK cells (1). Therefore, the role of IFNγ and its effect in the tumor should be considered. IFNγ is known to cause upregulation of MHC class I in tumor cells, which has been associated with NK cell inhibition (2,3). On the other hand, natural killer cell produced IFNγ can promote target cell cytolysis through Intercellular Adhesion Molecule 1 (ICAM1 or CD54) upregulation (4). ICAM1 binding to Lymphocyte Function associated Antigen 1 (LFA1) in the NK cells leads to strong adhesion required for lysis by NK cells (5). In this study we evaluated the role of IFNγ in several pediatric tumors. We tested tumors for expression of NK-ligands, including HLA class I and the adhesion molecule ICAM1, in presence and absence of IFNγ. We also evaluated the effect of exposing tumor cells to IFNγ in their sensitivity towards NK cell mediated lysis and conjugate formation. Untreated 50ng/mL IFNγ 48 hrs Tumor cells HLA-ABC CD54 (ICAM-1) B) C) HLA-ABC CD54 (ICAM-1) B) C) B) C) Figure 1. Cell lines Kasumi-1 (AML) and MOLT4 (T cell ALL) showed increased resistance towards NK cell mediated lysis after being exposed to IFNγ. HLA-ABC CD54 (ICAM-1) NB1643 untreatedNB1643 IFNγ Treated Absolute Time to First Contact Figure 2. Cell lines CCRF-CEM (T cell ALL) and Karpas-299 (Lymphoma) had unaffected sensitivity towards NK cell mediated lysis after being exposed to IFNγ. Figure 3. Cell lines NB1643 (Neuroblastoma) and BT-12 (Brain Tumor) showed increased sensitivity towards NK cell mediated lysis after being exposed to IFNγ. A. NK cell mediated lysis evaluated by calcein release assay for control (blue) and IFNγ exposed (red) tumor cells. B. ICAM-1 and HLA-ABC expression for control (blue) and IFNγ exposed (red) tumor cells evaluated by Cytometry by Time of Flight (CyTOF). C. Fold change in level of expression (Mean Mass Intensity) of ICAM1 and HLA-ABC after IFNγ exposure. A) Figure 4. A. Conjugate formation evaluated by Flow Cytometry for cell lines NB1643 (Neuroblastoma) and BT-12 (Brain Tumor). We observe increased conjugate formation between tumor cells and NK cells after the tumor has been exposed to IFNγ. B. Conjugate formation is decreased after blocking ICAM-1 (CD54) with monoclonal antibodies in IFNγ treated tumor cells. C. NK cell mediated lysis was decreased for IFNγ treated tumor cells after blocking ICAM-1 (CD54) with monoclonal antibodies. A) B) C) NB1643 untreated NB1643 IFNγ 10 minutes in contact (1:2) BT-12 untreated BT-12 IFNγ 30 minutes in contact (1:2) Figure 5. A. Timelapse Imaging was used to evaluate the kinetics of NK cells interaction with NB1643 (Neuroblastoma) cells. NK cell stained in green and tumor cell stained in red. Tumor cell death evaluated through Annexin V staining. B. Absolute time to first contact was evaluated for NK cells exposed to untreated or IFNγ treated tumor cells. C. Wells containing 1:1 E:T ratio were analyzed for time required to establish contact. In the first 12 minutes 50% of NK cells had already established contact with IFNγ treated tumor cells. However, when NK cells were exposed to untreated tumor cells it took at least 45 minutes for 50% of them to establish contact. D. Survival Curve shows decreased survival for IFNγ treated tumor cells compared to untreated cells. B) C) D) Acknowledgements IFNγ exposure can increase both ICAM-1 and HLA-ABC expression in the tumor. When HLA-ABC upregulation exceeds ICAM-1 upregulation, we observed decreased tumor killing mediated by NK cells. When HLA-ABC and ICAM-1 were not affected tumor killing by NK cells was not affected. When ICAM1 upregulation dramatically exceeds HLA-ABC upregulation we observed increased conjugate formation and increased NK cell cytotoxic activity. Blocking of ICAM-1 in this scenario decreased the conjugate formation and NK cell cytotoxic activity. Time-lapse Imaging in Nanowell Grids (TIMING) results showed that NK cells took less time to establish stable contact with IFNγ treated Neuroblastoma cells (NB1643) when compared to untreated tumor cells.