Abstract A97: A high-throughput RNAi screen unravels new immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes.
Authors: Khandelwal Nisit , Breinig Marco , Speck Tobias , Conrad Heinke , Umansky Ludmila , Boutros Michael , Beckhove Philipp 
Published: August 2014

Cytotoxic T lymphocytes (CTLs) form an indispensable component of body's defense against cancer and therefore lie at the heart of the cancer immunotherapy program. However, evasion and suppression of immune surveillance by tumor cells remains a reality and a major roadblock for the clinical success of cancer immunotherapy. In this study, we established a novel RNAi silencing screen based on a siRNA library, which contained multiple siRNA sequences to individually target more than 500 G-protein coupled receptors (GPCRs) in MCF7 breast cancer cells, to identify those tumor-associated receptors that promote tumor resistance to immunological destruction by CTLs.

Briefly, luciferase-tagged MCF7 cells were reverse transfected with the siRNA library, followed by co-culture with the reactive CTLs. Effect of individual gene knockdown on CTL-mediated tumor lysis was determined based on the luciferase activity of the remaining live cells in each well. Gene knockdowns that affected cell viability by themselves were excluded from the hit analysis. Z-score based normalization resulted in a hit list comprising of 79 candidates that negatively regulate CTL activity and 27 candidates that positively modulate the T cell response.

Using antigen-specific T cell clones in secondary assays, we have validated two of such high scoring hits, namely NiS-6 and NiS-1, that negatively regulate CTL response against tumor. siRNA-mediated knockdown of NiS-6 increases the susceptibility of both MCF7 and MDA-MBD-231 breast cancer cells to immunological destruction by survivin-specific T cells, as measured in standard chromium-release kill assays. Blocking of NiS-6 in MCF7s via siRNA also increases the interferon-gamma and granzyme B secretion by survivin-TCs in ELISpot readouts. Moreover, elevated levels of IL-2 and TNF-alpha, along with significantly diminished levels of immunosuppressive cytokine IL-10, were observed in the supernatant of the MCF7 and T cell coculture upon NiS-6 knockdown. Preliminary knockdown studies with NiS-1 receptor in tumor cells demonstrated that NiS-1 might be similarly involved in de-sensitizing tumor cells to CTL-mediated lysis.

Here we report a systematic and high throughput screening strategy for the identification of novel immune modulators associated with breast cancer. Based on our findings, we suggest a new and previously undefined role for the tumor-associated G-protein coupled receptors- NiS-6 and NiS-1 – in downregulating T cell responses. These surface receptors are therefore an attractive therapeutic target for the treatment of breast cancer in either a monotherpay setting or in conjunction with adoptive immunotherapy, so as to override tumor's acquired resistance to immunological destruction.

Citation Format: Nisit Khandelwal, Marco Breinig, Tobias Speck, Heinke Conrad, Ludmila Umansky, Michael Boutros, Philipp Beckhove. A high-throughput RNAi screen unravels new immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A97.