Abstract 4862: Reversal of NK cell exhaustion in advanced melanoma patients by Tim-3 blockade
Authors: Gallois Anne , Silva Ines Pires Da , Jimenez Sonia , Osman Iman , Bhardwaj Nina 
Published: May 2015

Introduction: Among the most promising approaches to activate therapeutic anti-tumor immunity is the blockade of immune checkpoints. CTLA-4 and PD-1 inhibitors have been recently shown to enhance anti-tumor responses and improve overall survival in various cancers including melanoma. Similarly, Tim-3 (T cell immunoglobulin- and mucin-domain-containing molecule-3) belongs to the group of immune checkpoints and has emerged as a key player in immune regulation. Tim-3 is co-expressed with PD-1 on human tumor-specific CD8+ T cells, and in vivo studies have shown that its blockade alone, or in combination with PD-1, is able to control tumor growth in several tumor models, including melanoma. Tim-3 is constitutively expressed on natural killer (NK) cells, key players in anti-tumor responses, but its precise role remains unclear. In this study, we examined the role of Tim-3 in NK cells from healthy donors and patients with metastatic melanoma.

Methods: A cohort of 83 melanoma patients, distributed by stage I, stage II and stage III/IV, was analyzed for Tim-3 expression on circulating NK cells, and associated demographic and clinical parameters. NK cells from patients with advanced melanoma (stage III/IV ; n=20) and healthy donor (n=20) were further evaluated for their expression of activating (NKG2D and NKp46) and inhibitory receptors (KIRB1 and KIRNKAT2) ; function (cytotoxicity, IFN-γ production and proliferation) ; and the intracellular expression of the transcription factor T-bet. NK cell cytotoxicity was measured by Lamp-1 expression, using K562 cells as target cells. IFN-γ production was measured after 4h stimulation with rhIL-12, and proliferation was quantified by CFSE after 6 days in the presence of rhIL-2. The same functional assays (cytotoxicity, IFN-γ production and proliferation) were performed after Tim-3 blockade.

Results: As the stage of melanoma advanced, we observed a pattern of gradually increasing Tim-3+ NK cells (p=0.01). Significantly, the expression of Tim-3 was higher in melanoma patients with poor prognostic factors, such as thickness &gt;1mm (p=0.006), mitotic rate &gt;=1/mm2 (p=0.03), ulceration (p=0.05) and presence of metastases (p=0.009). Compared to healthy donors, NK cells from metastatic melanoma patients were functionally impaired/exhausted according to their ability to proliferate (p=0.002), produce cytokines (p=0.03) and kill target cells (p=0.02). Tim-3 blockade was able to reverse this exhausted phenotype, as shown by increased NK cell cytotoxicity (p=0.002), cytokine production (p=0.007) and proliferation (p=0.009).

Conclusion: These data indicate that Tim-3 functions as an inhibitory receptor on NK cells in advanced cancer and supports the development of Tim-3-targeted therapies to restore anti-tumor immunity.

Citation Format: Anne Gallois, Ines Pires Da Silva, Sonia Jimenez, Iman Osman, Nina Bhardwaj. Reversal of NK cell exhaustion in advanced melanoma patients by Tim-3 blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4862. doi:10.1158/1538-7445.AM2014-4862