Purpose: Regulatory T (Treg) cells perform the immune suppressive function in cancer, but their suppressive mechanism in tumor microenvironment (TME) has not been clearly elucidated. We aim to identify the phenotype and functional mechanism of Treg cells in TME from cancer patients.

Experimental Design: We collected 72 malignant effusion (ME) and peripheral blood (PB) specimens from stage IV cancer patients and 10 tumor tissue (TM) and PB from lung cancer patients who underwent surgery. Lymphocytes from ME, TM, and PB were analyzed for subtype of Tconv and Treg cells, and their expressions of immune checkpoint (IC) molecules including programmed death (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), and T cell immunoglobulin containing molecule-3 (TIM-3) using by flow cytometry. To examine the functional role of PD-1 expressed Treg cells, TC-1 lung cancer mouse model was used.

Result: Upregulation of PD-1 on Treg cells in TM and ME compared to PB was even more distinguishable than that on CD4+ and CD8+ Tconv cells. To clarify the characteristics of tumor infiltrating Treg cells, we comprehensively examined the characteristics of Treg cells from tumor, peri-tumor, and PB in lung cancer patients by analyzing the expression of IC molecules including PD-1, TIM-3, CTLA-4, and TIGIT, of which PD-1 is the highest expressed on tumor infiltrating Treg cells. To investigate the mechanism by which Treg cells mediate immune suppression, we compared the suppressive activity of Treg cells expressing high and low levels of PD-1 by co-culturing each population with naïve CD8+ T cells with or without αCD3/CD28 stimulation. This was more potently inhibited in co-cultures with PD-1high tumor infiltrating Treg cells than in those with PD-1low Treg cells. PD-1 blocked tumor infiltrating Treg cells demonstrated a significantly decreased suppressive function in the proliferation of CD8+ cells and their IFN-γ production. These results implicate that PD-1 expressed tumor infiltrating Treg confers the suppressive function of proliferation in CD8+ T cells through PD-1: PD-L1 interaction.

Conclusion: Our study evidently demonstrated that upregulated PD-1 on tumor infiltrating Treg cells and their PD-1: PD-L1 interaction could be potential cause of T cell suppression, which might be helpful to completely understand their suppressive mechanism in cancer patients.

Citation Format: Hye Ryun Kim, Hyo Jin Park, Jimin Son, Hyo Sup Shim, Byoung Chul Cho, Sun Young Rha, Sang-Jun Ha. Upregulation of immune checkpoint molecules on Treg cells in tumor microenvironment reinforces immune exhaustion in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 713.