Elsevier

Cellular Immunology

Volume 278, Issues 1–2, July–August 2012, Pages 76-83
Cellular Immunology

Tumor-infiltrating regulatory T cells delineated by upregulation of PD-1 and inhibitory receptors

https://doi.org/10.1016/j.cellimm.2012.07.001Get rights and content

Abstract

Foxp3+ regulatory T (Treg) cells are dominant suppressor cells which regulate conventional T (Tconv) cells. Inside tumor microenvironment, Treg cells have been known to become potent in suppressing Tconv cell responses, thereby enabling tumor cells to circumvent immune response. However, the underlying mechanism by which tumor-infiltrating Treg cells display enhanced suppressive function is still unresolved. To understand characteristics and function of tumor-infiltrating Treg cells as well as Tconv cells in the tumor site, we analyzed their phenotypes either within tumor burden or at distant site of tumor using both heterotopic and orthotopic mouse cancer models. Compared to CD8+ T cells at distant site of tumor, tumor-infiltrating CD8+ T cells dramatically upregulated programmed death 1 (PD-1) and other inhibitory receptors, thereby being more exhausted functionally. Tumor-infiltrating CD4+ T cells also expressed higher level of PD-1 than CD4+ T cells at distant site of tumor but very surprisingly, upregulation of PD-1 occurred in CD4+Foxp3+ Treg as well as CD4+Foxp3 Tconv cells. Moreover, tumor infiltrating Treg cells upregulated other inhibitory receptors such as T cell immunoglobulin mucin 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and lymphocyte activation gene-3 (LAG-3). These results suggest that upregulation of PD-1 and other inhibitory receptors on tumor-infiltrating Treg cells is related with their enhanced suppressive function.

Highlights

► Tumor-infiltrating T cells express higher level of PD-1 than tumor-distant site ones. ► TILs include substantial population of regulatory cells such as Treg cells and MDSCs. ► PD-1 and inhibitory receptors are highly upregulated on tumor-infiltrating Treg cells.

Introduction

Evasion from cancer immune surveillance is common in many types of tumor. The ability of tumor cells to avoid the immune system’s detection gives them not only proliferative and survival advantages, but also its development and progression into metastatic stage. In order to escape the destruction response by the immune system, one mechanism that tumor cells employ is the suppression of antitumor immune response in the tumor microenvironment [1]. The prevalence characteristic of local immunosuppression is the display of effector T cells exhaustion which is constrained in a specific physiological area [2].

Effector T cells exhaustion was first observed in the mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV) [3]. During chronic infection of mice with LCMV, effector T cells such as CD8+ cytotoxic and CD4+ helper T cells have shown severe defects in their canonical effector functions including cytotoxicity and cytokine secretion as a response to stimulation by antigen presenting cells (APCs). Several studies suggested that the sustained expression of inhibitory receptors on the surface of these cells might be the underlying reason behind the effector T cells exhaustion phenotype [4]. Some evidences implied that effector T cells, especially CD8+ cytotoxic T cells upregulate the expression of programmed death-1 (PD-1) receptors in multiple solid tumor microenvironment and the blockage of the interaction between PD-1 with its ligands, PD-L1 and PD-L2 inhibits tumor growth and progression [5], [6], [7]. Although there is significant tumor regression observed, blocking the interaction between PD-1 and its ligands alone is not sufficient to restore the effector T cells functions and proliferation [8]. Thus, it is likely that multiple suppressive receptors are involved in shaping effector T cells exhaustion phenotype in tumor microenvironment.

Indeed, a recent study elucidated that another suppressive receptor, T cell immunoglobulin mucin 3 (TIM-3) is also being co-expressed with PD-1 in the majority of dysfunctional tumor infiltrating lymphocytes (TILs) population [4]. Another report also found that negative co-stimulatory receptor cytotoxic T-lymphocyte antigen 4 (CTLA-4) is being highly expressed in TILs [9]. A growing number of literatures also have observed that multiple inhibitory receptors such as T cell immunoglobulin mucin 3 (TIM-3) [4], [10], [11], lymphocyte activation gene 3 (LAG-3) [12], [13], and glucocorticoid-induced tumor necrosis factor receptor (GITR) [14], [15] are being expressed on elevated level in immunosuppressive environment. These data suggested that it is highly probable that these receptors synchronically suppress the function and proliferation of effector T cells, leading to the differentiation into exhausted T cells inside tumor microenvironment.

Recently, a number of studies showed the involvement of regulatory T (Treg) cells on the cancer immunosuppression modulation in multiple murine tumor models [16]. These data demonstrated that there is significant increase of Treg cells recruitment into these solid tumor tissues, compared to tumorigenic peripheral tissues. Thereby, they proposed that effector T cells exhaustion is the consequence of the recruitment of suppressive Treg cells to the primary tumor site. Despite the strong indication that TILs play major roles in immunosuppression response inside tumor microenvironment, the phenotypes of these Treg cells are still an enigma.

In this study, we analyzed tumor-infiltrating Treg cells in tumor burden from different murine tumor model. We demonstrated that there are remarkable distinctions between Treg cells from primary tumor site and those from periphery tissues. We observed that Treg cells within tumor burden substantially elevate the expression of multiple suppressive receptors such as PD-1, CTLA-4, TIM-3, LAG-3, and GITR, which of receptors were even co-expressed on Treg cells in TILs. In addition, our study revealed that inside primary tumor tissue, together with CD4+Foxp3+ Treg cells, there was also dramatic accumulation of myeloid derived suppressor cells (MDSCs) which are known to control the effector T cells.

Section snippets

Mice

Six- to seven-week-old male C3H/HeN mice were purchased from Central Lab, Japan (Seoul, Korea). All mice were maintained in a specific pathogen-free facility of the Yonsei University Medical College guidelines, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent experiment provided by the Assessment and Accreditation of laboratory Animal Care (AAALAC).

Tumor model

For preparation of heterotopic tumor-bearing model, 1 × 106 MIH-2 cells were implanted into the right

Functionally impaired phenotypes of T cells among TILs from heterotopic tumor model

To investigate the distribution of T cells in various tissues, we performed FACS analysis using lymphocytes which were isolated from spleen and tumor burden site of naïve and heterotopic tumor-bearing mice model. Mice were heterotopically transplanted with a hepatocellular carcinoma cell line, MIH-2, at the right thigh and sacrificed on day 45 after tumor transplantation. In terms of frequency, CD4+ and CD8+ T cells in splenocytes showed no significant difference between naïve and tumor-bearing

Discussion

In this study, we investigated the phenotype of Treg cells as well as CD4+ and CD8+ Tconv cells which co-exist in tumor site by utilizing both heterotopic and orthotopic tumor models. As expected, most of CD8+ T cells and less than half of CD4+ T cells in TILs expressed PD-1, which indicates a functional exhaustion of PD-1-expressing Tconv cells. Surprisingly, most of Treg cells among tumor-infiltrating CD4+ T cells expressed high level of PD-1 and other inhibitory receptors, which has not been

Acknowledgments

This work was supported by the Korean Health Technology R&D Project Grant funded by Ministry for Health, Welfare & Family Affairs (A101956) and the National Research Foundation of Korea (NRF) Grants funded by the Korea Government (MEST) (2010-0018544, 2009-0077426, and 2010-0027222).

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