B7-H1 and B7-H4 expression in colorectal carcinoma: Correlation with tumor FOXP3⁺ regulatory T-cell infiltration

Abstract

B7-H1 and B7-H4 are newly discovered members of the B7-CD28 family. They can inhibit T cell activation and proliferation and regulate T cell immune response negatively. Both B7-H1 and B7-H4 are expressed in many tumors and are classified as co-inhibitors of cell-mediated immunity. FOXP3⁺ regulatory T cells (Tregs) play an important role in the maintenance of tumor immunity tolerance. However, the implication of B7-H1 and B7-H4 expression and their interaction with Tregs infiltration in colorectal cancer are unknown. The present study aimed to determine the expression of B7-H1 and B7-H4 as well as Tregs infiltration in colorectal cancer and to explore the clinical and pathological implication of suppressor immune cells and molecules. Frozen sections and immunohistochemical assay were undertaken to assess B7-H1, B7-H4 expression and Tregs infiltration in fresh specimens collected from 56 patients with colorectal carcinoma. The results showed that expression of B7-H1 and B7-H4 in colorectal carcinoma tissues was significantly higher than in adjacent normal mucosa (P < 0.001). B7-H1 expression was positively correlated to the infiltration depth, lymph node metastasis and advanced Duke's stage (P < 0.05, P < 0.05 and P < 0.05, respectively), whereas B7-H4 expression was positively related to the infiltration depth and lymph node metastasis (P < 0.01 and P < 0.05, respectively). Furthermore, Tregs infiltration was more frequent in tumor tissue than in adjacent normal mucosa and was associated with poor differentiation and positive lymph node metastasis (P < 0.01, and P < 0.01, respectively). The statistical analysis indicated a significant correlation between Tregs infiltration and B7-H1 or B7-H4 expression respectively. These results suggest that over-expression of B7-H1 and B7-H4 has stronger prognostic significance and promote tumor tolerance, and they might contribute to Tregs development in the colorectal carcinoma tolerogenic milieu.

Introduction

Colorectal carcinoma (CRC) is one of the most frequent malignancies worldwide, and it is the second leading cause of cancer-related deaths in the world (Herszényi and Tulassay, 2010). Clinically, because of advances in surgical technology, radiotherapy and the use of powerful chemotherapeutics, the CRC prognosis has markedly improved. Nevertheless, 40–50% of CRC patients are confronted with recurrence and metastasis. Clearly it is necessary to explore the tumorigenesis in order to develop novel therapies for the treatment of CRC, and one possibility is an immunotherapeutic approach to correct the abnormal immunity (Koudougou et al., 2013).

The B7 protein family provides both stimulatory and inhibitory regulation of T cell responses, depending on which B7 ligand and receptor is engaged on the target cell (Carreno and Collins, 2002, Khoury and Sayegh, 2004). B7-H1 is an identified member of the B7 family as a co-stimulatory molecule. B7-H1 is expressed on immunocompetent cells such as T cells, B cells, dendritic cells (DCs), and macrophages (Yamazaki et al., 2005). With engagement of programmed cell death 1(PD-1) receptor, B7-H1 molecule presenting on DC influences the hindrance of T cell proliferation depending on T cell receptor (TCR) and the decrease in cytokine production (Mirza et al., 2010). Furthermore, the interaction of B7-H1–PD-1 plays a critical role in suppressing T cell-based immunity and has emerged as an essential mediator of tumor-associated immune suppression (Ichikawa and Chen, 2005). B7-H4 was identified in 2003 as a co-stimulatory molecule. It is also a ligand within the B7 family and it is vital in the process of hindering the proliferation of T cells and blocking the production of cytokines by Th1 and Th2 sub-populations (Sica et al., 2003, Zheng et al., 2012). B7-H4 mRNA expression was found to be widely distributed in the peripheral tissues. However, B7-H4 protein expression on the cells is primarily restricted to activated T cells, B cells, monocytes, and DCs (Sica et al., 2003, Choi et al., 2003). B7-H4 is not detected in the majority of normal tissues and cells, but is found in a variety of tumor tissues, mainly expressed in tumor cells and tumor infiltrating macrophages (Salceda et al., 2005, Sun et al., 2006, Tringler et al., 2006, Zang et al., 2007). Collectively, these findings suggest that B7-H1 and B7-H4 may function as a negative mediator of immune responses through different pathways in anti-tumor immunity (Ichikawa and Chen, 2005, Janakiram et al., 2012).

Regulatory T cells (Tregs) are a unique CD4⁺ helper T cell subset and are characterized by the CD4⁺CD25⁺ phenotype. Forkhead box protein 3 (FOXP3) belongs to the forkhead/winged-helix family of transcriptional regulators and appears to function as a master regulator in the development and control of Tregs. FOXP3 has been regarded as the most specific and reliable surface marker of Tregs (Fontenot et al., 2003, Hori et al., 2003, Khattri et al., 2003). Tregs have been found in a number of human tumors, and their presence has been associated with impeding immune escape against autologous tumor cells (Nomura and Sakaguchi, 2005). Furthermore, B7-H1 expression is associated with expansion of Tregs in the tumor microenvironment (Ni et al., 2012). Kristensen et al. (2013) showed that treatment with B7-H4-Ig fusion protein in vivo changes lymphocyte homeostasis and increases the functional activity of Tregs (Kristensen et al., 2013). Thus, our hypothesis is that there is a strong link between expression of inhibitory B7 molecules by the cancer cells and Tregs infiltrated into the tumor environment. In this report, we performed immunohistochemistry to characterize B7-H1 and B7-H4 expressions in human CRC. Tregs were also detected in the tumor tissues and the relationship between inhibitory B7 molecules and Tregs population was analyzed.