Immune checkpoints during the sequential steps of immune responses are crucial events to maintain self-tolerance and to protect tissues from damage caused by hyperimmune reactions during pathogenic inflammation (Pardoll 2012; Zou and Chen 2008). CTLA-4 is transiently expressed on effector T cells after activation and functions as an inhibitory receptor in the first immune checkpoint, which regulates antigen presentation from dendritic cells (DC) in primary or secondary lymph nodes (Zou and Chen 2008). When antigen is presented by DCs to naïve or memory T cells, T cells recognize antigen by binding to the T cell receptor (TCR) and epitope peptide complexed with major histocompatibility complex (MHC) expressed on the DC. Simultaneously, co-stimulatory receptor CD28, exclusively expressed on T cells, binds to CD80/CD86 on DCs and stimulates immune responses (Zou and Chen 2008). CTLA-4 shares identical ligands, CD80/CD86, with CD28, and counteracts the activity of CD28 (Schwartz 1992; Rudd et al. 2009). Although the mechanisms of T cell suppression by CTLA-4 are not clear, two different mechanisms have been demonstrated. CTLA-4 is thought to compete with CD28 in binding CD80 and/or CD86 because the binding affinity of CTLA-4 for both ligands is higher than that of CD28 (Linsley et al. 1994; Egen and Allison 2002). Another explanation is the activation of protein phosphatases, Src homolog domain-containing phosphatase 2 (SHP2, also known as PTPN11) and protein phosphatase 2 (PP2, also known as PP2A), which inhibit kinase signaling downstream of TCR and CD28 (Schwartz 1992). CTLA-4 is constitutively expressed on Tregs as well as CD8+ cytotoxic T cells and CD4+ T helper (Th) cells, and is a target gene of the forkhead transcription factor FOXP3 (Hori et al. 2003; Fontenot et al. 2003), the master gene of Treg. FOXP3 regulates the differentiation and functions of Treg (Hill et al. 2007; Gavin et al. 2007) and is one of the mechanisms of effector T cell suppression that downregulates CD80 and CD86 on APCs. This is caused by the binding of CTLA-4 on Treg counteracting CD28 binding CD80/CD86, resulting in the inhibition of antigen presentation to T cells (Vignali et al. 2008) (Fig. 17.1).

PD-1 is another important co-inhibitory receptor stably expressed on effector-phase T cells, which regulates T cell activation by interactions with PD-L1 and/or PD-L2 expressed on tumor cells, APC, and non-transformed cells in the tumor microenvironment (Dong et al. 2002; Konishi et al. 2004; Curiel et al. 2003; Kuang et al. 2009). PD-1 engagement inhibits kinase signaling downstream of TCR through phosphatase SHP2 (Dong et al. 2002). PD-1 is also highly expressed on Tregs and enhances the proliferation of Tregs in the presence of ligand (Freeman et al. 2000; Francisco et al. 2009).

As well as the molecules described above, various inhibitory receptors and ligands are involved in immune checkpoints. T cell membrane protein 3 (TIM3), LAG3 (also known as CD233), B and T Lymphocyte attenuator (BTLA; also known as CD272), adenosine A2a receptor (A2aR), and the family of killer inhibitory receptors are inhibitory receptors expressed on T cells. Galectin 9, MHC class II, herpesvirus entry mediator (HVEM), adenosine, and subsets of human leukocyte antigens (HLA) have been respectively identified as their ligands (Pardoll 2012). These receptors are also expressed on activated T cells, and the ligands are expressed on many types of tumor cells (Ngiow et al. 2011a; Grosso et al. 2007; Cedeno-Laurent and Dimitroff 2012; Fourcade et al. 2012; Baixeras et al. 1992). Furthermore, antibodies to these enhance anti-tumor immunity in mouse models and human in vitro experiments (Baghdadi et al. 2013; Ngiow et al. 2011b). LAG3 is expressed on Tregs as well as effector T cells and enhances Treg functions. A2aR functions in the development of Tregs by inducing FOXP3 expression in CD4+ T cells (Huang et al. 2004; Zarek et al. 2008).

Thus, co-inhibitory receptors/ligands regulate immune responses of effector T cells at sites of inflammation or tumors (Fig. 17.2), and drugs, such as antibodies and small molecules, which block receptor/ligand interactions are expected to be novel beneficial drugs for tumor therapy (Fig. 17.3). Clinical trials of anti-CTLA-4 humanized mAb, ipilimumab, and tremelimumab were conducted from 2000. Although objective clinical responses were observed in ~10 % of patients with melanoma using both antibodies, no survival benefit was observed in comparison with standard melanoma chemotherapy treatment (dacarbazine) in a randomized phase III trial for tremelimumab (Ribas 2010). However, in a randomized, three-arm phase III trial of patients with advanced melanoma using (1) gp100 peptide vaccine alone, (2) gp100 plus ipilimumab, or (3) ipilimumab alone, 3.5-month survival benefit was observed in the ipilimumab alone group compared with the gp100 peptide vaccine alone or gp100 plus ipilimumab groups (Hodi et al. 2010). Furthermore, Grade 3 or 4 immune-related adverse events (AEs) occurred in 10–15 % of patients treated with ipilimumab, including deaths associated with immune-related AEs. Ipilimumab was the first drug to demonstrate a survival benefit for metastatic melanoma, and was approved by the US Food and Drug Administration (FDA) for the treatment of advanced melanoma in 2010. Both humanized mAb to PD-1 (nivolumab/BS936558) and PD-L1 (BS936559) demonstrated very promising results in a phase I clinical trial for multiple tumors (Topalian et al. 2012; Brahmer et al. 2012). Objective response rates to BMS936558 were observed in 18 % of patients with non-small cell lung cancer (14 of 76 patients), 28 % of patients with melanoma (26 of 94 patients), and 27 % of patients with renal cell cancer (9 of 33 patients). Interestingly, no objective response was observed in 17 patients with PD-L1-negative tumors, although 9 of 25 patients (36 %) with PD-L1-positive tumors had an objective response. In a phase I trial using anti-PD-L1 (BMS936559), an objective response (a complete or partial response) was observed in 17 % of patients with melanoma (9 of 52), 12 % of patients with renal cell cancer (2 of 17), 10 % of patients with non-small cell lung cancer (5 of 49), and 6 % of patients with ovarian cancer (1 of 17). Furthermore, responses were durable for both antibodies. Responses lasting 1 year or more were shown in more than half of the patients with 1 year or more of follow-up. Immune-related AEs occurred in 14 % of patients treated with anti-PD-1 and 9 % of patients treated with anti-PD-L1. Interestingly, immune-related AEs frequently occurred in the skin and intestine in patients receiving any of ipilimumab, nivolumab, or BMS936559.

It is clarified that FOXP3 elicits Treg functions (Hori et al. 2003; Fontenot et al. 2003). Because FOXP3 knockout mice develop autoimmune diseases involving multiple organs, and patients with FOXP3 homozygous mutations develop immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome (Fontenot et al. 2003; Bennett et al. 2001), Tregs are considered to be crucial for the maintenance of self-tolerance and for maintaining immune balance to prevent damage of healthy cells from excessive immune reactions in cooperation with the immune checkpoint system described above. It was reported that Tregs are recruited and accumulate in tumor tissues by chemokine-induced chemotaxis via interaction between CCR4 and its ligands produced by cells of the tumor microenvironment (Curiel et al. 2004; Zou 2006). Hodgkin’s lymphoma (HL) is a representative example of Treg recruitment to the tumor site by chemokine-induced chemotaxis resulting in immunosuppression. HL is characterized by the presence of a small number of tumor cells in a rich background of T and B cells, macrophages, and other inflammatory cells (Küppers 2009). The question of why a very small number of HL cells can survive in the presence of a large excess of host immune cells was unclear. HL cells express high levels of the CCR4 ligand and thymus and activation-regulated chemokine (TARC)⁄CCL17 (van den Berg et al. 1999), and elevated serum levels of TARC⁄CCL17 were reported as an unfavorable prognostic factor in patients with HL (Weihrauch et al. 2005). Considering these findings, we have shown that HL tumor cells attracted CD4+CCR4+ T cells by interactions with TARC⁄CCL17 and macrophage-derived chemokine (MDC)⁄CCL22. Migratory CD4+ cells attracted by HL tumor cells were hyporesponsive to TCR stimulation and suppressed the activation and proliferation of effector CD4+ T cells in an autologous setting in vitro. Furthermore, double staining showed that HL cells in the affected lymph nodes were surrounded by a large number of lymphocytes expressing both CCR4 and FOXP3 (Ishida et al. 2006). Collectively, these findings imply that the migratory cells induced by HL function as Tregs to create a favorable environment for the tumor cells to escape from host immunity. It was proposed that Treg-mediated immunosuppression is a crucial tumor immune-evasion mechanism in ovarian, gastric, breast, and pancreatic cancers, and may be one of the main obstacles to successful tumor immunotherapy (Curiel et al. 2004; Zou 2006). Therefore, Tregs are a potential target for novel tumor therapy by inhibition of immunosuppression in the tumor site.