Distribution, characterization, and induction of CD8⁺ regulatory T cells and IL-17-producing CD8⁺ T cells in nasopharyngeal carcinoma

Undifferentiated nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) infection, which has a high incidence in Southern China and Southeast Asia. Conventional therapy is often ineffective for NPC patients with late-stage disease [1, 2]. Recently, immunotherapy has become a promising therapeutic option for various types of cancer with high immunogenicity, including NPC [3‐5]. The success of EBV-specific cytotoxic T lymphocyte (CTL) treatment has been reported in NPC. However, it has been difficult to obtain consistent results or stable clinical efficacy [6‐8]. One possibility is that immune-suppressive environments may be created in patients with NPC [9‐11]. Thus, a better understanding of the immune status of NPC patients, including the distribution of specific lymphocyte subsets and their functions, is crucial in developing more effective immunotherapy strategies.

CD8⁺ T cells that express high levels of the transcription factors Eomes and T-bet are usually destined to develop into cytotoxic effector cells that produce interferon (IFN) γ, granzyme B and perforin, however, CD8⁺ cells may also give rise to a regulatory lineage (Tcreg). The CD4⁺Foxp3⁺ regulatory T cells (Tregs), which have been recognized as a suppressor of antitumor immunity because of its natural suppressive effect on the proliferation and IL-2 secretion of naïve and effector T cells [12, 13], but the distribution, generation, characteristics, and function of Tcregs in cancer remain poorly understood, as does their pathogenic antigen specificity. Furthermore, interleukin (IL)-17-producing CD8⁺ T cells (Tc17 cells) have been identified in mice and humans, and their enrichment inside solid tumors has been reported [14]. However, the generation and function of Tc17 cells in cancer remain largely uncharacterized.

In this study, we aimed to investigate the distribution, characterization, and generation of CD8⁺ Tcregs and Tc17 cells in NPC patients. We observed an increase of Tcregs and decrease of Tc17 cells from peripheral blood mononuclear cells (PBMCs) and an accumulation of Tcregs and Tc17 cells in tumor tissues from 21 NPC patients. The Tcreg subset expressed CC chemokine receptor 6 (CCR6), cytotoxic T lymphocyte antigen 4 (CTLA4), and glucocorticoid-induced tumor necrosis factor (TNF)-related (GITR) proteins at high levels, resulting in a Treg-like phenotype. The Tc17 cells expressed high levels of CCR6 and low levels of CTLA4 and GITR protein, and they contained a high proportion of cells secreting TNFα, which is a Th1 cytokine. Moreover, the Tcregs from the tumor-infiltrating lymphocytes (TILs) secreted high levels of IL-10 and IFNγ but low levels of IL-2, IL-4, TNFα, and IL-17, resulting in a Tr1-like cytokine profile; Tcregs from PBMCs, in contrast, secreted high levels of IL-10 and low levels of transforming growth factor β (TGFβ), IL-2, IFNγ, TNFα, and IL-17. In addition, there was a significantly higher percentage of IFNγ-secreting cells among the Tc17 cells from the TILs than among those from the PBMCs, and this was true of cells from both NPC patients and healthy donors. Furthermore, the Tcregs from NPC patients possessed a suppressive function to the proliferation of CD4⁺ naive T cells, which was mainly mediated by a cell-to-cell contact-dependent mechanism. IFNγ failed to impair the suppressive function of Tcregs in vitro. Collectively, these data suggest that, in addition to CD8⁺ cytolytic effector cells, Tcregs and Tc17 cells with diverse functions are present in NPC patients. Additionally, this is the first demonstration of the existence of EBV antigen-induced Tcregs in patients with NPC.

The development and progression of cancer are associated strongly with the unique immunosuppressive characteristics of the tumor microenvironment, regardless of the chronic inflammatory response and infiltrating inflammatory cells that are often produced around tumor tissue [19‐22]. The increase and accumulation of CD4⁺ Tregs in circulating PBMCs and tumor tissues have been observed in many kinds of malignancy, including NPC [23‐26]. In a recent study, we showed by double immunohistochemical staining that there was a CD8⁺Foxp3⁺ TIL subset in NPC tissues in vivo[17].

In the present study, we showed that the percentage of CD8⁺ Tcregs increased in both circulating PBMCs and TILs of NPC patients when compared with PBMCs from healthy individuals (Figure 1, Figure 2A and 2B). This indicates that the accumulation of Tcregs in tumor tissue not only results from their recruitment from the peripheral blood to tumor tissue but also from the induction of CD8⁺ Tcreg precursor cells at the tumor site. As a consequence, this Tcreg subset should contain natural Tregs (nTregs) and induced Tregs (iTregs) together at tumor site. This hypothesis was supported by the following findings. First, the Tcregs in TILs expressed CD45RO and CD45RA, which implied that Tcregs contained both memory and naïve Tcreg cells. Second, the Tcregs in PBMCs showed high levels of expression of CCR6, a ligand of chemokine CCL20, which is usually overexpressed in the NPC tumor microenvironment and upregulated by the EBV-encoded LMP1 protein [27, 28]. Thus, these cells had migrated to the tumor tissue. Third, the Tcregs from the TILs expressed an IFNγ^highIL-10^highTGFβ^lowIL-2^lowTNFα^lowIL-17^low cytokine secretion phenotype (Tr1-like cytokine profile) (Figure 3). Moreover, we found that the Tcreg subset also contained some IL-17-producing cells (approximately 10%) and that the Tcregs from TILs expressed a significantly higher level of IFNγ than those from PBMCs of patients and controls. These results indicate that Tcregs in TILs of patients with NPC were not stable and did secrete some proinflammatory cytokines, such as IL-17 and IFNγ.

Our data demonstrate that Tcregs from TILs of NPC patients can suppress the proliferation of CD4⁺ naïve T cells and CD8⁺ effector T cells in vitro. This suppression was mainly mediated by cell-to-cell contact, and IFNγ did not affect the suppressive function of Tcregs in NPC (Figure 4). The results are supported by others' reports on the mechanism of suppression by Tcregs [29‐32], and they reveal unique functions for Tcregs in NPC tumor tissue. On one hand, Tcregs facilitate tumor growth by suppressing activated T cells; on the other hand, they secrete a large amount of the Th1 cytokine IFNγ, which inhibits tumor cell growth and promotes the differentiation of Th1 cells.

In recent years, attention has focused on the role of CD4⁺ Th17 cells in solid tumors, but their function remains largely unclear. Some researchers have suggested that Th17 cells promote tumor growth through an IL-6/STAT3 pathway, up-regulation of IL-8, and induction of tumor angiogenesis [33‐35]. Others believe that Th17 cells have an antitumor function and that the number of Th17 cells is associated with a better outcome for cancer patients. Additionally, Th17 cells amplified adoptive CTL immunotherapy in one mouse model [34, 35], and we have identified one CD4⁺ Th17 clone from an NPC patient that could inhibit melanoma growth in a NOD/SCID mouse model (unpublished data). However, the role of Th17 cells in NPC remains poorly understood.

In sum, the present study is the first to investigate the distribution, generation, and function of Tc17 cells in NPC. We found that the proportion of Tc17 cells was reduced significantly in PBMCs when compared to TILs from NPC patients or to PBMCs from healthy donors (Figure 2C and 2D). The Tc17 cells in TILs expressed only CD45RO and not CD45RA (Figure 3A), indicating that this cell population was derived from memory cells and migrated from the peripheral blood. In addition, the high levels of expression of CCR6 in the Tc17 cells from PBMCs also indicate that these cells were able to migrate to tumor tissue. Therefore, the proportion of Tc17 cells from PBMCs was reduced compared to TILs from paired NPC patients or PBMCs from healthy individuals.

Tc17 cells expressed high levels of IL-17 and TNFα, intermediate levels of IL-10, and low levels of IL-2. Similar to Tcregs, Tc17 cells in the TILs but not in the PBMCs of patients with NPC expressed high levels of IFNγ (Figure 3C). This was consistent with a recent report of a similar cytokine profile for Tc17 cells in human liver cancer [36]. Furthermore, other research groups have shown that Tc17 cells, after polarization in vitro, can inhibit the growth of B16 tumors in mouse models by secreting IFNγ in vivo[14, 37]. Most Tc17 cells are derived from CD8⁺ T cells that lacked the expression of T-bet and Eomes; however, the expression of T-bet and Eomes is important for IFNγ production and for the antitumor function of CD8⁺ T cells, especially in TILs [38]. We also found that in NPC patients, only Tc17 cells from TILs expressed high levels of IFNγ, which implied that Tc17 from TILs have a Th1 cell function and might have an antitumor function.

To address whether EBV antigens could induce Tcregs in NPC, we analyzed Tcregs specific for the EBV HLA-A2-restricted LMP1 and LMP2 epitopes by tetramer and Foxp3 staining. We identified CD8⁺ T cells and Tcregs that were specific for the LMP1 epitopes LLW and YLQ and the LMP2 epitope GLG both in the PBMCs and TILs from NPC patients (Figure 5). The percentage of LLW-, YLQ-, and GLG-specific CD8⁺ T cells and Tcregs was increased slightly in TILs as compared to PBMCs, but this increase was only significant for the LLW epitope antigen-specific CD8⁺ T cells and the YLQ and GLG epitope antigen-specific Tcregs. These findings demonstrate that EBV epitope-specific CD8⁺ T cells and Tcregs could home to or be induced in NPC tumor tissues, and some EBV epitope antigens, specifically YLQ and GLG, induced more antigen-specific Tcregs in tumor tissues than others. CD4⁺ Tregs that are specific for the LAGE1 and ARTC1 tumor antigens have been identified in melanoma, and EBNA1 P_561-573 and P_607-619 peptide-specific CD4⁺ Tregs have been identified in PBMCs from healthy donors [39, 40]. These reports indicate that some antigens activate Tregs preferentially. In sum, the present study is the first to identify EBV epitopes that can induce antigen-specific CD8⁺ Tcregs in PBMCs and TILs of NPC patients. Our results suggest that when EBV antigen- or peptide-specific CTL immunotherapy is established for NPC patients, antigen selection may affect clinical efficacy.

In summary, we determined for the first time that there were different subsets of CD8⁺ T cells, including Tcregs and Tc17 cells, among the circulating lymphocytes and TILs of patients with NPC. The results of our study suggest that Tcregs have diverse functions, and they also indicate a possible antitumor function for Tc17 cells in NPC tissues. Moreover, we revealed for the first time the presence of EBV antigen-specific Tcregs in NPC. Taken together, all these findings provide new insights into the composition and function of CD8⁺ T cells and will be helpful for the development of T-cell-based adoptive immunotherapy for NPC.