Tim-3 and Tim-4 as the potential targets for antitumor therapy

Both Tim-3 and Tim-4 belong to the T-cell immunoglobulin and mucin domain (Tim) gene family, which plays a critical role in immunoregulation. Tim-3 has been suggested as a negative regulator of anti-tumor immunity due to its function on inducing T cells exhaustion in cancer. In addition to its expression on exhausted T cells, Tim-3 also has been reported to up-regulate on nature killer (NK) cells and promote NK cells functionally exhausted in cancer. While Tim-3 selectively expression on most types of leukemia stem cells, it promotes the progression of acute myeloid leukemia. Recently, data from experimental models of tumor discovered that Tim-3 and Tim-4 up-regulation on tumor associated dendritic cells and macrophages attenuated the anti-tumor effects of cancer vaccines and chemotherapy. Moreover, co-blockage of Tim-3 and PD-1, Tim-3 and CD137, Tim-3 and carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) could enhance cell-mediated immunity in advanced tumor, and combined treatment with anti-Tim-3 and anti-Tim-4 mAbs further increase the efficacy of cancer vaccines. The therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy to improve the clinical efficacy of cancer immunotherapy.


Introduction
The T-cell immunoglobulin and mucin domain (Tim) gene family was discovered in 2001, which plays a critical role in immunoregulation. 1 The Tim gene family comprises of 8 members (TIM-1-8) on mouse chromosome 11B1.1 and 3 members (TIM-1, TIM-3, and TIM-4) on human chromosome 5q33.2. 2 Tim-3 is expressed on many types of immune cells, including T cells, dendritic cells (DCs), macrophages, nature killer cells (NK), cancer stem cells, and so on. 3 TIM-3 is previously known as a receptor for galectin-9 and phosphatidylserine (PS), it may induce the apoptosis of T cells, enhance the secretion of proinflammatory cytokines such as TNF by DCs and NK cells, and promote the phagocytosis of apoptotic cells by monocytes and macrophages through interaction with its ligands. 4,5 Recently, carcinoembryonic antigen cell adhesion molecule 1 (CEA-CAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition, was discovered as a heterophilic ligand for TIM-3, and their interaction had a crucial role in regulating autoimmunity. 6 Unlike Tim-3, Tim-4 is exclusively expressed on antigen-presenting cells (APCs), 7 and serves as a critical sensor for controlling the functions of naive and activated T cells and phagocytosis of apoptotic cells by APCs through interaction with PS. 8,9 Indeed, Tim-3 has been reported as a negative regulator of anti-tumor immunity. The expression of Tim-3 on T cells in cancer may induce T cell exhaustion, and promote the expansion of immunosuppressive CD4CFoxP3C regulatory T (Treg) cells and CD11bCGr-1C myeloid suppressor cells (MDSC). 10 Recent reports also showed that TIM-3, as a surface molecule, is selectively expressed on leukemia stem cells (LSCs) in acute myeloid leukemia (AML), and as such it can be a good target in eradicating AML stem cells, leaving normal hematopietic stem cells intact. [11][12][13] In addition, Tim-3 has also been reported to up-regulate on tumor associated dendritic cells (TADC) and macrophages (TAM), and attenuate the antitumor effects of cancer vaccines 4 (Fig. 1). The expression of Tim-4 on TAM could mediate degradation of dying tumor cells by autophagy, reduce antigen presentation and impaired cytotoxicity T lymphocyte (CTL) responses, and suppress the antitumor effects of chemotherapy. 14 Co-blockage of Tim-3 with other negative checkpoint regulators expressed on T cells, such as programmed death-1 (PD-1) and CD137, would enhance cell-mediated immunity in advanced tumor, and combined treatment with anti-Tim-3 and anti-Tim-4 mAbs further increase the efficacy of cancer vaccines.

Tim-3 on T cell subsets in tumor
Tim-3 was discovered nearly 10 y ago as a molecule expressed on IFN-g-producing CD4C T helper type 1 (Th1) and CD8C T cytotoxic type 1 (Tc1) cells, and induced T cell apoptosis through interaction with its ligand galectin-9. And then, several studies have identified TIM-3 as an important immune regulator in the tumor microenvironment due to its negative regulation on various T-cell subsets. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. CD4C or CD8C Tumor-Induced Senescent T cells may promote production of pro-inflammatory cytokines (TNF, IL-1b and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) by CD14C monocytes/macrophages (Mo/Ma), and Tim-3 and CD40 are involved in this modulation. 15 In addition, a synergistic action between negative checkpoint regulators expressed on T cells may also exist. The expression of negative checkpoint regulator PD-1 and TIM-3 impairing cellmediated immunity was observed in advanced melanoma and colorectal cancer. 16,17 Co-blockage of Tim-3 and PD-1 could enhance the expansion and function of tumor antigen-specific CD8C T cells in vitro and in vivo, induce tumor rejection in experimental models and enhance the vaccine effect in patients with advanced melanoma. 18,19 New treatment targeting TIM-3 and PD-1 on CD4C and CD8C T cells may provide a breakthrough treatment to cancer patients. In a murine model of ovarian cancer, co-blockage of Tim-3 and CD137 was able to prevent the tumor progression in advanced established tumor by increasing the number of CD4C and CD8C cells and decreasing immunosuppressive CD4CFoxP3C regulatory T (Treg) cells and CD11bCGr-1C MDSC. 20 Also, co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumor immune responses with improved elimination of tumors in mouse colorectal cancer models. 6 Other negative checkpoint regulators expression on tumor-infiltrating lymphocytes, such as cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3) and BTLA (B and T lymphocyte attenuator) also negatively regulate antitumor immunity in tumor microenvironments, the effects of co-blockage of them with Tim-3 still need further investigation. [21][22][23] Tim-3 and TIM-4 on TADC and TAM In turn, TAM promotes the growth of HCC by secretion of soluble factors such as interleukin-6 (IL-6). Additionally, Tim-3 inhibits the activation of tumor-specific CD8C T cells. Inhibition of Tim-3 could target HCC from 2 angles, first by blocking growth promotion mediated by TAM and second by releasing CD8C T cell cytotoxicity. 24,25 While Baghdadi et al also found that danger associated pattern molecules (DAMPs) such as HMGB1, HSP90 (heat shock protein 90), MSU (monosodium urate), S100A8, and ATP (apyrase) released from dying or stressed tumor cells treated with chemotherapeutic drugs could promote TIM-4 expression on macrophages and DCs. 14 Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading unnecessary or dysfunctional cellular organelles and proteins in all living cells, 26 and has an important role in cancer-cell resistance to anticancer therapies such as radiation, chemotherapy, and some other targeted therapies. [27][28][29] In the study of Baghdadi et al, they pointed out the mechanism of cancer-cell resistance to chemotherapy. After recognition of DAMPs from dying or stressed tumor cells by chemotherapy, TIM-4 up-regulated on TAM and directly interacted with AMPKa1, thus promoted phosphorylation of ULK1 at Ser555, recruited Atg13/FIP200 and initiated autophagic vesicle formation and activated autophagy mediated degradation of ingested tumors, leading to reduced antigen presentation and impaired cytotoxicity T lymphocyte (CTL) responses (Fig. 2). Consistently, blockage of the TIM-4-AMPKa1-autophagy pathway augmented the antitumor effect of chemotherapeutics by enhancing tumor-specific CTL responses. So, targeting of the TIM-4-AMPKa1 interaction may constitute a unique strategy for augmenting antitumor immunity and improving cancer chemotherapy. 14 Both TIM-3 and TIM-4 attenuate the antitumor effect of cancer vaccines, a combined treatment with anti-TIM-3 and anti-TIM-4 mAbs may further increase the efficacy of cancer vaccines and chemotherapy. In established B16 melanoma, combining anti-TIM-3 and anti-TIM-4 mAbs markedly increased vaccine-induced antitumor responses by increasing the numbers and effector functions of both NK cells and CD8C T cells. 30 The therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy to improve the clinical efficacy of cancer immunotherapy. Taken together, these findings identify TIM3 and Tim-4 as potential targets for inducing antitumor immunity in conjunction with DNA vaccines and/or immunogenic chemotherapy in clinical settings.

Other roles of Tim-3 in tumor
Tim-3 also functions as a human NK-cell co-receptor to enhance IFN-g production, which has important implications for the control of infectious disease and cancer. 31 Meanwhile, NK-cell responses may be negatively regulated when NK cells encounter target cells expressing cognate ligands of Tim-3. 32 For patients with metastatic melanoma, expression of Tim-3 on NK cells induce functionally impaired/exhausted NK cells, and Tim-3 blockage reversed this exhausted phenotype, thus Tim-3-targeted therapies may be an effective method to restore antitumor immunity. 33 In the studies of Kikushige et al, Tim-3 is selectively expressed on LSCs in most types of AML, with the exception of acute promyelocytic leukemia, not on normal haematopoietic stem cells (HSCs). Although it is not surprising in mouse models reconstituted with human AML LSCs or human haematopoietic stem cells, a human TIM-3 mouse IgG2a antibody with complementdependent and antibody-dependent cellular cytotoxic activities eradicates AML LSCs in vivo without affecting normal human hematopoiesis. [11][12][13] Thus, TIM-3 may serve as one of the promising targets to eradicate AML LSCs. Jajosky et al also pointed out that, RepSox (a reprogramming tool and inhibitor of transforming growth factor-b receptor 1) accelerated loss of Tim-3 from the surface of AML cells by inhibiting TGF-b signaling. 34 Tim-3 as human cancer prognostic factor The expression of TIM-3 on T cells was poor clinic pathological parameters such as nodal metastasis and advanced cancer stages in gastric cancer, 35 lung cancer, 36 cervical cancer 37 and ovarian cancer. 38 The ectopic expression of TIM-3 in tumor cells has been suggested as a potential, independent prognostic factor for patients with lung cancer, 36 cervical cancer 37 and prostate cancer. 39 The Tim-3/galectin-9 signaling pathway mediates Tcell senescence was also involved in patients with hepatitis B virus (HBV)-associated HCC, thus it has been a potential immunotherapeutic target. 40 In addition, polymorphisms in TIM-3 gene were showed to be associated with various cancers. Subjects carrying C4259TG genotype had a significantly higher risk of NSCLC, pancreatic cancer and renal cell carcinoma compared to the wide-type genotype. [41][42][43] TIM3-1516 genotypes GT or TT may differentially and interactively predispose cirrhosis and HCC in chronic HBV infection. 44 The relationship between TIM3-1516 polymorphic genotype and the distant metastasis of gastric cancer was also Figure 2. Tim-4 expression on tumor associated macrophages mediates degradation of dying tumor cells by autophagy. Danger associated pattern molecules (DAMPs) released from dying or stressed tumor cells treated with chemotherapeutic drugs such as high mobility group protein B1 (HMGB1), heat shock protein 90 (HSP90) and apyrase (ATP) promote TIM-4 expression on macrophage. After recognition of DAMPs released from dying or stressed tumor cells with chemotherapy, TIM-4 expression on TAM directly interacts with AMPKa1, thus promotes phosphorylation of ULK1 at Ser555, which is critical to recruit Atg13/FIP200 and initiate autophagic vesicle formation and activates autophagy mediated degradation of ingested tumors, leading to reduced antigen presentation and impaired antitumor immune responses.
found. 45 Overall, the results suggest that TIM-3 may play important roles in regulating the prognosis of these diseases.

Conclusion and future perspectives
Despite the fact that Tim-3 expression on various T cell subsets could negatively regulates the antitumor immunity in patients with cancer, the expression of Tim-3 on NK cells and TADC, and the expression of Tim-4 on TAM also attenuates the cell-mediated antitumor effects in established tumors. In addition, the expression of Tim-3 on LSCs also promotes the progression of AML. New treatment targeting TIM-3 and PD-1, Tim-3 and CD137, Tim-3 and CEACAM1, Tim-3 and other negative checkpoint regulators on T cells, Tim-3 on NK cells and LSCs, and combined chemotherapy or cancer vaccines with mAb to Tim-3 or Tim-4 may provide a breakthrough in the treatment for the patients with advanced cancer. Since polymorphisms in TIM-3 may play important roles in regulating the prognosis of various cancers, the mechanisms behind this still need further investigation.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Authors' Contributions
LC carried out collection and assembly of data, and manuscript writing. ZHR conceived of the review and helped to draft the manuscript, and gave final approval of manuscript. All authors read and approved the final manuscript.