TIM-3 in Cancer Treatment: The Potential Significance and Clinical Application

What is TIM-3？

T cell immunoglobulin and mucin domain 3 (TIM-3) is a crucial cell surface molecule found initially on CD4+ TH1 cells and CD8+ CTLs. Over time, its presence has been noted in various T cell subtypes, dendritic cells (DCs), natural killer (NK) cells, monocytes, macrophages, and mast cells. In certain cancers, TIM-3 can also be expressed on malignant cells [1-2].

TIM-3 is encoded by the havcr2 gene located on chromosome 5q33.2, a region associated with allergies and autoimmune diseases, containing genes like interleukin (IL)-4 and IL-5.

Gene Structure and Protein Structure of Tim-3

Structurally, TIM-3 is a single transmembrane (TM) molecule. It comprises an extracellular N-terminal IgV domain, a mucin domain with glycosylation sites, a link peptide with N-linked glycosylation sites, a TM domain, and a cytoplasmic tail at the C-terminus [3]. Although lacking classic inhibitory motifs like ITIM or ITSM, TIM-3 contains a conserved region with five tyrosine residues, including Y265 and Y272 in humans (Y256 and Y263 in mice) [4]. When TIM-3 interacts with its ligands, kinases like Itk, Fyn, and Lck phosphorylate these tyrosines, recruiting proteins with SH2 domains such as the p85 subunit of PI3K and PLC-γ1. TIM-3 activation enhances NFAT and NF-κB activation through interaction with ZAP-70 and SLP-76, components of the TCR signaling pathway. However, upon TCR induction [5], TIM-3 can also suppress AP-1 and NFAT activation, leading to impaired IL-2 production. HLA-B associated transcript 3 (Bat3) binds to TIM-3's cytoplasmic tail, preventing signal induction in the absence of TIM-3 ligands [6].

Fig.1 Structure of TIM-3 molecule and its ligands.[7]

Ligands and Functions of Tim-3

Tim-3 plays a pivotal role in modulating both adaptive and innate immune responses by interacting with specific ligands. Among the well-studied Tim-3 ligands are galectin-9 (Gal-9), phosphatidylserine (PtdSer), high-mobility group box-1 protein (HMGB1), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1).

High expression of Tim-3 on effector T cells signifies T cell exhaustion, leading to inhibited proliferation and reduced secretion of TNF-α and IFN-γ. The interaction between Tim-3 and its ligand, galectin-9 (Gal-9), triggers effector T cell apoptosis through the calcium-calpain-caspase-1 pathway [8]. On activated T cells, co-expression of CEACAM1 and Tim-3 forms a heterodimer, suppressing T cell function and diminishing its anti-tumor immunity. Within tumor loci, Tim-3+ Tregs exhibit superior suppressor function compared to Tim-3− Tregs. These Tim-3+ Tregs actively shape the tumor-immune microenvironment (TIME) by supporting the development of exhausted CD8+ T cells and restraining the expansion of pro-inflammatory cytokine-secreting CD4+ and CD8+ T cells [9].

Tim-3 upregulation on macrophages promotes their M2 polarization and enhances IL-6 secretion, thereby fostering tumor growth. Interestingly, Tim-3 exhibits diverse effects on dendritic cells (DCs) and natural killer cells (NKs) due to its interactions with different ligands. The immunoglobulin-like region of the TIM family specifically recognizes PtdSer [10]. Interaction between PtdSer and Tim-3 on DCs facilitates the clearance of apoptotic cells and cross-presentation. Conversely, the interaction of HMGB1 and Tim-3 on DCs suppresses nucleic acid-mediated innate immune responses within the tumor site [11].

Tim-3 is expressed on mature NK cells, and its interaction with Gal-9 enhances IFN-γ production in NKs. However, a study in chronic hepatitis B yielded contradictory results [12]. Notably, the Tim-3/Gal-9 pathway in tumor-infiltrating lymphocytes (TIL cells) has been extensively studied in hepatocellular carcinoma (HCC).

Tim-3 Mediates Immune Tolerance in Autoimmunity and Alloimmunity

In earlier research, TIM-3 was identified as a crucial regulator in both mouse models and human patients, influencing the dynamics of autoimmune diseases. Studies indicated that treatment with TIM-3 monoclonal antibodies exacerbates the severity of experimental autoimmune encephalomyelitis (EAE) [13]. Patients with multiple sclerosis (MS) exhibited lower TIM-3 expression on T cells and higher interferon (IFN)-γ production. Notably, blocking TIM-3-mediated immune tolerance with TIM-3-Ig fusion proteins or anti-TIM-3 monoclonal antibodies accelerated autoimmune diabetes, emphasizing TIM-3's role as an immune checkpoint molecule. These findings underscore TIM-3's significance in immune regulation.

Similar insights were gained in the context of transplantation. Administration of TIM-3-Ig fusion proteins disrupted transplantation tolerance, a function reliant on donor-specific CD4+CD25+ regulatory T cells [14]. Numerous studies have reaffirmed TIM-3's critical role in suppressing the rejection of transplanted skin, pancreatic islets, heart, and bone marrow allografts, highlighting its vital contribution to immune tolerance [15]. Despite these advances, the precise in vivo mechanisms of TIM-3 signaling and the immune cells expressing TIM-3 in mediating immune tolerance remain areas of ongoing research and exploration.

TIM-3 in Immune Tolerance to Tumors

In recent studies, TIM-3, found abundantly on tumor antigen-specific T cells and tumor infiltrating lymphocytes (TIL), has emerged as a pivotal player in tumor immunity. The increased expression of TIM-3 has been linked to the exhaustion of CD8+ T cells in melanoma; however, this exhaustion can be reversed by TIM-3 monoclonal antibodies (mAbs) [16]. Notably, in non-small cell lung cancer (NSCLC) patients, TIM-3 is predominantly observed in tumor-infiltrating CD4+ and CD8+ T cells, with minimal presence in peripheral blood T cells. Within CD4+ TIL, TIM-3 is notably present in Foxp3+ CD4+ regulatory T cells, and a higher frequency of CD4+TIM-3+ TIL correlates with poorer survival rates. This phenomenon is consistent across various cancers, where TIM-3 is expressed both in TIL and peripheral blood T cells [17-18].

In preclinical models, TIM-3 monoclonal antibodies exhibit varying antitumor effects when used alone. For instance, TIM-3 is upregulated in TIL in mouse tumor models such as CT26 colon adenocarcinoma, 4T1 mammary adenocarcinoma, and B16F10 melanoma [19]. When administered alone, TIM-3 mAbs do not inhibit CT26 tumor growth; however, their combination with PD-1 mAbs proves highly effective. TIM-3 mAbs decelerate tumor progression in models like MC38 colon carcinoma, WT3 sarcoma, CT26 colon adenocarcinoma, and TRAMP-C1 prostate tumor. Combining TIM-3 mAbs with CTLA4 mAbs or PD-1 mAbs amplifies antitumor effects, establishing TIM-3 monoclonal antibodies as a promising immunotherapy option. Furthermore, their synergy with PD-1 mAbs and/or CTLA4 mAbs holds the potential to further enhance the efficacy of cancer immunotherapy.

The Clinical Significance of TIM-3

The clinical significance of TIM-3 lies in assessing its expression level and functional status in immune-related diseases, as well as its feasibility as a potential therapeutic target.

By detecting the expression level of TIM-3, its involvement in the immune response can be assessed. In certain diseases, such as tumors, autoimmune diseases, and infectious diseases, the expression of TIM-3 may be abnormally regulated, leading to immune dysfunction. Therefore, detecting the expression level of TIM-3 can help doctors evaluate the progression and prognosis of the disease and guide the formulation of treatment strategies.

In addition, TIM-3 is a potential therapeutic target, and the development of its inhibitors has also attracted much attention. By inhibiting the function of TIM-3, the ability of immune cells to recognize and attack tumor cells or pathogens can be enhanced, thereby improving the therapeutic effect. Therefore, clinical detection of the expression level and functional status of TIM-3 can provide an important reference for the development and evaluation of the efficacy of TIM-3 inhibitors.

In summary, the clinical significance of TIM-3 lies in assessing its expression level and functional status in immune-related diseases, as well as its feasibility as a potential therapeutic target.

Signaling Pathway of TIM-3

Understanding the signaling pathway of TIM-3 is a complex yet crucial endeavor in immunology. While much remains to be discovered, existing knowledge sheds light on several key aspects. TIM-3 interacts with HLA-B-associated transcript 3 (Bat3) and SH2 (Src homology 2) domain-containing protein Fyn through conserved tyrosines Y256 and Y263 in its cytoplasmic tail. Upon T-cell activation, TIM-3 is recruited to the immunological synapse, where Bat3 binds to its cytoplasmic tail, recruiting the active form of Lymphocyte-specific protein tyrosine kinase (Lck) [20]. However, engagement with ligands leads to phosphorylation of the conserved tyrosine residues, releasing Bat3 and enabling TIM-3's inhibitory function.

Ligands such as galectin-9 and carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) trigger phosphorylation of Y256 and Y263 by the tyrosine kinase Interleukin-2-inducible T-cell Kinase (ITK), subsequently releasing Bat3. Moreover, a long-non-coding RNA known as Lnc-Tim-3 was found to bind TIM-3 and regulate its activity. In dysfunctional CD8+ T cells from hepatocellular carcinoma (HCC) patients, increased Lnc-Tim-3 expression led to the release of Bat3, diminishing T-cell activation and antitumor immunity [21].

Notably, Bat3 expression levels play a pivotal role in TIM-3-mediated signaling. Enhanced Bat3 expression blocks inhibitory signaling and enhances effector T-cell function, while reduced Bat3 expression strengthens TIM-3-mediated inhibitory signals. This phenomenon has been observed in CD8+ tumor-infiltrating lymphocytes (TILs) isolated from colorectal carcinomas, underscoring the importance of Bat3 in TIM-3 regulation [22]. It is worth noting that while Bat3-mediated regulation of TIM-3 signaling is well-documented in T cells, further research is needed to explore whether similar downstream signaling mechanisms apply to other cells such as dendritic cells (DCs). Initial studies suggest that Tim-3 ligation on DCs activates SH2 domain-containing signal transducers, namely Bruton’s tyrosine kinase and c-Src, leading to inhibition of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and subsequent inhibition of DC activation [22].

Fig.2 Model of Tim-3 signaling in T cells[23]

TIM-3 Protein

View Beta LifeScience's TIM-3 Proteins

Synonym : CD366 FLJ14428 HAVcr-2 Havcr2 HAVR2_HUMAN Hepatitis A virus cellular receptor 2 Kidney injury molecule 3 KIM 3 KIM3 T cell immunoglobulin and mucin domain containing 3 T cell immunoglobulin mucin 3 T-cell immunoglobulin and mucin domain-containing protein 3 T-cell immunoglobulin mucin family member 3 T-cell immunoglobulin mucin receptor 3 T-cell membrane protein 3 Tim 3 TIM-3 TIM3 TIMD-3 TIMD3

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