The TIM3/Gal9 signaling pathway: An emerging target for cancer immunotherapy

The discovery and identification of noninvasive biomarkers help identify cancer in the early stages and develop treatment strategies as well. Identification of non-invasive biomarkers would be beneficial to patients as they could bypass invasive surgical procedures like biopsies. Numerous ongoing studies aim to identify the relevant biomarkers to predict the sensitivity, potency, and efficacy of the immune checkpoint therapy; however, the perplexing mechanism of immune checkpoints make biomarker identification more challenging. While the role of TIM3 and Gal9 in cancer development has been recognized and widely studied, TIM3 and Gal9 as biomarkers are still being explored. We have summarized the recent studies on the possible use of TIM3 and Gal9 as prognostic and predictive biomarkers in various diseases. High TIM3 expression on tumor-infiltrating T cells and increased Gal9 level in tumor cells in the TME are associated with numerous tumors. For example, high levels of plasma exosomal TIM3 and Gal9 are related to advanced tumor stages, large tumor size, and metastasis in NSCLC [ 95 ]. Upregulated Gal9 expression on tumor cells and TIM3 expression on CD8 + T cells are observed in gastric cancer [ 121 ], glioma [ 89 ], chronic lymphocytic leukemia [ 122 ], and esophageal squamous cell carcinoma [ 96 ]. Increased infiltration of TIM3 + T cells in tumors and increased Gal9 expression by tumor cells are inversely proportional to overall survival in cancer patients. The presence of tumor DNA in blood circulation has been reported in various cancers [ 123 , 124 ]. Isolation of tumor DNA from blood samples to assess DNA methylation or histone distributions of TIM3 and Gal9 genes could be used for prognostic or diagnostic purposes. A study performed in primary breast cancer patients to examine the promoter CpG profile in various immune checkpoint receptor genes found TIM3 promoter to be hypomethylated, whereas the gene body of TIM3 was hypermethylated. In addition, the distribution of repressive histone H3k27me3 was also found to be reduced in the TIM3 promoter region [ 125 ]. Similarly, in melanoma patients, DNA methylation of the TIM3 and Gal9 promoter region was inversely correlated with the mRNA expression of TIM3 and Gal9. mRNA expression of TIM3 and Gal9 is positively correlated with tumor infiltrating leucocyte fraction, but DNA methylation of TIM3 and Gal9 gene is negatively correlated with infiltrating leucocyte fraction in the tumor environment [ 126 ]. In addition to cancers, TIM3 and Gal9 expressions are prominent in other diseases like systemic lupus erythematosus, antiphospholipid syndrome [ 127 ], allograft rejection dysfunction [ 128 ], endometriosis [ 129 ], and, hepatitis C [ 130 ]. Gal9 levels in the serum can be used clinically as a stable biomarker in detecting IFN signature in systemic lupus erythematosus and antiphospholipid syndrome [ 127 ]. Li et al. showed that the serum levels of soluble TIM3 (sTIM3) and soluble Gal9 (sGal9) were found to be elevated in patients with allograft rejection, making sTIM3 and sGal9 promising biomarker for detection of allograft rejection dysfunction [ 128 ]. Elevated Gal9 levels were observed in the serum in endometriosis patients. Recently, a pilot study suggested that Gal9 is a promising biomarker for the diagnosis of endometriosis with a sensitivity of 94% and a specificity of 93.75% [ 129 ]. By contrast, a meta-analysis showed that CA125, a gold standard biomarker for endometriosis, only exhibit a sensitivity of 52 % and a specificity of 93% [ 131 ]. These results indicate that Gal9 could be a sensitive biomarker of endometriosis and other gynecological disorders. Additionally, increased Gal9 level is found in the blood of patients with hepatitis C infection, making it a useful predictive biomarker for hepatitis C [ 130 ]. Although the TIM3/Gal9 pathway is not fully understood, the higher level of sTIM3 and sGal9 is observed in the plasma during cancers and other diseases which can be exploited as potential biomarkers.

Galectins are soluble proteins composed of a characteristic structure, called the carbohydrate recognition domain (CRD), which binds to β galactose [ 30 , 31 ]. Based on the number of CRDs, galectins are subdivided into the prototype, tandem-repeat type, and chimeric-type galectins [ 32 ]. Gal9 is a member of a ‘tandem-repeat’ type of galectin superfamily that consists of two non-homologous CRDs joined by a flexible peptide linker (14 to 56 amino acids in length) that plays important roles in protein-protein interactions, membrane insertions, and presentation of the CRDs [ 33 ]. The peptide linker contains a metalloproteinase site which can assist with the secretion of Gal9 into the extracellular matrix [ 34 , 35 ]. Gal9 is highly expressed in epithelial cells, endothelial cells, T cells, B cells, mast cells, and macrophages [ 31 , 36 ]. Gal9 has an important function in eosinophil activation, hemagglutination and anti-metastasis, T cell apoptosis via the TIM3/Gal9 pathway, T cell homeostasis and production of cytokines, and differentiation of Tregs and Th17 cells, making Gal9 a regulator of immune responses [ 37 ]. Four receptors with an affinity for Gal9 have been reported ( Table 1 ). Among the receptors, TIM3 is a fully characterized receptor for Gal9 [ 3 ]. The TIM3/Gal9 interaction shows inhibitory or stimulatory effects in the immune system depending on the cell type that expresses TIM3. In normal physiology, TIM3 expressed on T cell subsets (Th1, Th17, and Tc1) and macrophages show inhibitory effects upon Gal9 binding, whereas TIM3 expressed on NK cells and DCs show stimulatory effects when it binds to Gal9 [ 38 , 39 ]. The other receptors for Gal9 are Dectin-1, 4–1BB, and CD40. Dectin-1 is highly expressed on macrophages and exerts an immunosuppressive effect after binding to Gal9 [ 40 ]. Similarly, the Gal9/CD40 interaction is known to prevent the proliferation and survival effects of CD40 on CD4 lo CD40 + effector T cells [ 41 ]. Gal9 binds to 4–1BB (an activating immune checkpoint protein) and promotes the clustering of 4–1BB, enhancing the proinflammatory pathway and immune response. [ 42 ].

In addition to cancers, TIM3/Gal9 interaction has also been documented in autoimmune diseases (multiple sclerosis, rheumatoid arthritis, and Type 1 diabetes), viral infections, and protozoal infections. In a clinical study, patients with multiple sclerosis were treated with glatiramer or IFNβ. Increased TIM3 expression was observed in both glatiramer and IFNβ-treated patients compared to untreated patients, suggesting the recovery and maintenance of a basal level of TIM3 [ 109 ]. TIM3/Gal9 interaction causes T cell exhaustion and death, which decreases autoreactive T cells and improves autoimmune diseases. Type 1 diabetes is an autoimmune disease, characterized by activation of innate immunity, proliferation of autoreactive CD4 + and CD8 + T cells that may damage β cells [ 110 ]. Gal9 activates DCs to induce the production of TNFα, and these activated DCs play a crucial role in the activation of Th 1 and autoimmune response. Treating patients with RMT3–23 (anti-TIM3 mAb) resulted in decreased production of TNFα by DCs and inhibits the downstream Th1 functions. This result demonstrates that the TIM3/Gal9 pathway blockade on DCs could be a potential therapeutic target in type 1 diabetes mellitus [ 110 , 111 ]. Significant downregulation of TIM3 and Gal9 is found in peripheral blood cells in patients suffering from thyroid-associated ophthalmopathy (TAO), an autoimmune disease. It is thought that Th1, and Th17 play a role in the pathogenesis of TAO [ 112 ]. mRNA expression of TIM3 and Gal9 was found to be significantly lower in TAO patients compared to healthy subjects. In an in vitro study, treatment using Gal9 blocking antibody blocked TIM3/Gal9 downstream function and resulted in increased IFNγ, TNFα, and IL-17 levels showing TIM3/Gal9 has a negative correlation with disease severity. The TIM3/Gal9 pathway suppresses T cell-mediated cytokine release in TAO fibroblasts, suppresses the Akt/NFKB pathway, and alleviates TAO [ 113 ]. The TIM3/Gal9 interaction has shown to suppress inflammation and immune responses in TAO. In chronic hepatitis B infection, higher TIM3 expression on NK cells was observed. TIM3/Gal9 binding inhibits the cytotoxic function of NK cells, leading to the compromised antiviral immunity of the host. Blockade of the TIM3/Gal9 axis helped restore the cytotoxic function of NK cells [ 19 ]. Another study in a hepatitis B virus mouse model showed that TIM3/Gal9 coupling lowered the INFγ production and led to the exhaustion of CD8 + T cells. This resulted in the inhibition of antiviral immunity of infiltrating CD8 + T cells. [ 114 ]. Also, plasma endotoxin and the cytokine IL-10 are responsible for overexpression of TIM3 and PD-1 on acute alcoholic hepatitis leading to the suppression of host immunity [ 115 ]. Co-blockade of TIM3 and PD-1 pathways have shown to generate a synergistic effect in anti-viral immunity of host against hepatitis B virus and chronic hepatitis B infection [ 116 ]. In a murine malaria model, the number of Gal9- and TIM3-positive cells are increased in the lungs, liver, and spleen, suggesting an important role of the TIM3/Gal9 pathway in the damage of these organs [ 117 , 118 ]. In a clinical study, the plasma level of Gal9 was found higher in severe malaria patients compared to uncomplicated cases. Moreover, Gal9 is associated with the severity of malaria. It is postulated that Gal9 inhibits immune response in malaria patients by binding to TIM3 [ 119 ]. As a result, malarial infection could be treated by blocking the TIM3/Gal9 pathway. In a PbANKA-infected mouse model, TIM3/Gal9 pathway blockade with anti-TIM3 antibody showed an increase in the mRNA level of cytokines, including IFNγ, TNFα, IL-10, and IL-4. This finding suggests that antibody-mediated blockade of TIM3 contributes to the therapeutic clearance of malaria infection [ 120 ].

Altogether, in CD4 + and CD8 + T cells, the TIM3/Gal9 interaction results in reduced production of cytokines (IFNγ, IL-2 and TNFα), inhibits T cell proliferation, and induces apoptosis of T cells, resulting in suppression of Th1 like immunity. The TIM3/Gal9 interaction is believed to have stimulatory or inhibitory activity in innate immunity depending on the type of innate immune cells. This pathway is critical for maintaining the immunity against self-antigens and important for preventing the onset of autoimmunity. In the TME, the interaction of TIM3 with its ligand Gal9 is believed to inhibit both adaptive and innate immunity, which favors tumor progression. Although there are perplexing physiological discoveries on the stimulatory and inhibitory effect of the TIM3/Gal9 pathway on different types of immune cells, these findings suggest that the TIM3/Gal9 pathway is involved in the pathogenesis of various types of cancers. Collective studies have highlighted the inhibitory role of the TIM3/Gal9 signaling pathway in the progression of tumors to their advanced stage. The inhibition of TIM3 interaction with Gal9 could be a potential therapeutic approach for the treatment of various cancers. In addition, monotherapy using a single immune checkpoint inhibitor is likely to develop resistance. As a result, co-blockade of more than one immune checkpoint protein, such as blocking the TIM3/Gal9 and PD-1/PD-L1 pathways simultaneously, reduces the possibility of developing drug resistance. More studies are needed to unravel the complete molecular mechanism of the TIM3/Gal9 pathway to broaden its therapeutic applications for cancer therapy. Furthermore, overexpression of TIM3 or Gal9 in the TME and elevated serum levels of TIM3 or Gal9 can be used as a predictive or prognostic biomarker to help the optimization and selection of appropriate therapeutic options in cancers. The identification of TIM3 and Gal9 as biomarkers in the serum could accelerate the development of non-invasive and early-stage cancer diagnoses.

Immune checkpoint proteins are stimulatory or inhibitory regulators that play a key role in maintaining immune homeostasis and preventing the onset of autoimmunity [ 1 ]. Stimulatory checkpoint proteins like the cluster of differentiation 28 (CD28), OX40, glucocorticoid-induced tumor necrosis factor receptor (GITR), CD137, and CD27 enhances T cell functions, whereas inhibitory checkpoint proteins such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), B and T lymphocyte attenuator (BTLA), V-domain immunoglobulin suppressor of T cell activation (VISTA), and lymphocyte-activation gene 3 (LAG3) suppress T cell immunity [ 2 ]. Immune checkpoint proteins help in maintaining a balance between positive and negative signals mediated by T cells. In cancers, inhibitory immune checkpoints are often activated, which protect the tumor cells from immune surveillance [ 1 ]. Targeting immune checkpoint proteins is, therefore, one of the most active areas of cancer research. Particularly, checkpoint inhibitors targeting the PD-1/PD-L1 axis and CTLA-4 have been approved for different types of cancer. Among these checkpoint proteins, TIM3 has attracted much attention and is being vigorously studied. TIM3 biology and its suppressing role of antitumor immunity upon interaction with its ligands like Galectin 9 (Gal9) [ 3 ], phosphatidylserine (Ptdser) [ 4 ], high mobility group box 1 (HMGB1), [ 5 ] and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) [ 6 ] are being actively studied. A recent review by Wolf Y et al. focused on the four ligands of TIM3, the role of TIM3 in cancer and chronic viral infections, and ongoing clinical trials of anti-TIM3 antibodies [ 7 ]. Another review by Solinas C et al. described TIM3 biology in different immune cells, T helper cells (Th1 and Th17), regulatory T cells (Tregs), dysfunctional cytotoxic T lymphocytes (CD8 + T cells), natural killer (NK) cells, and dendritic cells (DCs) along with several anti-TIM3 antibodies in clinical trials [ 8 ]. These articles discussed the heterogeneous biological role of TIM3 in cancer and the application of TIM3 as a therapeutic target regardless of its specificity to any particular ligand. Our review focuses on the role of the TIM3/Gal9 interaction in various cancers and implications of this pathway in cancer immunotherapy. As the binding site of Gal9 on TIM3 is different from other ligands, the anti-TIM3 antibodies that have been investigated in clinical trials may not block the TIM3/Gal9 pathway. A handful of anti-TIM3 mAbs (TSR022, Sym023, ICAGN02390, BGB-A425, and MBG453) that prominently block the TIM3/Ptdser interaction are investigated in clinical trials. Among them, LY3321367 is the only anti-TIM3 mAb that partially blocks the TIM3/Gal9 interaction [ 9 – 13 ]. Even though Gal9 is the most relevant ligand for TIM3 [ 8 ], drugs that specifically block the TIM3/Gal9 pathway have not entered clinical trials yet. As a result, there is a great need to fully understand the TIM3/Gal9 interaction and its role in tumor pathogenesis. The current review highlights the importance of the TIM3/Gal9 pathway in cancer development, and how the blockade of this pathway can be exploited for the treatment of different types of cancer. We also discuss the potential applications of the TIM3/Gal9 pathway in other diseases along with the possibilities of using TIM3 or Gal9 as a prognostic or predictive biomarker for cancer.

Unlike other immune checkpoint proteins such as PD-1, CTLA-4, TIGIT, and LAG-3, which have an inhibitory motif in their cytoplasmic tail, TIM3 has five tyrosine residues in its cytoplasmic tail. Of the five tyrosine residues, Y265 and Y272 (Y256 and Y263 in mice) play an important role in TIM3 signal transduction [ 47 ]. While the exact molecular mechanism of TIM3 signaling is not fully elucidated, it has been demonstrated that before TIM3 ligand engagement, the tyrosine residues present in the intracellular tail of TIM3 are necessary for the augmentation of T cell receptor (TCR) activation suggesting the coupling of TIM3 and TCR pathways [ 48 ]. TIM3 when not bound to any of its ligands, its cytoplasmic tail containing Y256 and Y263, interacts with HLA-B associated transcript 3 (Bat3), [ 49 ] which facilitates the recruitment of catalytically activated Lck [ 7 ], a member of the Src family protein kinase ( Figure 1 ). The formed Lck clusters phosphorylate the CD3 domain of TCR, leading to the recruitment and activation of protein kinase Zap 70 [ 50 ], which phosphorylates the linker adaptor protein (LAT) [ 51 , 52 ]. Phosphorylated LAT recruits and activates the signaling effector phospholipase Cγ1 (PLCγ1), which participates in the production of second messengers like inositol 1,4,5-triphosophate (IP3) and diacylglycerol (DAG) [ 53 , 54 ]. These second messengers subsequently activate NFAT, MEK/ERK, and NF-kB pathways, resulting in T cell response and modulation, cell proliferation, and production of interleukin-2 (IL-2), Tumor Necrosis Factor (TNFα), and interferon γ (IFNγ) [ 53 , 55 ] ( Figure 2A ). Interaction between TIM3 and Gal9 induces phosphorylation of the cytoplasmic tail at Y265 and Y272, [ 47 ] resulting in the release of bound Bat3 from the cytoplasmic tail [ 49 ] ( Figure 2B ). Bat3-released TIM3 binds to Fyn, one of the Src family protein kinases that share the same binding site with Bat3 [ 49 ]. Fyn recruitment in the cytoplasmic tail of TIM3 [ 7 ] phosphorylates the phosphoprotein associated with glycosphingolipid microdomains 1 (PAG), which promotes the recruitment of the C-terminal c-Src kinase (Csk). Csk then phosphorylates the C-terminal tyrosine of Lck, which negatively regulates the Lck activity and leads to the inhibition of T cell functions [ 56 – 58 ]. Davidson et al. have reported that PAG-associated Fyn is involved in increasing the Ca ++ flux intracellularly, which weakens the TCR signaling and leads to T cell anergy [ 59 ]. Thus, the TIM3/Gal9 signaling pathway mediates events like inhibition of T cell proliferation, reduction in cytokine production and potentially resulting in T cell death. The TIM3/Gal9 interaction also affects proximal TCR signaling. The conserved tyrosine residues present in the cytoplasmic tail of TIM3 are also responsible for proximal signaling events. The TIM3/Gal9 interaction enhances the clustering of tyrosine receptor phosphatases CD45 and CD148 present in the immunological synapse [ 43 ]. The receptor phosphatase CD45 dephosphorylates the tyrosine residues Y505 and Y394 of Lck and reduces its catalytic activity [ 60 ]. Similarly, CD148 hypophosphorylates LAT and PLCγ1, leading to the inhibition of the downstream T cell functions [ 61 ]. In conclusion, the TIM3/Gal9 interaction inhibits immunity mediated by Th1, Th17, and Tc1 cells through inhibiting the production of IFNγ, TNFα, and IL-2 ( Figure 3A , 3B and 3D ) ( Table 2 ). In Tregs, numerous studies have reported the downstream effects of the TIM3/Gal9 interaction, but the exact intermediate steps of this pathway are yet to be explored. The TIM3/Gal9 interaction is believed to maintain and regulate Tregs function and development. One study has shown that TIM3/Gal9 ligation results in overexpression of cytokines like IL-10 and TGFβ, which facilitates the proliferation of CD4 + CD25 + Foxp3 + Treg cells. The proliferation of Tregs then suppresses the activity of effector T cells [ 62 ]. A study by Yan et al. has shown that TIM3 expression is significantly higher in CD4 + Foxp3 + T cells (70%) as compared to CD4 + Foxp3 − T cells (20%). They also found that Foxp3 is expressed in tumor-infiltrating TIM3 + CD4 + T cells which exhibit Treg properties in the tumor microenvironment [ 63 ]. A similar study using CT26 cells demonstrated that 50% of CD4 + Foxp3 + T cells express TIM3, whereas only 10% of CD4 + Foxp3 − TILs express TIM3. In line with this in vitro study, an in vivo study showed that TIM3 + Tregs produced two-fold production of IL-10 and had twice the immune-suppressive activity compared to TIM3 − Tregs in B16 melanoma and CT26 colon carcinoma [ 64 ]. Additionally, Tregs also express Gal9 ( Figure 3C ), which may bind to TIM3 expressed on Th1/Th17 cells, and suppress T cell-mediated immunity and induce apoptosis of Th1/Th17 cells [ 65 , 66 ]. In tumor-associated Tregs, TIM3 is overexpressed as a marker of infiltrating Tregs [ 63 ]. TIM3 + Tregs are prominent in the tumor nest but are significantly low in peripheral blood. The TIM3/Gal9 interaction was found to enhance the immunosuppressive function of Tregs and participate in the development of allograft tolerance [ 67 , 68 ]. The TIM3/Gal9 interaction enhances the immunosuppressive activity of Tregs in the tumor microenvironment (TME) by increasing the secretion of IL-10 and TGFβ. The expression of TIM3 on innate immune cells like NK cells, DCs, macrophages, and monocytes is prominent ( Table 2 ). For example, TIM3 is highly expressed in mature CD56 dim CD16 + NK cells and acts as a maturation marker [ 69 ]. Even though the role of the TIM3/Gal9 pathway in NK cells is still not fully understood, Jost et al. has shown that Gal9 binding to TIM3 present on NK cells has a stimulatory effect when NK cells are pre-treated with cytokines IL-12, IL-15 and IL-18. But constant exposure to Gal9 leads to the downregulation of TIM3 on NK cells ( Figure 4A ) [ 70 ]. In addition, Gleason et al. reported that coupling of TIM3 and Gal9 increases IFNγ production in NK92 NK cells, suggesting that TIM3 is an NK-cell coreceptor to enhance IFNγ production [ 39 ]. In contrast, during pregnancy, the TIM3/Gal9 pathway plays an important role in the differentiation of NK cells to decidual NK (dNK)-like cells to promote fetal tolerance by maintaining the anti-inflammatory function of dNK cells [ 71 ]. The binding of Gal9 to TIM3 expressed on dNK cells inhibits lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines and perforin, thus maintaining a normal pregnancy [ 72 ]. In addition, the TIM3/Gal9 pathway in NK cells also has been reported in other diseases. For instance, blockade of the TIM3/Gal9 interaction leads to increased IFNγ production resulting in increased cytotoxicity of NK cells in chronic hepatitis B virus infection [ 19 ]. Under normal physiological condition, the binding of Gal9 to TIM3 present on NK cells induces immune-stimulatory activity, whereas, in diseased conditions like cancers or viral infections, the TIM3/Gal9 interaction leads to overactivation of NK cells, resulting in exhaustion of NK cells. The possible reason could be that the TIM3/Gal9 pathway may crosstalk with other pathways like IL-2/IL-2 Receptor (IL-2R) and LPS/Toll-like Receptor 4 (TLR4) to produce immune-stimulatory or inhibitory effects. In monocytes and macrophages (M/M ϕ ), Gal9 bound TIM3 crosstalks with TLR4 receptors, hence increasing the production of IL-10 but reducing the production of cytokines like TNFα, IL-6, and IL-12 ( Figure 4B and 4C ) [ 45 , 73 ]. Similar findings were reported in human CD14 + M/M ϕ , TIM3 acts as a brake that controls TLR4 and TLR7, thus inhibiting TLR-mediated IL-2 production [ 44 ]. In active monocytes, the inhibition of LPS/TLR4 pathway is found to be reversed after treatment with mAbs that block the TIM3/Gal9 interaction [ 74 ]. Zhang and colleagues demonstrated that the TIM3/Gal9 interaction inhibits the function of macrophages by decreasing the production of inflammatory mediators. Furthermore, TIM3/Gal9 binding, in an autocrine fashion, regulates M1/M2 polarization via LPS stimulation. Short term LPS exposure in macrophages upregulates TIM3/Gal9 signaling, subsequently inhibiting M1 polarization. Conversely, long term LPS stimulation results in downregulation of TIM3/Gal9 pathway, resulting in inhibition of M2 polarization [ 75 ]. The upregulation of TIM3 expression is correlated with M2 polarization while downregulation is correlated with the M1 polarization of macrophages ( Figure 4C ). However, a study by Ma et al. reported a different finding regarding the TIM3/Gal9 interaction on M/M ϕ . Trans ligation of Gal9 with TIM3 negatively regulates M/M ϕ and decreases the TLR-mediated IL-12 expression, whereas cis ligation of Gal9 with TIM3 increases the expression of IL-12 and IL-23 via STAT3 phosphorylation resulting in enhanced inflammatory responses [ 76 ]. In DCs, the TIM3/Gal9 interaction synergizes with TLRs to promote inflammation [ 18 ]. Although the role of the TIM3/Gal9 interaction in DCs has not been fully elucidated, ligation of TIM3 with another ligand HMGB1 suppresses DC function. A recent study has shown that within the TME, Gal9 binds to TIM3 present on DCs, inhibiting anti-tumor immunity by suppression of the CXCL9 expression [ 77 ]. The TIM3/Gal9 interaction on DC is assumed to be in synergy with the TLRs and activates their innate immunity ( Figure 4D ) [ 18 ]. In summary, several studies have demonstrated the inhibitory role of TIM3/Gal9 interaction as it down-regulates Th1-mediated anti-tumor immunity as well as inhibits the autoimmune and alloimmune responses [ 3 , 78 ]. On the contrary, Gal9 binding with TIM3 expressed on innate immune cells like DCs and NK cells enhances the immune response promoting inflammation. [ 18 , 79 ]. These intriguing findings complicate the comprehension of the TIM3/Gal9 pathway. Further studies are necessary to understand the definitive molecular mechanism of the TIM3/Gal9 pathway in the innate immune system as well as the adaptive immune system.