Tim-3 Interaction Research Articles

The effect of Toxoplasma gondii infection on galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy

BackgroundToxoplasma gondii infection causes adverse pregnancy outcomes by affecting the expression of immunotolerant molecules in decidual immune cells. Galectin-9 (Gal-9) is widely expressed in decidual macrophages (dMφ) and is crucial for maintaining normal pregnancy by interacting with the immunomodulatory protein T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3). However, the effects of T. gondii infection on Gal-9 expression in dMφ, and the impact of altered Gal-9 expression levels on the maternal–fetal tolerance function of decidual natural killer (dNK) cells, are still unknown.MethodsPregnancy outcomes of T. gondii-infected C57BL/6 and Lgals9−/− pregnant mice models were recorded. Expression of Gal-9, c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and Forkhead box protein O1 (FOXO1) was detected by western blotting, flow cytometry or immunofluorescence. The binding of FOXO1 to the promoter of Lgals9 was determined by chromatin immunoprecipitation–polymerase chain reaction (ChIP-PCR). The expression of extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), cAMP-response element binding protein (CREB), phosphorylated CREB (p-CREB), T-box expressed in T cells (T-bet), interleukin 10 (IL-10), and interferon gamma (IFN-γ) in dNK cells was assayed by western blotting.ResultsToxoplasma gondii infection increased the expression of p-JNK and FOXO1 in dMφ, resulting in a reduction in Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK, inhibited the expression of p-CREB and IL-10, and promoted the expression of T-bet and IFN-γ in dNK cells. In the mice model, knockout of Lgals9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy.ConclusionsToxoplasma gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 contributed to dysfunction of dNK via Gal-9/Tim-3 interaction. This study provides new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.Graphical

PEG-Sheddable Nanodrug Remodels Tumor Microenvironment to Promote Effector T Cell Infiltration and Revise Their Exhaustion for Breast Cancer Immunotherapy.

Low infiltration of cytotoxic T lymphocytes and their exhaustion manifest the two concurrent main hurdles for achieving effective tumor immunotherapy of triple-negative breast cancer. It is found that Galectin-9 blockage can revise the exhaustion of effector T cells, meanwhile the repolarization of protumoral M2 tumor-associated macrophages (TAMs) into tumoricidal M1-like ones can recruit effector T cells infiltrating into tumor to boost immune responses. Herein, a sheddable PEG-decorated and M2-TAMs targeted nanodrug incorporating Signal Transducer and Activator of Transcription 6 inhibitor (AS) and anti-Galectin-9 antibody (aG-9) is prepared. The nanodrug responds to acidic tumor microenvironment (TME) with the shedding of PEG corona and the release of aG-9, exerting local blockade of PD-1/Galectin-9/TIM-3 interaction to augment effector T cells via exhaustion reversing. Synchronously, targeted repolarization of M2-TAMs into M1 phenotype by AS-loaded nanodrug is achieved, which promotes tumor infiltration of effector T cells and thus synergizes with aG-9 blockade to boost the therapeutic efficacy. Besides, the PEG-sheddable approach endows nanodrug with stealth ability to reduce immune-related adverse effects caused by AS and aG-9. This PEG sheddable nanodrug holds the potential to reverse the immunosuppressive TME and increase effector T cell infiltration, which dramatically enhances immunotherapy in highly malignant breast cancer.

Intestinal Lamina Propria CD4+ T Cells Promote Bactericidal Activity of Macrophages via Galectin-9 and Tim-3 Interaction during Salmonella enterica Serovar Typhimurium Infection.

The intestinal immune system is crucial for protection from pathogenic infection and maintenance of mucosal homeostasis. We studied the intestinal immune microenvironment in a Salmonella enterica serovar Typhimurium intestinal infection mouse model. Intestinal lamina propria macrophages are the main effector cells in innate resistance to intracellular microbial pathogens. We found that S Typhimurium infection augmented Tim-3 expression on intestinal lamina propria CD4+ T cells and enhanced galectin-9 expression on F4/80+ CD11b+ macrophages. Moreover, CD4+ T cells promoted the activation and bactericidal activity of intestinal F4/80+ CD11b+ macrophages via the Tim-3/galectin-9 interaction during S Typhimurium infection. Blocking the Tim-3/galectin-9 interaction with α-lactose significantly attenuated the bactericidal activity of intracellular S Typhimurium by macrophages. Furthermore, the Tim-3/galectin-9 interaction promoted the formation and activation of inflammasomes, which led to caspase-1 cleavage and interleukin 1β (IL-1β) secretion. The secretion of active IL-1β further improved bactericidal activity of macrophages and galectin-9 expression on macrophages. These results demonstrated the critical role of the cross talk between CD4+ T cells and macrophages, particularly the Tim-3/galectin-9 interaction, in antimicrobial immunity and the control of intestinal pathogenic infections.

Memory Regulatory T cells Increase Only In Inflammatory Phase of Chronic Hepatitis B Infection and Related to Galectin-9/Tim-3 interaction

CD4+Foxp3+ regulatory T cells (Tregs) are the main immune suppressors with subpopulation of inflamed-tissue related memory Tregs (mTregs) and non-related resting Treg (rTregs). Previously, Treg was proposed to be the cause of chronicity of hepatitis B virus (HBV) infection but with controversies. We then investigated the role of mTregs in distinct immune phases of chronic HBV infection, especially the non-inflammatory versus inflammatory phases. It was found mTregs but not rTregs increased only in the inflammatory phase and correlated with serum alanine aminotransferase (ALT) level. These mTregs accumulated in the inflamed liver, expressed significantly higher Tim-3, CCR4, CCR5 and fewer CCR7, and possessed potent suppressive function. These mTregs mainly originated from natural Tregs because of high Helios expression. Hierarchical clustering analysis showed higher frequency of mTreg was concordant with higher serum ALT and galectin-9 levels. Furthermore, galectin-9 could expand mTregs through galectin-9/Tim-3 interaction. In conclusion, increased mTregs are found only in inflammatory phase of chronic HBV infection. Galectin-9, associated with liver inflammation, contributes to the expansion of mTregs through galectin-9/Tim-3 interaction. Therefore, this expansion of mTregs only reflects as an immune regulatory mechanism to limit the on-going liver damages rather than the cause of chronicity of HBV infection.

Intestine lamina propria CD4+T cells promote the bactericidal activity of macrophages via galectin-9 and Tim-3 interaction during Salmonella typhimurium infection (MUC5P.760)

Abstract The intestinal immune system is crucial for protection the host from pathogenic infection and maintenance of mucosal homeostasis. The intestine lamina propria macrophages are main effector cells in innate resistance to intracellular microbial pathogens. In the present study, we found that Salmonella typhimurium infection augmented the Tim-3 expression on intestinal lamina propria CD4+T cells and enhanced galectin-9 expression on F4/80+CD11c+macrophages. Moreover, CD4+T cells promote the activation and bactericidal activity of F4/80+CD11c+macrophages via galectin-9 and Tim-3 interaction during Salmonella typhimurium infection. Blockade the interaction of Tim-3 and galectin-9 with a-Lactose significantly attenuated the killing of intracellular bacteria by macrophages. Furthermore, the interaction of Tim-3 and galectin-9 also promoted the formation and activation of inflammasome, which leads to the cleavage of caspase-1 and IL-1β. The secretion of active IL-1β further improved bactericidal activity of macrophages and expression of galectin-9 on macrophages. These results demonstrated the critical role of the cross-talk between CD4+T cell and macrophage, in particularly the interaction of Tim-3 and galectin-9, in antimicrobial immunity and control intestinal pathogen infection.

Galectin-9 ameliorates respiratory syncytial virus-induced pulmonary immunopathology through regulating the balance between Th17 and regulatory T cells

Respiratory syncytial virus (RSV) infections are characterized by lung inflammation, mucus hypersecretion, and hyperresponsiveness. CD4+ T cells play a pivotal role in the development of RSV-induced lung pathology. Thus targeting the activation of CD4+ T cell subsets and enhancing regulatory functions of CD4+ T cells could be an effectively therapeutic approach. In the present study, we showed that RSV-induced lung inflammation can be suppressed by lectin family member Galectin-9 (Gal-9), which is identified as a T-cell immunoglobulin- and mucindomain-containing molecule-3 (Tim-3) ligand (L) and the Gal-9/Tim-3 interaction acts as a specific inhibitor of T helper(Th)1 and Th17 immune responses. Tim-3 expression was up-regulated in RSV-infected mice compared to non-infected controls. Therefore, we constructed a recombinant adenoviral (rAAV) 9-Gal-9 adenoviral plasmid, and administered it intranasally into RSV-infected mice for five times at every other day until day 8 post-infection. We found that Gal-9 administration significantly decreased viral load, inhibited mucus production, and diminished severity of lung pathology which were all induced by RSV infection. Complicated mechanisms were involved in these inhibitory effects, including inhibition of Th17 cell production, induction of regulatory cell expansion, as well as alteration of CD8 T-cell apoptosis. Our findings suggest that regulating the function of the Gal-9/Tim-3 pathway will be an effective and safe approach to treat RSV infection in lungs.

Galectin-9 Signaling through TIM-3 Is Involved in Neutrophil-Mediated Gram-Negative Bacterial Killing: An Effect Abrogated within the Cystic Fibrosis Lung

The T cell Ig and mucin domain-containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment.

Taming Dendritic Cells with TIM-3: Another Immunosuppressive Strategy Used by Tumors

Evaluation of: Chiba S, Baghdadi M, Akiba H et al. Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1. Nat. Immunol. 13, 832-842 (2012). The identification of TIM-3 expression on tumor-associated dendritic cells (TADCs) provides insight into another aspect of tumor-mediated immunosuppression. The role of TIM-3 has been well characterized on tumor-infiltrating T cells; however, its role on TADCs was not previously known. The current paper demonstrated that TIM-3 was predominantly expressed by TADCs and its interaction with the nuclear protein HMGB1 suppressed nucleic acid-mediated activation of an effective antitumor immune response. The authors were able to show that TIM-3 interaction with HMGB1 prevented the localization of nucleic acids into endosomal vesicles. Furthermore, chemotherapy was found to be more effective in anti-TIM-3 monoclonal antibody-treated mice or mice depleted of all DCs, which indicated that a significant role is played by TADCs in inhibiting tumor regression. Taken together, these findings identify TIM-3 as a potential target for inducing antitumor immunity in conjunction with DNA vaccines and/or immunogenic chemotherapy in clinical settings.

Galectin-9 binding to Tim-3 renders activated human CD4+ T cells less susceptible to HIV-1 infection

AbstractGalectin-9 (Gal-9) is a tandem repeat-type member of the galectin family and is a ligand for T-cell immunoglobulin mucin domain 3 (Tim-3), a type-I glycoprotein that is persistently expressed on dysfunctional T cells during chronic infection. Studies in autoimmune diseases and chronic viral infections show that Tim-3 is a regulatory molecule that inhibits Th1 type immune responses. Here we show that soluble Gal-9 interacts with Tim-3 expressed on the surface of activated CD4+ T cells and renders them less susceptible to HIV-1 infection and replication. The Gal-9/Tim-3 interaction on activated CD4+ T cells, leads to down-regulation of HIV-1 coreceptors and up-regulation of the cyclin-dependent kinase inhibitor p21 (also known as cip-1 and waf-1). We suggest that higher expression of Tim-3 during chronic infection has evolved to limit persistent immune activation and associated tissue damage. These data demonstrate a novel mechanism for Gal-9/Tim-3 interactions to induce resistance of activated CD4+ T cells to HIV-1 infection and suggest that Gal-9 may play a role in HIV-1 pathogenesis and could be used as a novel microbicide to prevent HIV-1 infection.

Influence of Galectin-9/Tim-3 Interaction on Herpes Simplex Virus-1 Latency

After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)-expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1-infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8(+) T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.

An influence of galectin-9/Tim-3 interaction on Herpes simplex virus-1 latency (105.14)

Abstract In this report, we show that the majority of CD8+T cells in the trigeminal ganglion (TG) of mice ocularly infected with HSV-1 express Tim-3, a molecule that delivers negative signals to CD8+T cells when it engages its ligand, Galectin 9 (Gal-9). Curiously Gal-9 glycoprotein is also up regulated in the TG both during the time when replicating virus is present as well during early viral latency. Accordingly it is conceivable that the efficiency of ganglionic CD8+T cell function could be influenced by Gal-9/Tim-3 interaction. In support of this we show that the activity of peptide stimulated CD8+T cells recovered from the TG of WT mice produced less IFN-γ, TNF-α and Granzyme B molecules in comparison to TG CD8+T cells of Gal-9KO mice. Differences in the number of productively and latently infected neuronal cells were also evident between WT and Gal-9KO mice. In vitro studies demonstrated that TG cell cultures of WT HSV-1 infected mice exposed to recombinant Gal-9 in the latent phase caused apoptosis of some CD8+T cells and induce viral reactivation. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8+T cells can influence the outcome of latent HSV-1 infection. Neuronal cell function is likely to be preserved when host Gal-9 levels are low, since inhibitory effects on latency controlling CD8+T cells will be minimal.

Galectin-9 regulates T helper cell function independently of Tim-3

β-Galactoside-binding lectin 9 (galectin-9) is a tandem repeat-type member of the galectin family. It was initially characterized as an eosinophil chemoattractant and an inducer of apoptosis in thymocytes. Subsequently, galectin-9 was identified as a ligand for transmembrane immunoglobulin mucin domain 3 (Tim-3), a type I glycoprotein induced on T cells during chronic inflammation. Work in autoimmune diseases and chronic viral infections have led to the current hypothesis that the function of Tim-3 is to limit immune responses. However, it is still not known to what degree these effects are due to the galectin-9/Tim-3 interaction. In this study, we show that galectin-9 is not limited to the role of a pro-apoptotic agent, but that it can also induce the production of pro-inflammatory cytokines from T helper cells. This effect is dose-dependent and does not require Tim-3. These findings suggest that the effects of galectin-9 on T cells are more complex than previously thought and are mediated by additional receptors apart from Tim-3.

Galectin-9/TIM-3 Interaction Regulates Virus-Specific Primary and Memory CD8+ T Cell Response

In this communication, we demonstrate that galectin (Gal)-9 acts to constrain CD8+ T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with α-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8+ T cell response to HSV particularly in the acute phase. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8+ T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved direct inhibitory effects on TIM-3+ CD8+ T effector cells as well as the promotion of Foxp3+ regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

Regulation of virus-specific acute and memory CD8+ T cell response by Galectin-9/TIM-3 interaction (137.5)

Abstract We demonstrate that galectin-9 (Gal-9) acts to constrain CD8+ T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with α-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8+ T cell response to HSV. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8+ T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved apoptosis of TIM-3+ Teffectors as well as the promotion of Foxp3+ regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance primary and memory responses.

Blood diffusion and Th1-suppressive effects of galectin-9–containing exosomes released by Epstein-Barr virus–infected nasopharyngeal carcinoma cells

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is the third most frequent virus-associated human malignancy. How this tumor escapes immune recognition despite the expression of several viral antigens has remained poorly understood. Our previous in vitro studies have shown that NPC cells release exosomes containing high amounts of galectin-9, a ligand of the membrane receptor Tim-3, which is able to induce apoptosis in mature Th1 lymphocytes. Here, we sought to determine whether galectin-9-carrying exosomes were produced in NPC patients and whether such exosomes might play a role in the immune evasion of NPC cells. We report that galectin-9-containing exosomes are selectively detected in plasma samples from NPC patients and mice xenografted with NPC tumors. The incorporation into exosomes protects galectin-9 against proteolytic cleavage but retains its Tim-3-binding capacity. Importantly, NPC exosomes induce massive apoptosis in EBV-specific CD4(+) cells used as a model of target T cells. This effect is inhibited by both anti-Tim-3 and antigalectin-9 blocking antibodies. These results indicate that blocking galectin-9/Tim-3 interaction in vivo might alleviate the Th1-suppressive effect of NPC exosomes and sustain antitumoral T-cell responses and thereby improve clinical efficacy of immunotherapeutic approaches against NPC.