NKG2A is a late immune checkpoint on CD8 T cells and marks repeated stimulation and cell division.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-ß in vitro, although TGF-ß signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.

Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma.

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been described as a transmembrane protein, expressed on the surface of various T cells as well as different cells of innate immunity. It has since been associated with Th1 mediated autoimmune diseases and transplantation tolerance studies, thereby indicating a possible role of this receptor in counter-regulation of Th2 immune responses. In the present study we therefore directly examined the role of Tim-3 in allergic inflammation and respiratory tolerance. First, Tim-3-/- mice and wild type controls were immunized and challenged with the model allergen ovalbumin (OVA) to induce an asthma-like phenotype. Analysis of cell numbers and distribution in the bronchoalveolar lavage (BAL) fluid as well as lung histology in H&E stained lung sections demonstrated a comparable degree of eosinophilic inflammation in both mouse strains. Th2 cytokine production in restimulated cell culture supernatants and serum IgE and IgG levels were equally increased in both genotypes. In addition, cell proliferation and the distribution of different T cell subsets were comparable. Moreover, analysis of both mouse strains in our respiratory tolerance model, where mucosal application of the model allergen before immunization, prevents the development of an asthma-like phenotype, revealed no differences in any of the parameters mentioned above. The current study demonstrates that Tim-3 is dispensable not only for the development of allergic inflammation but also for induction of respiratory tolerance in mice in an OVA-based model.

Tim-3 suppresses autoimmune hepatitis via the p38/MKP-1 pathway in Th17 cells.

T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) mediates T-cell suppression in various autoimmune diseases, such as chronic inflammatory liver disease. However, the regulatory effect of Tim-3 on Th17 cells in autoimmune hepatitis (AIH) is incompletely understood. Here, we studied the expression and function of Tim-3 in T cells in AIH patients and in a Con A (concanavalin A)-induced mouse AIH model. We report that the frequency of CD4+ Tim-3+ T cells in peripheral blood samples of AIH patients was lower than that in the control group. The p38/MKP-1 and p-JNK pathways were activated, and the expression of interleukin-17A protein was elevated in patients with AIH. Furthermore, the extent of pathological damage in the livers of mice with a blocked Tim-3 signaling pathway (anti-Tim-3 group) was markedly increased and correlated with elevated alanine aminotransferase and aspartate aminotransferase levels. In addition, the frequency of CD4+ IL-17+ T (Th17) cells in the anti-Tim-3 group was increased, while that in mice with blocked p38 activity was decreased. Finally, the expression of MKP-1 (p-p38) gradually increased in the control, Con A, and anti-Tim-3 groups, but the levels of interleukin-17A were decreased in the p38-blocked group. In summary, our results suggest that Tim-3 suppresses AIH by regulating Th17 cells through the p38/MKP-1 pathway.

The role of Tim-3/Galectin-9 pathway in T-cell function and prognosis of patients with human papilloma virus-associated cervical carcinoma.

The interaction between Tim-3 on T cell and its ligand, Galectin-9, negatively regulates cellular immune responses. However, the role of Tim-3/Galectin-9 pathway in the immune evasion of cervical cancer remains unknown. This study is to investigate the expression, function, and regulation of Tim-3/Galectin-9 signaling pathway in human papilloma virus (HPV) positive cervical cancer. Flow cytometry showed that Tim-3 expression on T cell and Galectin-9 expression on monocytes in HPV positive cervical cancer patients were significantly higher compared to cervical intraepithelial neoplasia and benign uterine fibroids Tim-3 + CD4+ Th1 cells and Tim-3 + CD8+ T cells in HPV positive cervical cancer patients were significantly reduced after surgery. Serum TGF-ß and IL-10 levels were positively correlated with Tim-3 + Treg cells, while IFN-γ and IL-2 were negatively correlated with Tim-3 + Th1 cells. Additionally, Tim-3 + CD4+ T cells were positively correlated with Galectin-9 + monocytes. Survival curve analysis showed that Tim-3 + CD4+ T cells were negatively correlated with patient survival, and closely related to FIGO stage, degree of differentiation, and lymph node metastasis of HPV positive cervical cancer. In vitro experiments showed that by blocking the Tim-3/Galectin-9 pathway, the proliferation of T cells and their ability to express IFN-γ, IL-2, perforin, and granzyme B was significantly restored. In conclusion, high levels of Tim-3 and Galectin-9 in HPV positive cervical cancer patients play roles in the progression of disease by promoting Treg cells to inhibit the cytotoxic function of Th1 and CD8+ T cells. Tim-3/Galectin-9 may serve as a new immunotherapy target for patients with HPV positive cervical cancer.

TIM-3 and CEACAM1 do not interact in cis and in trans.

TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells.

Overexpression of TIM-3 in Macrophages Aggravates Pathogenesis of Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder and lacks effective treatments because of unclear mechanisms. Aberrant function of alveolar macrophages is directly linked to pulmonary fibrosis. Here, we show TIM-3 (T-cell immunoglobulin domain and mucin domain-3), a key regulator of macrophage function, aggravates pulmonary fibrosis. TIM-3 mRNA of patients with IPF was analyzed based on the Gene Expression Omnibus and Array Express databases. Lung pathology and profibrotic molecules were assessed in a bleomycin (BLM)-induced pulmonary fibrosis model using wild-type (WT) and TIM-3 transgenic (TIM-3-TG) mice. Macrophage cells, RAW264.7, were then applied to investigate the effect of macrophage TIM-3 under BLM exposure in vitro. Macrophage depletion and adoptive-transfer experiments were finally performed to examine lung morphology and profibrotic molecules. TIM-3 expression was increased both in patients with IPF and in our BLM-induced mouse model. TIM-3-TG mice developed more serious pathological changes in lung tissue and higher expressions of TGF-ß1 (transforming growth factor-ß1) and IL-10 than WT mice. After BLM treatment, TGF-ß1 and IL-10 expression was significantly decreased in RAW264.7 cells after TIM-3 knock-out, whereas it was increased in TIM-3-TG peritoneal macrophages. The scores of pulmonary fibrosis in WT and TIM-3-TG mice were significantly reduced, and there was no difference between them after macrophage depletion. Furthermore, WT mice receiving adoptive macrophages from TIM-3-TG mice also had more serious lung fibrosis and increased expression of TGF-ß1 and IL-10 than those receiving macrophages from WT mice. Our findings revealed that overexpressed TIM-3 in alveolar macrophages aggravated pulmonary fibrosis.

Dysfunction of natural killer cells mediated by PD-1 and Tim-3 pathway in anaplastic thyroid cancer.

The survival rate of anaplastic thyroid cancer (ATC) remains about 7% to 14%. The natural killer (NK) cells are a critical component of antitumor immunity, and their composition and function in thyroid cancer patients are investigated in this study. In healthy controls and early stage thyroid cancer patients, >90% of circulating NK cells were CD56loCD16hi and fewer than 10% were CD56hiCD16hi/lo. However, the frequency of the CD56hiCD16hi/lo NK subset was significantly higher in more advanced thyroid cancer patients and further increased in ATC patients. Two members of the inhibitory KIR family, CD158a and CD158b, was significantly higher in CD56hiCD16hi/lo NK cells than in CD56loCD16hi NK cells, while NKG2D, an activator of NK cells, was significantly lower in CD56hiCD16hi/lo NK cells than in CD56loCD16hi NK cells. We also found that the CD56hiCD16hi/lo NK cells presented higher PD-1, higher Tim-3, and lower cytotoxicity against the human ATC cell line CAL-62, than the CD56loCD16hi NK cells. The expression of exhaustion markers and reduction in cytotoxicity was further exacerbated in more advanced thyroid cancer patients and in ATC patients. Interestingly, PD-1 and Tim-3 blockade was effective at reinvigorating both the more impaired CD56hiCD16hi/lo NK cells and the less impaired CD56loCD16hi NK cells from ATC patients. Together, our study identified a dysfunction of NK cells in more advanced thyroid cancer patients and ATC patients, and presented actionable targets for future development of immunotherapies in thyroid cancers.

Immune checkpoint receptors: homeostatic regulators of immunity.

Alcoholic liver disease (ALD) is an escalating global problem accounting for more than 3 million deaths annually. Bacterial infections are diagnosed in 25-47% of hospitalized patients with cirrhosis and represent the most important trigger for acute decompensation, multi-organ failure, septic shock and death. Current guidelines recommend intensive antibiotic therapy, but this has led to the emergence of multi-drug resistant bacteria, which are associated with increased morbidity and mortality rates. As such, there is a pressing need to explore new paradigms for anti-infective therapy and host-directed immunomodulatory therapies are a promising approach. Paradoxically, cirrhotic patients are characterised by heightened immune activity and exacerbated inflammatory processes but are unable to contend with bacterial infection, demonstrating that whilst immune effector cells are primed, their antibacterial effector functions are switched-off, reflecting a skewed homeostatic balance between anti-pathogen immunity and host-induced immunopathology. Preservation of this equilibrium physiologically is maintained by multiple immune-regulatory checkpoints and these feedback receptors serve as pivotal regulators of the host immunity. Checkpoint receptor blockade is proving to be effective at rescuing deranged/exhausted immunity in pre-clinical studies for chronic viral infection and sepsis. This approach has also obtained FDA approval for restoring anti-tumor immunity, with improved response rates and good safety profiles. To date, no clinical studies have investigated checkpoint blockade in ALD, highlighting an area for development of host-targeted immunotherapeutic strategies in ALD, for which there are no current specific treatment options. This review aims at framing current knowledge on immune checkpoints and the possibility of their therapeutic utility in ALD-associated immune dysfunctions.

[Tim-3: a novel biomarker and therapeutic target in oncology]. / Tim-3 - Biomarqueur et cible thérapeutique en cancérologie.

T cells harboring multiple co-inhibitory molecules lose their anti-tumoral functionality. PD-1 is a clinically approved target in cancer therapy, but its expression alone does not mean dysfunctionality. The expression of Tim-3 on numerous cell types (T cell, Treg, dendritic cell, myeloid cells) favors tumor escape to immune cells. Within many tumors, PD-1/Tim-3 coexpressing CD8-T cells lose their ability to secrete cytokines (IFNγ, IL-2, TNFα) and their intratumoral infiltration correlates with a bad prognosis. Tim-3 recently appeared as a potential biomarker of anti-PD-1 resistance. Combined blockade of PD-1 and Tim-3 axis demonstrated potent clinical efficacy in preclinical models and reinforced the rationale of using an anti-Tim-3 to override tumor resistance.

The immunological effect of Galectin-9/TIM-3 pathway after low dose Mifepristone treatment in mice at 14.5 day of pregnancy.

The abortifacient Mifepristone (RU486) has proven to be a safe, effective, acceptable option for millions of women seeking abortion during the first and second trimester of pregnancy although its precise mechanism of action is not well understood. The main objective of this study was to investigate the impact of low dose Mifepristone administration on placental Galectin-9 (Gal-9) expression, as well as its effect on the cell surface expression of Gal-9, TIM-3 and CD107a molecules by different T and NK cell subsets. A model of Mifepristone-induced immunological changes was established in syngeneic pregnant BALB/c mice. RU486-induced alteration in placental Gal-9 expression was determined by immunohistochemistry. For immunophenotypic analysis, mid-pregnancy decidual lymphocytes and peripheral mononuclear cells were obtained from Mifepristone treated and control mice at the 14.5 day of gestation. TIM-3 and Gal-9 expression by peripheral and decidual immune cells were examined by flow cytometry. Our results revealed a dramatically decreased intracellular Gal-9 expression in the spongiotrophoblast layer of the haemochorial placenta in Mifepristone treated pregnant mice. Although low dose RU486 treatment did not cause considerable change in the phenotypic distribution of decidual and peripheral immune cells, it altered the Gal-9 and TIM-3 expression by different NK and T cell subsets. In addition, the treatment significantly decreased the CD107a expression by decidual TIM-3+ NK cells, but increased its expression by decidual NKT cell compared to the peripheral counterparts. These findings suggest that low dose Mifepristone administration might induce immune alterations in both progesterone dependent and independent way.

Tumor cell-intrinsic Tim-3 promotes liver cancer via NF-κB/IL-6/STAT3 axis.

T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), mediating immune exhaustion in tumor microenvironment, has become a promising target for tumor therapy. However, the exact mechanisms for tumor cell-intrinsic Tim-3 in tumor development and its potential contribution in Tim-3-targeted therapy strategy have not been elucidated yet. In this study, we showed that human liver cancer tissues contained high ratio of Tim-3-expressing hepatocytes, and cytokines rich in tumor microenvironment and HBV involved in Tim-3 upregulation in malignant hepatocytes. We demonstrated that hepatocyte-specific Tim-3 overexpression enhances tumor cell growth, whereas Tim-3 inhibition on malignant hepatocytes by anti-Tim-3 antibodies or RNAi suppresses tumor growth both in vitro and in Tim-3 knockout mice. Mechanistically, the hepatocyte-Tim-3 receptor activates NF-κB phosphorylation, which in turn stimulates IL-6 secretion and STAT3 phosphorylation. Our results identify tumor cell-intrinsic functions of Tim-3 in tumorigenesis and suggest that blocking Tim-3 in tumor cells might contribute to the clinical efficacy of anti-Tim-3 antibody treatment in the future tumor therapy.

miR-330-5p/Tim-3 axis regulates macrophage M2 polarization and insulin resistance in diabetes mice.

Obesity is associated with a state of low-grade inflammatory response in adipose tissue, and contributes to the development of type 2 diabetes. Immune cells such as macrophages can infiltrate adipose tissue and are responsible for the majority of inflammatory cytokine production. Therefore, adipose tissue promotes macrophage infiltration, resulting in local inflammation and insulin resistance. Tim-3 negatively regulates IFN-γ secretion and influences the ability to induce T cell tolerance in diabetes. MicroRNA contributes to the development of immunological tolerance and involves in macrophage polarization. However, the potential of Tim-3 to regulate macrophage polarization and the related microRNA has not been reported. In this experiment, 8-week-old C57BL/6 mice were fed a high-fat diet for 8 weeks. The adipose tissue macrophages were isolated, miR-330-5p and Tim-3 levels, and M1/M2 polarization were analyzed. In addition, insulin tolerance tests was detected. The results demonstrated that miR-330-5p levels increased but Tim-3 levels decreased, leading to M1 polarization and insulin tolerance in diabetes mice. In addition, inhibition of miR-330-5p enhanced Tim-3 levels, leading to M2 polarization and insulin tolerance attenuation in diabetes mice. Furthermore, we detected the inverse relationship between miR-330-5p and Tim-3. We found that Tim-3 mRNA contained conserved miR-330-5p binding sites in its 3'UTR, and miR-330-5p could directly regulate Tim-3 expression through these 3'UTR sites. Our study demonstrated that miR-330-5p served as a regulator of the M2 polarization and miR-330-5p/Tim-3 axis potentially down-regulated insulin resistance in diabetes, probably through enhancing the M2 polarization of macrophage.

The Potential Role of Inhibitory Receptors in the Treatment of Psoriasis.

Psoriasis is a common autoimmune disorder that affects the skin. Approximately 30% of individuals with psoriasis will develop inflammatory arthritis, often in the setting of human leukocyte antigen B27. Both forms of disease are thought to be the result of prolonged inflammation mediated by T lymphocytes, dendritic cells, and keratinocytes. While there are treatments aimed at immunomodulation, targeting T cell co-inhibitory receptors signaling pathways may provide therapeutic benefit. This review will discuss in detail four T cell co-inhibitory receptors and their potential application for the treatment of psoriasis and psoriatic arthritis.

Tim-3, Lag-3, and TIGIT.

Co-inhibitory receptors play a key role in regulating T cell responses and maintaining immune homeostasis. Their inhibitory function prevents autoimmune responses but also restricts the ability of T cells to mount effective immune responses against tumors or persistent pathogens. T cells express a module of co-inhibitory receptors, which display great diversity in expression, structure, and function. Here, we focus on the co-inhibitory receptors Tim-3, Lag-3, and TIGIT and how they regulate T cell function, maintenance of self-tolerance, their role in regulating ongoing T cell responses at peripheral tissues, and their synergistic effects in regulating autoimmunity and antitumor responses.

Combined Blockade of T Cell Immunoglobulin and Mucin Domain 3 and Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Results in Durable Therapeutic Efficacy in Mice with Intracranial Gliomas.

BACKGROUND Glioblastoma multiforme (GBM) evades immune surveillance by inducing immunosuppression via receptor-ligand interactions between immune checkpoint molecules. T cell immunoglobulin and mucin domain 3 (Tim-3) is a key checkpoint receptor responsible for exhaustion and dysfunction of T cells and plays a critical role in immunosuppression. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been recently identified as a heterophilic ligand for Tim-3. MATERIAL AND METHODS We established an intracranial GBM model using C57BL/6 mice and GL261 cells, and treated the mice with single or combined monoclonal antibodies (mAbs) against Tim-3/CEACAM1. The CD4+, CD8+, and regulatory T cells in brain-infiltrating lymphocytes were analyzed using flow cytometry, and the effector function of T cells was assessed using ELISA. We performed a rechallenge by subcutaneous injection of GL261 cells in the "cured" (>90 days post-orthotopic tumor implantation) and naïve mice. RESULTS The mean survival time in the control, anti-Tim-3, anti-CEACAM1, and combined treatment groups was 29.8, 43.4, 42.3, and 86.0 days, respectively, with 80% of the mice in the combined group becoming long-term survivors showing immune memory against glioma cells. Infiltrating CD4+ and CD8+ T cells increased and immunosuppressive Tregs decreased with the combined therapy, which resulted in a markedly elevated ratio of CD4+ and CD8+ cells to Tregs. Additionally, plasma IFN-γ and TGF-ß levels were upregulated and downregulated, respectively. CONCLUSIONS Our data indicate that combined blockade of Tim-3 and CEACAM1 generates robust therapeutic efficacy in mice with intracranial tumors, and provides a promising option for GBM immunotherapy.

Synergistic effect of IL-12 and IL-18 induces TIM3 regulation of γδ T cell function and decreases the risk of clinical malaria in children living in Papua New Guinea.

BACKGROUND: γδ T cells are important for both protective immunity and immunopathogenesis during malaria infection. However, the immunological processes determining beneficial or detrimental effects on disease outcome remain elusive. The aim of this study was to examine expression and regulatory effect of the inhibitory receptor T-cell immunoglobulin domain and mucin domain 3 (TIM3) on γδ T cells. While TIM3 expression and function on conventional αß T cells have been clearly defined, the equivalent characterization on γδ T cells and associations with disease outcomes is limited. This study investigated the functional capacity of TIM3+ γδ T cells and the underlying mechanisms contributing to TIM3 upregulation and established an association with malaria disease outcomes. METHODS: We analyzed TIM3 expression on γδ T cells in 132 children aged 5-10 years living in malaria endemic areas of Papua New Guinea. TIM3 upregulation and effector functions of TIM3+ γδ T cells were assessed following in vitro stimulation with parasite-infected erythrocytes, phosphoantigen and/or cytokines. Associations between the proportion of TIM3-expressing cells and the molecular force of infection were tested using negative binomial regression and in a Cox proportional hazards model for time to first clinical episode. Multivariable analyses to determine the association of TIM3 and IL-18 levels were conducted using general linear models. Malaria infection mouse models were utilized to experimentally investigate the relationship between repeated exposure and TIM3 upregulation. RESULTS: This study demonstrates that even in the absence of an active malaria infection, children of malaria endemic areas have an atypical population of TIM3-expressing γδ T cells (mean frequency TIM3+ of total γδ T cells 15.2% ± 12). Crucial factors required for γδ T cell TIM3 upregulation include IL-12/IL-18, and plasma IL-18 was associated with TIM3 expression (P = 0.002). Additionally, we show a relationship between TIM3 expression and infection with distinct parasite clones during repeated exposure. TIM3+ γδ T cells were functionally impaired and were associated with asymptomatic malaria infection (hazard ratio 0.54, P = 0.032). CONCLUSIONS: Collectively our data demonstrate a novel role for IL-12/IL-18 in shaping the innate immune response and provide fundamental insight into aspects of γδ T cell immunoregulation. Furthermore, we show that TIM3 represents an important γδ T cell regulatory component involved in minimizing malaria symptoms.

Checkpoint inhibitors in hematological malignancies.

Inhibitory molecules such as PD-1, CTLA-4, LAG-3, or TIM-3 play a role to keep a balance in immune function. However, many cancers exploit such molecules to escape immune surveillance. Accumulating data support that their functions are dysregulated in lymphoid neoplasms, including plasma cell myeloma, myelodysplastic syndrome, and acute myeloid leukemia. In lymphoid neoplasms, aberrations in 9p24.1 (PD-L1, PD-L2, and JAK2 locus), latent Epstein-Barr virus infection, PD-L1 3'-untranslated region disruption, and constitutive JAK-STAT pathway are known mechanisms to induce PD-L1 expression in lymphoma cells. Clinical trials demonstrated that PD-1 blockade is an attractive way to restore host's immune function in hematological malignancies, particularly classical Hodgkin lymphoma. Numerous clinical trials exploring PD-1 blockade as a single therapy or in combination with other immune checkpoint inhibitors in patients with hematologic cancers are under way. Although impressive clinical response is observed with immune checkpoint inhibitors in patients with certain cancers, not all patients respond to immune checkpoint inhibitors. Therefore, to identify best candidates who would have excellent response to checkpoint inhibitors is of utmost importance. Several possible biomarkers are available, but consensus has not been made and pursuit to discover the best biomarker is ongoing.

Elevated Galectin-9 Suppresses Th1 Effector Function and Induces Apoptosis of Activated CD4+ T Cells in Osteoarthritis.

T cell immunoglobulin and mucin domain 3 (Tim-3) is a critical regulatory molecule found on activated Th1 cells, exhausted CD8+ T cells, and resting monocytes/macrophages. Galectin-9 (Gal-9) is an identified ligand for Tim-3. Interaction between Tim-3 and Gal-9 is thought to inhibit Th1 responses. The regulation and function of Tim-3 and Gal-9 in osteoarthritis (OA) have not been intensively investigated. We found that in peripheral blood, CD4+ T cells, but not CD8+ T cells or CD14+ monocytes, from OA patients presented significantly elevated Tim-3 and Gal-9 expression compared to those from healthy controls (HC). The CD4+ T cells from OA did not present altered Th1, Th2, and Th17 composition in the peripheral blood, but secreted less Th1 cytokine interleukin 2 (IL-2) and interferon gamma (IFN-γ) after activation. Further investigation demonstrated that Gal-9 induced high levels of apoptosis in activated CD4+ T cells from OA patients. Inhibition of Gal-9 resulted in significantly higher IL-2 and IFN-γ expression that was directly correlated with the number of non-apoptotic cells. In the synovial fluid, both secreted Gal-9 and surface Gal-9 levels were significantly higher in less-severe grade 2 OA patients than in more-severe grade 4 OA patients. Surface Tim-3 was also higher in synovial fluid CD8+ T cells and CD14+ monocytes from grade 2 OA patients and lower in grade 4 OA patients. Together, these results suggested that Tim-3 and Gal-9 could downregulate T cell inflammation in OA, and could be utilized as a novel therapeutic strategy.

Effects of T cell immunoglobulin and mucin domain-containing molecule-3 signaling molecule on human monocyte-derived dendritic cells with hepatitis B virus surface antigen stimulation in vitro.

The aim of the present study was to investigate the in vitro effects of hepatitis B virus surface antigen (HBsAg) on the immune function of human monocyte-derived dendritic cells (MD­DCs), and the moderating role of T cell immunoglobulin and mucin domain­containing molecule­3 (Tim­3) signaling molecule. The monocytes, obtained from healthy adult peripheral blood, were incubated with recombinant human granulocyte­macrophage colony­stimulating factor and interleukin (IL)­4 to induce DCs. DC­associated cell markers were detected using flow cytometry. MD­DCs were treated with HBsAg (5 µg/ml) in vitro for 48 h and subsequently, cell markers, lymphocyte stimulatory capacity, signaling protein and downstream cytokines were assessed. In addition, a Tim­3 monoclonal antibody was used to inhibit the Tim­3 signaling pathway, and subsequently the immune responses of MD­DCs to HBsAg stimulation were determined using the aforementioned method. The cell phenotype expressions of MD­DCs were all significantly increased with cluster of differentiation (CD)11c at 70.09±0.57%, human leukocyte antigen­DR at 79.83±2.12%, CD80 at 48.33±7.34% and CD86 at 44.21±5.35%. The treatment of MD­DCs with HBsAg resulted in a CD80 and CD86 enhanced expression, enhanced lymphocyte stimulatory capacity, upregulated expression of Tim­3 and nuclear factor­κB (NF­κB), as well as enhanced cytokine secretion of IL­6, IL­10 and interferon (IFN)­Î³. However, a reduced immune response of MD­DCs in response to HBsAg stimulation was observed when the Tim­3 signaling pathway was inhibited prior to stimulation. The expression of NF­κB was decreased and the cytokine secretion level of IL­6, IL­10 and IFN­Î³ were downregulated. The treatment with HBsAg in vitro resulted in an enhanced immune response of MD­DCs, which may be positively regulated by the Tim-3 signaling molecule.