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We investigated expressions of PD-L1, LAG-3, TIM-3, and OX40L as immune checkpoint proteins, and MSI (repetitive short-DNA-sequences due to defective DNA-repair system) status were analyzed with immunohistochemistry from tissue blocks. Of 83 patients, PD-L1 expression was observed in 18.1% (n = 15) of the patients. None of the patients exhibited LAG-3 expression. TIM-3 expression was 4.9% (n = 4), OX40L was 22.9% (n = 19), and 8.4% (n = 7) of the patients had MSI tumor. A low-to-intermediate positive correlation was observed between PD-L1 and TIM-3 expressions (rho: 0.333, p < 0.01). Although PD-L1 expression was higher in grade 3 NET/NEC, MSI status was prominent in grade 1/2 NET. [ABSTRACT FROM AUTHOR]

The mechanisms by which retinoic acid-inducible gene I (RIG-I), a critical RNA virus sensor, is regulated in many biological and pathological processes remain to be determined. Here, we demonstrate that T cell immunoglobulin and mucin protein-3 (Tim-3), an immune checkpoint inhibitor, mediates infection tolerance by suppressing RIG-I-type I interferon pathway. Overexpression or blockade of Tim-3 affects type I interferon expression, virus replication, and tissue damage in mice following H1N1 infection. Tim-3 signaling decreases RIG-I transcription via STAT1 in macrophages and promotes the proteasomal dependent degradation of RIG-I by enhancing K-48-linked ubiquitination via the E3 ligase RNF-122. Silencing RIG-I reversed Tim-3 blockage-mediated upregulation of type I interferon in macrophages. We thus identified a new mechanism through which Tim-3 mediates the immune evasion of H1N1, which may have clinical implications for the treatment of viral diseases. [ABSTRACT FROM AUTHOR]

With the success of immune checkpoint inhibitors (ICI), such as anti-programmed death-1 (PD-1) antibody for solid tumors and lymphoma immunotherapy, a number of clinical trials with ICIs have been attempted for acute myeloid leukemia (AML) immunotherapy; however, limited clinical efficacy has been reported. This may be due to the heterogeneity of immune microenvironments and various degrees of T cell exhaustion in patients and may be involved in the IFN-γ pathway. In this study, we first characterized the percentage of PD-1+ and T cell immunoglobulin mucin-domain-containing-3 (Tim-3) +IFN-γ+ T cells in peripheral blood (PB) in AML compared with healthy individuals (HIs) by flow cytometry and further discussed the possibility of the reversal of T cell exhaustion to restore the secretion capacity of cytokines in T cells in AML based on blockade of PD-1 or Tim-3 (anti-PD-1 and anti-Tim-3 antibody) in vitro using a cytokine protein chip. A significantly increased percentage of PD-1+, Tim-3+, and PD-1 +Tim-3+ IFN-γ+ T cells was observed in PB from patients with AML in comparison with HIs. Moreover, higher PD-1+IFN-γ+CD3+/CD8+ T cell levels were associated with poor overall survival in AML patients. Regarding leukemia cells, the percentage of Tim-3 in CD117+CD34+ AML cells was positively correlated with PD-1 in IFN-γ+CD4+ T cells. Furthermore, blocking PD-1 and Tim-3 may involve multiple cytokines and helper T cell subsets, mainly Th1 and Treg cells. Blockade of PD-1 or Tim-3 tends to restore cytokine secretion to a certain extent, a synergistic effect shown by the co-blockade of PD-1 and Tim-3. However, we also demonstrated the heterogeneity of secretory cytokines in ICI-treated T cells in AML patients. [ABSTRACT FROM AUTHOR]

Genetic variants in the T cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) gene have been reported to be associated with the risk of cancers and patients' outcomes. The aims of this study were to explore the role of TIM-3 polymorphisms in the risk of colorectal cancer (CRC) and the prognosis of CRC patients in a northern Chinese population. Two polymorphisms of TIM-3 were genotyped using polymerase chain reaction and ligase detection reaction in 364 CRC patients and 372 healthy control subjects. The levels of TIM-3 mRNA were investigated in 65 CRC tissues by quantitative real-time PCR. The results showed that neither rs10053538 nor rs10515746 was associated with susceptibility to CRC. However, the CA+AA genotypes of rs10053538 were related to an advanced clinical stage and increased risk of lymph nodemetastasis (P =.046 and 0.024, respectively). Multivariate analyses performed after adjusting for clinical variables showed that patients with the CA+AA genotypes of rs10053538 exhibited a significantly shorter disease-free survival (DFS) and overall survival (OS) time compared with those carrying the CC genotype (HR = 1.91, 95% CI = 1.04–3.51; HR = 2.61, 95% CI = 1.35–5.03). In addition, the expression of TIM-3 mRNA was significantly increased in the CRC tissues of patients carrying the rs10053538 CA+AA genotypes compared with patients carrying the CC genotype (P =.019). The rs10053538 may serve as an independent molecular marker for predicting the clinical outcome of CRC patients in the study population. [ABSTRACT FROM AUTHOR]

The immune system is important for elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. Anti-leukemia immune response can be inhibited by various mechanisms leading to immune evasion and disease relapse. Selected markers of immune escape were analyzed on AML cells from leukapheresis at diagnosis (N = 53). Hierarchical clustering of AML immunophenotypes yielded distinct genetic clusters. In the absence of DNMT3A mutation, NPM1 mutation was associated with decreased HLA expression and low levels of other markers (CLIP, PD-L1, TIM-3). Analysis of an independent cohort confirmed decreased levels of HLA transcripts in patients with NPM1 mutation. Samples with combined NPM1 and DNMT3A mutations had high CLIP surface amount suggesting reduced antigen presentation. TIM-3 transcript correlated not only with TIM-3 surface protein but also with CLIP and PD-L1. In our cohort, high levels of TIM-3/PD-L1/CLIP were associated with lower survival. Our results suggest that AML genotype is related to blast immunophenotype, and that high TIM-3 transcript levels in AML blasts could be a marker of immune escape. Cellular pathways regulating resistance to the immune system might contribute to the predicted response to standard therapy of patients in specific AML subgroups and should be targeted to improve AML treatment. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) with t(8;21) is a heterogeneous disease and needs to be stratified. Both, cancer cells and immune cells participate in tumor initiation, growth and progression and might affect clinical outcomes. TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3), an immune checkpoint molecule, is expressed not only on immune cells but also on leukemic stem cells (LSCs) in AML. This prompted us to investigate the prognostic significance of TIM-3 in t(8;21) AML. A total of 47 t(8;21) AML patients were tested for TIM-3 expression by multi-parameter flow cytometry at diagnosis. 35 of these, who received chemotherapy alone or along with allogeneic hematopoietic stem cell transplantation were followed up. The expression pattern of TIM-3 on T-cells and NK (natural killer) cells as a whole (T + NK) and LSCs were evaluated independently. High percentage of T + NK − TIM-3+ and CD34+CD38−TIM-3+ cells were significantly associated with a high 2-year cumulative incidence of relapse (CIR) (p = 0.028, 0.016). Further, concurrent high frequencies of T + NK-TIM-3+ and CD34+CD38-TIM-3+ cells at diagnosis were significantly associated with a high 2-year CIR (p < 0.0001) and this together with c-KIT D816 mutation were the independent adverse prognostic factors for relapse (hazard ratio (HR)=2.5, [95% confidence interval (CI), 1.1–6.0], p = 0.04; HR = 46.5, [95% CI, 2.7–811.5], p = 0.009). In conclusion, the expression pattern of TIM-3 on both T and NK cells and LSCs at diagnosis had prognostic significance in t (8;21) AML. [ABSTRACT FROM AUTHOR]

Introduction: Dysfunction of the immune system is a hallmark of cancer. Through increased understanding of the complex interactions between immunity and cancer, immunotherapy has emerged as a treatment modality for different types of cancer. Promising activity with immunotherapy has been reported in numerous malignancies, but challenges such as limited response rates and treatment resistance remain. Furthermore, outcomes with this therapeutic approach in hematologic malignancies are even more limited than in solid tumors. T-cell immunoglobulin domain and mucin domain 3 (TIM-3) has emerged as a potential immune checkpoint target in both solid tumors and hematologic malignancies. TIM-3 has been shown to promote immune tolerance, and overexpression of TIM-3 is associated with more aggressive or advanced disease and poor prognosis.Areas Covered: This review examines what is currently known regarding the biology of TIM-3 and clinical implications of targeting TIM-3 in cancer. Particular focus is given to myeloid malignancies.Expert Opinion: The targeting of TIM-3 is a promising therapeutic approach in cancers, including hematologic cancers such as myeloid malignancies which have not benefited much from current immunotherapeutic treatment approaches. We anticipate that with further clinical evaluation, TIM-3 blockade will emerge as an important treatment strategy in myeloid malignancies. [ABSTRACT FROM AUTHOR]

The prognosis of hepatocellular carcinoma (HCC) is extremely poor, of which hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) accounts for the majority in China. Immune checkpoint inhibitors have become an effective immunotherapy method for the treatment of HCC, but they are mainly used for T cells. NK cells play a vital role as the first line of defense against tumors. Therefore, we explored the characteristic expression pattern of immune checkpoints on NK cells of HBV-HCC patients. We analyzed the correlation between the co-expression of TIGIT and TIM-3 and the clinical progress of patients with HBV-HCC. The co-expression of TIGIT and TIM-3 on NK cells is elevated in patients with HBV-HCC. TIGIT+TIM-3+NK cells showed exhausted phenotypic characteristics and displayed dysfunction manifested as weakened killing function, reduced cytokine production, and proliferation function. TIGIT+TIM-3+NK cells participate in NK cells function exhaustion and are closely related to the disease progression of patients with HBV-HCC, suggesting a new target for future immunotherapy. [ABSTRACT FROM AUTHOR]

Objectives: T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) is considered as a negative regulator of T-cell driven immune response. This study is planned to investigate the prognostic role of pre-transplant soluble TIM-3 (sTIM-3) levels in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Methods: Pre-transplant serum sTIM-3 levels were measured in 177 allo-HSCT recipients [median age: 36(16–66) years; male/female: 111/66]. Results: Pre-transplant sTIM-3 levels were significantly higher in acute myeloid leukemia (AML) patients compared to acute lymphoblastic leukemia (ALL) patients (p = 0.01). Pre-transplant sTIM-3 levels were significantly lower in patients with abnormal cytogenetics (p = 0.017). Pre-transplant sTIM-3 levels were significantly higher in patients who developed viral hemorrhagic cystitis (p = 0.034). A positive correlation was demonstrated between sTIM-3 levels and acute graft versus host disease (GvHD) grade (p = 0.013; r = 0.299). Overall survival (OS) was not statistically different between low- and high-TIM-3 groups (%35.2 vs %20.4; p > 0.05). Primary diagnosis (p = 0.042), sinusoidal obstruction syndrome (p < 0.001), acute GvHD (p = 0.001), chronic GvHD (p = 0.009) and post-transplant relapse (p = 0.003) represented significant impact on OS. Discussion: Increased sTIM-3 levels in AML patients seem to be compatible with the previous reports. The inhibitor role of TIM-3 in cellular immune response may be a possible explanation for the association of sTIM-3 with viral infections and GvHD. However, the main challenge remains to be the ambiguous association of pre-transplant sTIM-3 levels and post-transplant complications, as allo-HSCT recipients are expected to represent donor genetic features in the post-transplant setting. Conclusion: Further studies are warranted to clarify the particular role of sTIM-3 in the allo-HSCT setting. [ABSTRACT FROM AUTHOR]

Purposes: The pH in the umbilical artery at delivery provides information on the fetal environment and is related to postnatal outcomes. The ability to predict fetal acidemia at delivery would improve clinical management and neonatal well-being. We hypothesized that an alteration in maternal immunity would accompany placental changes that precede a decrease in pH in the fetal circulation in twin gestations.Methods: Peripheral blood mononuclear cells (PBMCs), obtained from 86 women with twin pregnancies, were lysed and assayed for concentrations of T-cell immunoglobulin mucin domain 3 (Tim-3) and galectin-9 (gal-9) by ELISA. Tim-3-gal-9 interaction is a primary mechanism promoting immune suppression. At delivery, the pH of arterial cord blood was determined.Results: In eight women (9.3%), the pH in the placental arteries from both twins was <7.15, indicating fetal acidosis. In the remaining 78 women the arterial pH was ≥7.15 in both twins. The median Tim-3 level was 361 pg/ml when arterial pH was <7.15 and 199 pg/ml when pH was ≥7.15 (p = .003). Similarly, gal-9 was 31.2 versus 12.4 ng/ml when pH was <7.15 or ≥7.15, respectively (p = .001). A Tim-3 concentration >260 pg/ml predicted arterial pH <7.15 with a sensitivity of 87.5%, specificity of 79.5% and negative predictive value of 98.4%. A gal-9 level >18.4 predicted arterial pH <7.15 with a sensitivity of 100%, specificity of 73.8% and a negative predictive value of 100%.Conclusion: We conclude that elevations in Tim-3 and gal-9 in PBMCs during gestation predict the subsequent occurrence of a pH <7.15 in the fetal arteries at delivery in twin gestations. [ABSTRACT FROM AUTHOR]

T cell immunoglobulin and mucin domain-3 (TIM-3) is an immune checkpoint that regulates normal immune responses but can be exploited by tumor cells to evade immune surveillance. TIM-3 is primarily expressed on immune cells, particularly on dysfunctional and exhausted T cells, and engagement of TIM-3 with its ligands promotes TIM-3-mediated T cell inhibition. Antagonistic ligand-blocking anti-TIM-3 antibodies have the potential to abrogate T cell inhibition, activate antigen-specific T cells, and enhance anti-tumor immunity. Here we describe M6903, a fully human anti-TIM-3 antibody without effector function and with high affinity and selectivity to TIM-3. We demonstrate that M6903 blocks the binding of TIM-3 to three of its ligands, phosphatidylserine (PtdSer), carcinoembryonic antigen cell adhesion-related molecule 1 (CEACAM1), and galectin 9 (Gal-9). These results are supported by an atomic resolution crystal structure and functional assays, which demonstrate that M6903 monotherapy enhanced T cell activation. This activation was further enhanced by the combination of M6903 with bintrafusp alfa, a bifunctional fusion protein that simultaneously blocks the transforming growth factor-β (TGF-β) and programmed death ligand 1 (PD-L1) pathways. M6903 and bintrafusp alfa combination therapy also enhanced anti-tumor efficacy in huTIM-3 knock-in mice, relative to either monotherapy. These in vitro and in vivo data, along with favorable pharmacokinetics in marmoset monkeys, suggest that M6903 as a monotherapy warrants further pre-clinical assessment and that M6903 and bintrafusp alfa may be a promising combination therapy in the clinic. [ABSTRACT FROM AUTHOR]

The immune checkpoint blockade (ICB) immunotherapy has prolonged overall survival for cancer patients but the response rates are low. The resistance to ICB is likely due to compensatory upregulation of additional immune inhibitory molecules. In this study, we first systematically examined Tim-3 expression in immune cells in mouse tumors and found that Tim-3 was specifically up-regulated in a large number of Treg, conventional CD4+, CD8+ T cells, dendritic cell 1 (DC1), and macrophage 1 (M1) in the tumor microenvironment (TME). Interestingly, Tim-3+ T cells in the TME were phenotypically effector but not "exhausted" T cells because Tim-3+ PD-1+ CD8+ T cells had a higher number of mitochondria, greater levels of glycolysis, and higher tumor-specific cytolytic activities compared to Tim-3− PD-1− CD8+ T cells. The combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor activity but also increased the expression of Lag-3 and GITR in TIL, demonstrating cross-regulation between multiple checkpoint molecules. Furthermore, we found that the antitumor efficacy with triple combination of Tim-3, PD-1, and Lag3 mAbs was much greater than any two antibodies. Mechanistically, we demonstrated that simultaneous targeting of Tim-3, PD-1, and Lag-3 cooperatively increased the levels of granzyme B and tumor-specific cytolytic activities of CD8+ TIL. Our data indicate that multiple checkpoint molecules are coordinately upregulated to inhibit the function of hyperactivated T cells in the TME and requirement for the simultaneous blockade of PD-1, Tim-3 and Lag3 for cancer treatment. [ABSTRACT FROM AUTHOR]

Background: Polymorphisms in T-cell immunoglobulin and mucin domain 3 (TIM-3) gene have been implicated in susceptibility to autoimmune diseases (ADs) with inconsistent results. The aim of this study was to perform meta-analyses for clarifying the relationship between them. Methods: All relevant case-control studies were searched in PubMed, Embase, Wanfang, and China National Knowledge Infrastructure. Meta-analysis was conducted in the dominant and allelic models, and checked for heterogeneity and publication bias. The Odds ratio (OR) and 95% confidence interval (CI) was used to assess the strength of the associations. Results: Seventeen studies with four TIM-3 polymorphisms (+4259A>C, −574G>T, −1516G>T, and −1541C>T) were identified, involving 3,399 cases and 3,911 controls. TIM-3 −1516G>T polymorphism showed significant associations with ADs risk among Chinese; however, the significant finding was unstable in sensitivity analysis. In the overall population, TIM-3 + 4259A>C polymorphism demonstrated stable significant associations with ADs risk in the dominant (OR = 1.57, 95%CI = 1.13–2.18, P = 0.007) and allelic (OR = 1.51, 95%CI = 1.10–2.08, P = 0.01) models, and with rheumatoid arthritis (RA) risk in the dominant (OR = 2.09, 95%CI = 1.60–2.73, P < 0.00001) and allelic (OR = 1.95, 95%CI = 1.51–2.51, P < 0.00001) models. Cumulative meta-analyses confirmed that these significant results were robust. Concerning TIM-3 −574 G > T or −1541C>T polymorphism, no significant associations were detected. Conclusion: These findings reveal that TIM-3 + 4259A>C might be a potential susceptible predictor of ADs and especially RA. Further functional and clinical investigation between these diseases and TIM-3 polymorphisms is warranted. [ABSTRACT FROM AUTHOR]

Background: Despite years of research, the treatment of acute kidney injury (AKI) remains a significant challenge. Animal studies presented causal links between elevated regulatory T cell (Treg) response and better prognosis in AKI. Previous studies in mice and humans showed that TIM-3+ Treg cells were more potent than TIM-3- Treg cells. In this study, we investigated the role of TIM-3 in Treg in AKI patients. Methods: Peripheral blood from AKI patients and healthy controls were gathered, and TIM-3+ Treg subset was examined. Results: Compared to healthy controls, the AKI patients presented a significant upregulation in the frequency of circulating CD4+CD25+ T cells; however, the majority of this increase was from the CD4+CD25+TIM-3- subset, and the frequency of CD4+CD25+TIM-3+ T cells was downregulated in AKI patients. In both healthy controls and AKI patients, the CD4+CD25+TIM-3+ T cells expressed higher levels of Foxp3, and were more potent at expressing LFA-1, LAG-3, CTLA-4, IL-10 and TGF-β. In addition, the CD4+CD25+TIM-3+ T cells from both healthy controls and AKI patients presented higher capacity to suppress CD4+CD25- T cell proliferation than the CD4+CD25+TIM-3- T cells. Interestingly, the total CD4+CD25+ T cells from AKI patients presented significantly lower inhibitory capacity than those from healthy controls, indicating that the low frequency of CD4+CD25+TIM-3+ T cells was restricting the efficacy of the Treg responses in AKI patients. Conclusions: We demonstrated that TIM-3 downregulation impaired the function of Treg cells in AKI. The therapeutic potential of CD4+CD25+TIM-3+ T cells in AKI should be investigated in future studies. [ABSTRACT FROM AUTHOR]

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog with binding affinity to MHC class II molecules. It is thought that LAG-3 exerts a bimodal function, such that co-ligation of LAG-3 and CD3 could deliver an inhibitory signal in conventional T cells, whereas, on regulatory T cells, LAG-3 expression could promote their inhibitory function. In this study, we investigated the role of LAG-3 expression on CD4+ T cells in patients with long bone fracture. We found that LAG-3+ cells represented approximately 13% of peripheral blood CD4+ T cells on average. Compared to LAG-3− CD4+ T cells, LAG-3+ CD4+ T cells presented significantly higher Foxp3 and CTLA-4 expression. Directly ex vivo or with TCR stimulation, LAG-3+ CD4+ T cells expressed significantly higher levels of IL-10 and TGF-β than LAG-3− CD4+ T cells. Interestingly, blocking the LAG-3-MHC class II interaction actually increased the IL-10 expression by LAG-3+ CD4+ T cells. The frequency of LAG-3+ CD4+ T cell was positively correlated with restoration of healthy bone function in long bone fracture patients. These results together suggested that LAG-3 is a marker of CD4+ T cells with regulatory function; at the same time, LAG-3 might have limited the full suppressive potential of Treg cells. [ABSTRACT FROM AUTHOR]

T-cells play an important role in immunity but when these cells are overexposed to specific antigens, their function may decline. This state is usually referred to as exhaustion and the T-cells show reduced proliferation and functions such as cytokine release. T-cell exhaustion has been observed in several cancers as well as in chronic infections such as tuberculosis (TB). In chronicMycobacterium tuberculosis(Mtb)infection, T-cells may express the exhaustion phenotype and show a progressive loss of secretion of IL-2, IFN-γ and TNF-α. In some cancers and chronic infection models, blocking the exhaustion phenotype can be achieved with the so-called checkpoint inhibitors. This results in tumor control and more effective immunity. However, in the case of TB, the T-cell exhaustion results are quite ambiguous. Hence, there is a need to investigate and explain the contribution of checkpoint at a molecular level to the outcome of events in chronic TB. Such information could help to guide the success of new therapies against chronic TB. This review highlights the mechanism through which T-cells undergo exhaustion and the approaches that can avert such events. This will help to design immunotherapies that can reinvigorate T-cell potency to protect patients from TB. [ABSTRACT FROM AUTHOR]

T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) is an important immune regulatory molecule in cancer immune system. However, expression and function of Tim-3 in monocytes/macrophages in cancer progression mainly remain unclear. In this study, we analyzed Tim-3 levels in peripheral blood mononuclear cells (PBMCs) from 62 gastric cancer patients and 45 healthy controls using flow cytometry and then associated Tim-3 levels with clinical pathological data from patients. We found Tim-3 level was significantly upregulated in monocytes from gastric cancer patients compared with those from healthy controls, and that upregulated Tim-3 levels associated with depth of tumor invasion and tumor lymph node metastasis and advanced clinical stages of gastric cancer patients. Furthermore, tumor-bearing mouse experiments revealed that Tim-3 level on monocytes/macrophages associated with xenograft formation and growth. In addition, culture of monocytes from healthy controls with gastric cancer cell-conditioned medium upregulated Tim-3 expression, but IL-10, TNF-α, IFN-γ, or GMCSF treatment or T-bet, Eomes, and T-bet/Eomes double gene knockout did not affect Tim-3 levels in blood monocytes/macrophages from human or mouse, respectively. Gal-9/Tim-3 signal was able to significantly stimulate monocyte to secrete IL-6, IL-8, and IL-10, but not IL-1β, IL-12p70, or TNF-α in presence of LPS. In conclusion, our study demonstrated that Tim-3 expressed by monocyte/macrophages might be an important mechanism in gastric cancer progression. [ABSTRACT FROM AUTHOR]

Aim: To investigate the clinical significance of Tim-3 (T-cell immunoglobulin- and mucin-domain-containing molecule 3) expression in natural killer (NK) cells from patients with gastric cancer. Materials and methods: Sixty-two patients with gastric cancer and 32 healthy controls were recruited for this study. Tim-3 expression in peripheral blood samples was analyzed using flow cytometry. The expression pattern of Tim-3 on NK cells was also confirmed using a gastric cancer-bearing mouse model. To further investigate the mechanisms that regulate Tim-3 expression,T-bet−/−, Eomes−/−, andEomes/T-betdouble knockout mice were utilized. Additionally, we statistically analyzed the clinical significance of Tim-3 expression on NK cells. Results: We found that the levels of Tim-3 in NK cells obtained from patients with gastric cancer were significantly higher than the levels in healthy controls. Clinical analyses showed that Tim-3 levels on NK cells were associated with advanced tumor stage. In a tumor-bearing mouse model, Tim-3 levels in NK cells increased with tumor growth, indicating that tumor progression could induce Tim-3 expression in NK cells. Finally, we report that T-bet is a key factor involved in regulating Tim-3 expression. Conclusion: Our data indicate that Tim-3 expression on NK cells is regulated by T-bet, and that Tim-3 levels correlate with advanced stages of gastric cancer. [ABSTRACT FROM PUBLISHER]

Objective and design: We investigated the involvement of Th17 cells and T-cell immunoglobulin and mucin domain 3 (TIM-3) in Guillain-Barré syndrome (GBS) in comparison to healthy subjects. Materials and subjects: Peripheral blood samples were obtained from 29 healthy subjects and 29 GBS patients. Treatment: Peripheral blood mononuclear cells (PBMCs) and CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAbs, in the absence or presence of anti-TIM-3 mAb. Methods: mRNA levels of TIM-3 and the transcription factor retinoic acid-related orphan receptor γt (RORγt) were determined by RT-PCR and were expressed relative to β-actin mRNA (housekeeping gene). Serum IFN-γ and IL-17 levels were determined by ELISA. Results: Compared to controls, relative TIM-3 mRNA levels were lower in both stimulated and unstimulated PBMCs from GBS patients. Unstimulated GBS CD4+ T cells and GBS CD4+ T cells stimulated with anti-CD3 and CD28 mAbs had higher relative RORγt mRNA expression compared to controls. GBS CD4+ T cells secreted significantly more IFN-γ and IL-17 in the presence of anti-TIM-3 mAb. GBS patients had (1) higher numbers of Th17, but not Th1 or Th2 cells in peripheral blood and (2) higher serum concentrations of IFN-γ and IL-17 compared to controls. Conclusion: TIM-3 may inhibit Th17 cell activation, thereby modulating their cytokine secretion patterns. Th17 cell differentiation, IL-17 levels, and TIM-3 regulation may be involved in the pathogenesis of GBS. [ABSTRACT FROM AUTHOR]

T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is preferentially expressed on Th1-helper type T-cells and functions to repress the Th1-mediated immune response. However, the role of Tim-3 during the inflammatory pathogenesis of asthma remains unclear. This study determines the expression level of Tim-3 in CD4+ T-cells within the peripheral blood and bronchoalveolar lavage fluid (BALF) isolated from a murine model of atopic asthma and explores the potential role of Tim-3 during the inflammatory response. Mice were randomly divided into normal control, asthma day 1, and asthma day 7 groups, and peripheral blood T lymphocytes and BALF cells were collected. The ratio of Tim-3+/CD4+ cells among the total CD4+cell populations from peripheral blood and BALF was determined by flow cytometry, and the expression of the Tim-3 mRNA was determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In contrast with the normal control group, the ratio of Tim-3+/CD4+:CD4+ cells and the level of Tim-3 mRNA in both the peripheral blood T lymphocytes and BALF cells among the asthma day 1 and asthma day 7 groups were significantly increased ( p < 0.01), and those in the asthma day 7 group were higher than the asthma day 1 group ( p < 0.05). There was also a positive correlation between the ratio of Tim-3+/CD4+:CD4+ detected in BALF and that the ratio detected in peripheral blood T lymphocytes (r = 0.84, p < 0.01). Therefore, the expression of Tim-3 is increased in CD4+ T-cells following airway challenge and likely affects asthma-induced inflammation by repressing the Th1-mediated immune response. [ABSTRACT FROM AUTHOR]

Purpose : To assess the expression of T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) in the spleens of BALB/c mice undergoing anterior chamber-associated immune deviation (ACAID). Methods : ACAID was generated after intracameral (i.c.) injection of ovalbumin (OVA) into BALB/c mice and evaluated by assessing the delayed-type hypersensitivity (DTH) response following a subsequent subcutaneous (s.c.) injection of OVA emulsified in complete Freund's adjuvant (CFA) on Days 0, 3, 7, 14, 21 and 28. Tim-3 mRNA levels were detected using real-time RT-PCR, and the frequency of CD4 + Tim-3 + T cells in splenocytes as well as the coexpression of Tim-3 with CD25 on CD4 + T cells were assessed by flow cytometry. Results : A significantly suppressed DTH response was observed on Days 7, 14, 21, and 28, but not on Days 0 and 3 during the development of ACAID. The levels of Tim-3 mRNA and the frequency of CD4 + CD25 + Tim-3 + T cells in the splenocytes reached a peak on Day 3, declined on Day 7, and returned to basal levels thereafter. Conclusions : A transient upregulation of Tim-3 expression was observed in the early stage of ACAID, suggesting its possible involvement in the development of ACAID. [ABSTRACT FROM AUTHOR]

We identified novel mechanisms whereby TIM-3 suppresses innate immunity as induced by nucleic acids. Interaction of TIM-3 with HMGB1 inhibits the recruitment of nucleic acids to the endosomal compartment of dendritic cells, impairing the transduction of innate immune signals. Thus, TIM-3 is an effective target for enhancing the immunogenicity of nucleic acids in the context of cancer therapy. [ABSTRACT FROM AUTHOR]

Nivolumab, a monoclonal antibody targeting PD-1, is currently approved for metastatic non-small cell lung cancer (mNSCLC) treatment after failure of first-line chemotherapy. However, only a quarter of patients benefit from this therapy with objective clinical response. In this context, there is an unmet need for improved understanding of resistance mechanisms. Thus, we studied a prospective cohort of mNSCLC (n = 61) treated in second or third-line with nivolumab. We analyzed various blood myeloid and lymphoid markers by flow cytometry (176 variables) at baseline, and after 15 and 30 days of therapy. By attempting to link the evolution of peripheral lymphoid, myeloid cells and anti-PD-1 response, we observed that accumulation of lymphoid cells and monocytic MDSC (mMDSC) expressing, respectively, Tim-3 and galectin-9 is implicated in resistance to PD-1 blockade both for patients with primary or acquired secondary resistance to anti-PD-1. In vitro, anti-Tim-3 blocking antibody reverses resistance to anti-PD-1 in PBMC from lung cancer patients and high levels of blood mMDSC negatively impact on anti-PD-1 efficacy. Together, these data underline that the galectin-9/Tim-3 pathway and mMDSC are key mechanisms of primary or secondary resistance to anti-PD-1 and could be a new target for immunotherapy drug combinations. [ABSTRACT FROM AUTHOR]

Suppression of immune reactivity by increased expression of co-inhibitory receptors has been discussed as a major reason as to why the immune system fails to control tumor development. Elucidating the co-inhibitory expression pattern of tumor-infiltrating lymphocytes in different cancer types will help to develop future treatment strategies. We characterized markers reflecting and affecting T-cell functionality by flow cytometry on lymphocytes isolated from blood, ascites and tumor from advanced ovarian cancer patients (n = 35). Significantly higher proportions of CD4+ and CD8+ T-cells expressed co-inhibitory receptors LAG-3, PD-1 and TIM-3 in tumor and ascites compared to blood. Co-expression was predominantly observed among intratumoral CD8+ T-cells and the most common combination was PD-1 and TIM-3. Analysis of 26 soluble factors revealed highest concentrations of IP-10 and MCP-1 in both ascites and tumor. Correlating these results with clinical outcome revealed the proportion of CD8+ T-cells without expression of LAG-3, PD-1 and TIM-3 to be beneficial for overall survival. In total we identified eight immune-related risk factors associated with reduced survival. Ex vivo activation showed tumor-derived CD4+ and CD8+ T-cells to be functionally active, assessed by the production of IFN-γ, IL-2, TNF-α, IL-17 and CD107a. Blocking the PD-1 receptor resulted in significantly increased release of IFN-γ suggesting potential reinvigoration. The ovarian tumor environment exhibits an inflammatory milieu with abundant presence of infiltrating immune cells expressing inhibitory checkpoints. Importantly, we found subsets of CD8+ T-cells with double and triple expression of co-inhibitory receptors, supporting the need for multiple checkpoint-targeting agents to overcome T-cell dysfunction in ovarian cancer. [ABSTRACT FROM AUTHOR]

Tumor-infiltrating lymphocytes (TILs) are predominantly present in breast cancer patients with estrogen receptor negative tumors, among whom increasing levels correlate with favorable outcomes. Nevertheless, currently available immune checkpoint inhibitors appear to benefit only a small number of women with breast cancer. Upregulation of additional immune checkpoint markers is one mechanism of resistance to current inhibitors that might be amenable to targeting with newer agents. T-cell Immunoglobulin and Mucin domain-containing molecule 3 (TIM-3) is an immune checkpoint receptor that is an emerging target for cancer immunotherapy. We investigated TIM-3 immunohistochemical expression in 3,992 breast cancer specimens assembled into tissue microarrays, linked to detailed outcome, clinico-pathological parameters and biomarkers including CD8, PD-1, PD-L1 and LAG-3. We scored and reported absolute counts for TIM-3+ intra-epithelial and stromal TILs (iTILs and sTILs), and find that breast cancer patients with TIM-3+ iTILs (≥ 1) represent a minority of cases (11%), with a predilection for basal-like breast cancers (among which 28% had TIM-3+ iTILs). TIM-3+ sTILs (≥ 2) represented 20% of cases and included more non-basal cases. The presence of TIM-3+ iTILs highly correlates with hematoxylin and eosin-stained stromal TILs and with other immune checkpoint markers (PD-1+ iTILs, LAG-3+ iTILs and PD-L1+ tumors). In prognostic analyses, early breast cancer patients with TIM-3+ iTILs have significantly improved breast cancer-specific survival whereas TIM-3+ sTILs did not reach statistical significance. In multivariate analyses, the presence of TIM-3+ iTILs is an independent favorable prognostic factor in the whole cohort as well as among ER negative patients. Our study supports TIM-3 as a target for breast cancer immunotherapy. [ABSTRACT FROM AUTHOR]

High mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1β) production by healthy leukocytes, leading to IL-1β-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis. [ABSTRACT FROM AUTHOR]

Bothin vivodata in preclinical cancer models andin vitrodata with T cells from patients with advanced cancer support a role for Tim-3 blockade in promoting effective anti-tumor immunity. Consequently, there is considerable interest in the clinical development of antibody-based therapeutics that target Tim-3 for cancer immunotherapy. A challenge to this clinical development is the fact that several ligands for Tim-3 have been identified: galectin-9, phosphatidylserine, HMGB1, and most recently, CEACAM1. These observations raise the important question of which of these multiple receptor:ligand relationships must be blocked by an anti-Tim-3 antibody in order to achieve therapeutic efficacy. Here, we have examined the properties of anti-murine and anti-human Tim-3 antibodies that have shown functional efficacy and find that all antibodies bind to Tim-3 in a manner that interferes with Tim-3 binding to both phosphatidylserine and CEACAM1. Our data have implications for the understanding of Tim-3 biology and for the screening of anti-Tim-3 antibody candidates that will have functional propertiesin vivo. [ABSTRACT FROM AUTHOR]

Background: Researches on immunotherapy of glioma has been increasing exponentially in recent years. However, autoimmune-like side effects of current immune checkpoint blockade hindered the clinical application of immunotherapy in glioma. The discovery of the TIM-3, a tumor-specific immune checkpoint, has shed a new light on solution of this dilemma. We aimed at investigating the role of TIM-3 at transcriptome level and its relationship with clinical practice in glioma. Methods: A cohort of 325 glioma patients with RNA-seq data from Chinese Glioma Genome Atlas (CGGA project) was analyzed, and the results were well validated in TCGA RNA-seq data of 699 gliomas. R language was used as the main tool for statistical analysis and graphical work. Results: TIM-3 was enriched in glioblastoma (the most malignant glioma) and IDH-wildtype glioma. TIM-3 can act as a potential marker for mesenchymal molecular subtype according to TCGA transcriptional classification scheme in glioma. TIM-3 was closely related to immune functions in glioma, especially T cell mediated immune response to tumor cell and T cell mediated cytotoxicity directed against tumor cell target. Moreover, TIM-3 and PD-L1 played almost exactly the same inflammatory activation functions in glioma. Clinically, high expression of TIM-3 was an independent indicator of poor prognosis. Conclusion: The expression of TIM-3 is closely related to the pathology and molecular pathology of glioma. Meanwhile, in glioma TIM-3 plays a specific role in T cell tumor immune response. Therefore, TIM-3 is a promising target for immunotherapeutic strategies, providing an alternative treatment when glioma gains resistance to antibodies of PD-1/PD-L1. [ABSTRACT FROM AUTHOR]

Despite the success of immune checkpoint blockade in melanoma, the majority of patients do not respond. We hypothesized that the T and NK cell subset frequencies and expression levels of their receptors may predict responses and clinical outcome of anti-CTLA-4 treatment. We thus characterized the NK and T cell phenotype, as well as serum levels of several cytokines in 67 melanoma patients recruited in Italy and Sweden, using samples drawn prior to and during treatment. Survival correlated with low expression of the inhibitory receptor TIM-3 on circulating T and NK cells prior to and during treatment and with the increased frequency of mature circulating NK cells (defined as CD3−CD56dimCD16+) during treatment. Survival also correlated with low levels of IL-15 in the serum. Functional experimentsin vitrodemonstrated that sustained exposure to IL-15 enhanced the expression of PD-1 and TIM-3 on both T and NK cells, indicating a causative link between high IL-15 levels and enhanced expression of TIM-3 on these cells. Receptor blockade of TIM-3 improved NK cell-mediated elimination of melanoma metastasis cell linesin vitro. These observations may lead to the development of novel biomarkers to predict patient response to checkpoint blockade treatment. They also suggest that induction of additional checkpoints is a possibility that needs to be considered when treating melanoma patients with IL-15. [ABSTRACT FROM AUTHOR]

Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors that are expressed on tumor-infiltrating lymphocytes (TIL) in tumor-bearing mice and humans. As anti-PD-1 single agent response rates are only <20% in head and neck squamous cell carcinoma (HNSCC) patients, it is important to understand how multiple inhibitory checkpoint receptors maintain suppressed cellular immunity. One such receptor, Tim-3, activates downstream proliferative pathways through Akt/S6, and is highly expressed in dysfunctional TIL. We observed that PD-1 and Tim-3 co-expression was associated with a more exhausted phenotype, with the highest PD-1 levels on TIL co-expressing Tim-3. Dampened Akt/S6 phosphorylation in these PD-1+Tim-3+TIL, when the PD-1 pathway was ligated, suggested that signaling cross-talk could lead to escape through Tim-3 expression. Indeed, PD-1 blockade of human HNSCC TIL led to further Tim-3 upregulation, supporting a circuit of compensatory signaling and potentially permitting escape from anti-PD-1 blockade in the tumor microenvironment. Also, in a murine HNC tumor model that is partially responsive to anti-PD-1 therapy, Tim-3 was upregulated in TIL from persistently growing tumors. Significant antitumor activity was observed after sequential addition of anti-Tim-3 mAb to overcome adaptive resistance to anti-PD-1 mAb. This increased Tim-3-mediated escape of exhausted TIL from PD-1 inhibition that was mediated by phospho-inositol-3 kinase (PI3K)/Akt complex downstream of TCR signaling but not cytokine-mediated pathways. Taken together, we conclude that during PD-1 blockade, TIL upregulate Tim-3 in a PI3K/Akt-dependent manner, providing further support for dual targeting of these molecules for more effective cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors highly expressed on tumor infiltrating T lymphocytes (TIL). PD-1 inhibits T cell activation and type-1 T cell responses, while Tim-3 is proposed to mark more extensively exhausted cells, although the mechanisms underlying Tim-3 function are not clear. Trials of anti-PD-1 therapy have identified a large subset of non-responder patients, likely due to expression of alternative checkpoint molecules like Tim-3. We investigated the phenotypic and functional characteristics of T cells with differential expression of PD-1 (high/low) and Tim-3 (positive/negative), using TIL directly isolated from head and neck squamous cell carcinomas (HNSCC). Unexpectedly, we found that expression of Tim-3 alone does not necessarily mark TIL as dysfunctional/exhausted. In Tim-3-TIL, PD-1 levels correlate with T cell dysfunction, with a PD-1low/intermedphenotype identifying recently activated and still functional cells, whereas PD-1hiTim-3−T cells are actually exhausted. Nonetheless, PD-1intermedcells are still potently suppressed by PD-L1. PD-1 expression was associated with reduced phosphorylation of ribosomal protein S6 (pS6), whereas Tim-3 expression was associated with increased pS6. Using a novel mouse model for inducible Tim-3 expression, we confirmed that expression of Tim-3 does not necessarily render T cells refractory to further activation. These results suggest the existence of PD-1 and Tim-3 crosstalk in regulating antitumor T cell responses, with important implications for anti-PD-1 immunotherapy. [ABSTRACT FROM PUBLISHER]

T cell Ig mucin-3 (Tim-3), an immune checkpoint inhibitor, shows therapeutic potential. However, the molecular mechanism by which Tim-3 regulates immune responses remains to be determined. In particular, very little is known about how Tim-3 works in innate immune cells. Here, we demonstrated that Tim-3 is involved in the development of tumor-promoting M2 macrophages in colon cancer. Manipulation of the Tim-3 pathway significantly affected the polarization status of intestinal macrophages and the progression of colon cancer. The Tim-3 signaling pathway in macrophages was explored using microarray, co-immunoprecipitation, gene mutation, and high-content analysis. For the first time, we demonstrated that Tim-3 polarizes macrophages by directly binding to STAT1 via residue Y256 and Y263 in its intracellular tail and inhibiting the STAT1-miR-155-SOCS1 signaling axis. We also identified a new signaling adaptor of Tim-3 in macrophages, and, by modulating the Tim-3 pathway, demonstrated the feasibility of altering macrophage polarization as a potential tool for treating this kind of disease. [ABSTRACT FROM PUBLISHER]

The immune receptor Tim-3 is often highly expressed in human acute myeloid leukemia (AML) cells where it acts as a growth factor and inflammatory receptor. Recently, it has been demonstrated that Tim-3 forms an autocrine loop with its natural ligand galectin-9 in human AML cells. However, the pathophysiological functions of Tim-3 in human AML cells remain unclear. Here, we report for the first time that Tim-3 is required for galectin-9 secretion in human AML cells. However, this effect is cell-type specific and was found so far to be applicable only to myeloid (and not, for example, lymphoid) leukemia cells. We concluded that AML cells might use Tim-3 as a trafficker for the secretion of galectin-9 which can then be possibly used to impair the anticancer activities of cytotoxic T cells and natural killer (NK) cells. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) infection is one of the most important etiologic causes of oropharyngeal head and neck squamous cell carcinoma (HNSCC). Patients with HPV-positive HNSCC were reported to have a better clinical outcome than patients with HPV-negative cancers. However, little is known about the possible causes of different clinical outcomes. In this study, we analyzed a detailed immune profile of tumor samples from HNSCC patients with respect to their HPV status. We analyzed the characteristics of immune cell infiltrates, including the frequency and distribution of antigen-presenting cells and naïve, regulatory and effector T cells and the cytokine and chemokine levels in tumor tissue. There was a profound difference in the extent and characteristics of intratumoral immune cell infiltrates in HNSCC patients based on their HPV status. In contrast to HPV-negative tumor tissues, HPV-positive tumor samples showed significantly higher numbers of infiltrating IFNγ+CD8+T lymphocytes, IL-17+CD8+T lymphocytes, myeloid dendritic cells and proinflammatory chemokines. Furthermore, HPV-positive tumors had significantly lower expression ofCox-2mRNA and higher expression of PD1 mRNA compared to HPV-negative tumors. The presence of a high level of intratumoral immune cell infiltrates might play a crucial role in the significantly better response of HPV-positive patients to standard therapy and their favorable clinical outcome. Furthermore, characterization of the HNSCC immune profile might be a valuable prognostic tool in addition to HPV status and might help identify novel targets for therapeutic strategies, including cancer immunotherapy. [ABSTRACT FROM PUBLISHER]

Natural killer (NK) cells are innate immune cells that become progressively exhausted in advanced stage cancer, crippling their ability to execute antitumor functions. We previously characterized the nature of NK cell exhaustion in metastatic melanoma patients, reporting a correlation with high expression of TIM-3. Blockade of this immune checkpoint molecule reversed the exhausted phenotype and improved NK cell function. [ABSTRACT FROM PUBLISHER]