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Objective:To explore the expression levels, clinical significances and prognostic evaluation value of T-cell immunoglobulin mucin-3 (Tim-3) and galectin-9 (Gal-9) in bone marrow cells of patients with newly diagnosed acute lymphocytic leukemia (ALL).Methods:Bone marrow samples from 30 newly diagnosed ALL patients admitted to First Affiliated Hospital of Xinjiang Medical University from September 2016 to September 2018 were selected, and peripheral blood samples from 20 healthy volunteers during the same period in the First Affiliated Hospital of Xinjiang Medical University were treated as the controls. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect mRNA relative expression levels of Tim-3 and Gal-9. Differences in mRNA expression of Tim-3 and Gal-9 among ALL patients with varied clinicopathological characteristics were compared. Overall survival (OS) analysis was performed by using the Kaplan-Meier method, Cox proportional hazards model was used to make univariate and multivariate survival analysis.Results:mRNA relative expression levels of Tim-3 and Gal-9 in 30 newly diagnosed ALL patients were higher than those in the healthy control group (2.86±0.47 vs. 0.45±0.05, t = 21.65, P<0.05; 9.79±0.58 vs. 0.96±0.23, t = 63.24, P<0.05). mRNA relative expression level of Tim-3 had statistically significant differences in patients with different ages, France-America-Britain (FAB) Cooperative Group classification, hazard grades and central nervous system invasion (all P<0.01). There were statistically significant differences in mRNA relative expression level of Gal-9 for patients with different ages, FAB Cooperative Group classification, white blood cell count (WBC), central nervous system invasion and NOTCH1 mutation (all P<0.01). All patients were grouped by mRNA relative expression levels of Tim-3 and Gal-9, and patients in high Tim-3 expression group (≥2.86) had worse overall survival (OS) compared with that for patients in low Tim-3 expression group (<2.86) ( P = 0.048). Patients in high Gal-9 expression group (≥9.79) had worse OS compared with that for patients in low Gal-9 expression group (<9.79) ( P = 0.031). Moreover, the OS in Tim-3 and Gal-9 both high expression group was worse than that in Tim-3 and Gal-9 both low expression group and in the low expression group of either of them (all P<0.05). There was no statistically significant difference in OS between the high Tim-3 expression with low Gal-9 expression group and the high Gal-9 expression with low Tim-3 expression group ( P > 0.05). Multivariate Cox regression analysis revealed that peripheral blood WBC≥11.4×10 9/L, BCR-ABL gene mutation, central nervous system invasion, and high expression of Tim-3 and Gal-9 were independent risk prognostic factors of OS for newly diagnosed ALL patients (all P<0.05) . There was a positive correlation between the expression levels of Tim-3 and Gal-9 ( r = 0.788, P<0.01). Conclusions:The high expression of Tim-3 and its ligand Gal-9 are independent effecting factors of poor prognosis in newly diagnosed ALL patients. The expression levels of Tim-3 and Gal-9 can be served as a potential prognostic indicator for ALL patients.
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Objective • To detect T cell immunoglobulin mucin 3 (Tim-3) and galectin 9 (Gal-9) expression as well as CD3+ T cells and CD8+ T cells infiltration in the tumor tissues of adenocarcinoma of the esophagogastric junction (AEG), and analyze their correlations with the patients' clinical characteristics and survival prognosis. Methods • A retrospective case study was used to collect clinical data and follow-up data of 116 AEG patients who were admitted to Renji Hospital, Shanghai Jiao Tong University School of Medicine from Dec. 2005 to Dec. 2013. Tim-3, Gal-9, CD3, and CD8 protein expressions were detected by immunohistochemistry in the tumor tissues, and the clinical characteristics and prognosis were compared among the patients with different levels of protein expression and T cells infiltration. Results • The results of immunohistochemistry showed that Tim-3 mainly expressed in the infiltrating immune cells, and Gal-9 mainly expressed in the tumor cells. The analysis on the clinical characteristics revealed that Tim-3 expression level was related to the Siewert classification (P=0.030) and CD8+ T cells infiltration level was related to the tumor TNM stage (P=0.042). The results of survival analysis showed that the patients with high level of CD8+ T cells infiltration had a better survival prognosis (P=0.047). However, there was no difference in the prognosis among the patients with different Tim-3 and/or Gal-9 expression levels or with different CD3+ T cell infiltration levels. Conclusion • AEG patients with high level of CD8+ T cells infiltration usually have earlier TNM stages and better prognosis. There is no significant difference in the prognosis of AEG patients with different Tim-3/Gal-9 expression levels.
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Background: The occurrence of tumor is closely related to the function of immune system. As an effector cell of innate immunity, the function of γδ T cells is reported to be regulated by co-stimulatory molecules. T-cell immunoglobulin mucin-3 (Tim-3) and programmed death-1 (PD-1), two critical inhibitory co-stimulatory molecules, may affect the immune function of T lymphocytes via binding with their ligands, thus mediating the immune escape of tumor cells. Aims: To investigate the expressions and clinical significance of Tim-3 and PD-1 on γδ T cells in peripheral blood of colon cancer patients. Methods: Peripheral blood samples of 44 colon cancer patients were collected preoperatively at the First Affiliated Hospital of Soochow University from Dec. 2017 to Jun. 2018. Forty healthy volunteers were served as controls. The peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Expressions of Tim-3 and PD-1 on γδ T cells were detected by flow cytometry, and their correlations with tumor clinicopathological characteristics were analyzed. Results: The proportions of Tim-3+, PD-1+ and Tim-3+PD-1+ γδ T cells in peripheral blood of colon cancer patients were significantly higher than those of healthy volunteers (P0.05). Conclusions: Tim-3 and PD-1 are highly expressed on γδ T cells in peripheral blood of colon cancer patients and associated with the clinicopathological stage of tumor. Expressions of Tim-3 and PD-1 on peripheral blood γδ T cells might be the promising objective indicators for evaluating the development and progression of colon cancer.
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Objective To investigate the meaning and molecular mechanisms of Galectin-9/ T-cell immunoglobulin mucin-3 (Tim-3) pathway on lipopolysaccharide (LPS) induced murine macrophage M1/M2 subtype polarization.Methods The murine peritoneal macrophages RAW264.7 were cultured in vitro until the cells had matured with 80％-90％ fusion rate. ① The cells were cultured in serum-free medium and treated with 0 (blank control), 0.01, 0.1, 1, 10 and 100 mg/L LPS for 24 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) or Western Blot was used to determine the expressions of M1 macrophage markers such as interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and M2 macrophage markers such as arginase-1 (Arg-1), leukocyte differentiation antigen 206 (CD206), as well as Tim-3 and Galectin-9 in the cells. ② The other mice peritoneal macrophages were divided into blank control group (cultured in serum-free DMEM medium for 24 hours), LPS treatment group (cultured in serum-free DMEM medium containing 0.1 mg/L LPS for 24 hours) and α-lactose pretreatment group (pretreated with serum-free DMEM containing 40μmol/L Galectin-9 signal antagonist 1 hour before LPS stimulation). Over closed Galectin-9 signal was used to verify the role of Galectin-9 in macrophage M1/M2 subtype polarization.Results ① After stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L) for 24 hours, the expression of M1 markers was only slightly increased such as iNOS mRNA or not significantly changed such as IL-6 mRNA in macrophages, while the expressions of M2 markers such as Arg-1 mRNA and CD206 mRNA were significantly increased and peaked at LPS concentrations of 0.1 mg/L and 0.01 mg/L [compared with blank control group:Arg-1 mRNA (2-ΔΔCt) was 1.85±0.07 vs. 1.00±0.02, CD206 mRNA (2-ΔΔCt) was 2.03±0.11 vs.1.00±0.05, both P < 0.01]. With the increase of LPS concentration, the expressions of IL-6 mRNA and iNOS mRNA continued to increase, while the expressions of Arg-1 mRNA and CD206 mRNA were gradually decreased, and the macrophage M1/M2 subtype polarization status changed. At the same time, the level of Tim-3 protein in macrophages was significantly up-regulated after stimulation with 0.01 mg/L LPS as compared with that of blank control group (Tim-3/GAPDH:0.84±0.04 vs. 0.69±0.02,P < 0.01), peaked at LPS concentrations of 0.1 mg/L, and then decreased with increasing LPS concentration. The intracellular Galectin-9 and supernatant secreted Galectin-9 (s-Galectin-9) protein levels showed no significant change after stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L), while then gradually decreased with the increase of LPS concentration. ② Compared with blank control group, the mRNA expressions of M1 marker iNOS and M2 markers Arg-1 and CD206 were significantly increased in LPS treatment group, but IL-6 mRNA level was not changed significantly. The mRNA levels of IL-6 and iNOS were further elevated after pretreatment with α-lactose as compared with that of the LPS treatment group [IL-6 mRNA (2-ΔΔCt): 1.44±0.02 vs. 1.14±0.11, iNOS mRNA (2-ΔΔCt):2.45±0.04 vs. 2.01±0.08, bothP < 0.01], while the mRNA levels of Arg-1 and CD206 were significantly decreased [Arg-1 mRNA (2-ΔΔCt): 0.75±0.01 vs. 1.85±0.02, CD206 mRNA (2-ΔΔCt): 0.58±0.02 vs. 2.03±0.14, bothP < 0.01]. Meanwhile, the blocking of Galectin-9 signaling could also reduce the extracellular s-Galectin-9 (compared with LPS treatment group: s-Galectin-9/GAPDH was 0.10±0.01 vs. 0.23±0.02,P < 0.01), down-regulated the expressions of Tim-3 and Galectin-9 (Tim-3/GAPDH: 0.28±0.01 vs. 0.43±0.01, Galectin-9/GAPDH: 0.21±0.01 vs. 0.43±0.01, bothP < 0.01).Conclusions LPS regulates macrophage M1/M2 subtype polarization via Galectin-9/ Tim-3 signaling pathway. Low-doses of LPS can limit the development of inflammation by accommodating the expression and secretion of Galectin-9 to polarize macrophages to M2. High-doses of LPS promotes the development of inflammation by down-regulating the expression and secretion of Galectin-9 to polarize macrophages to M1.
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Objective To purify the anti-T cell immunoglobulin mucin ( TIM)-3 monoclonal antibody 4E8 and examine its biological function in vitro. Methods The mouse monoclonal antibody against human TIM-3, clone 4E8, was obtained by standard protocol for monoclonal antibody purification. The cell lines expressing human TIM-3 molecule were obtained by cell transfection technique. We ex-amined the ability of 4E8 binding to human TIM-3 by flow cytometry. The ability of 4E8 blocking the binding of fusion protein TIM-3 Ig-huFc with phosphatidylserine( PtdSer) , the apoptotic cell surface TIM-3 ligand, was also analyzed by flow cytometry. Mixed lympho-cyte reaction ( MLR) and ELISA assays were used to determine the effect of TIM-3 monoclonal antibody ( 4E8) on IFN-γsecretion in CD4+ T cells. Results 4E8 specifically bound to human TIM-3 but could not block the binding of TIM-3 to Ptdser. Compared with the negative control (IFN-γ secretion: 958.3±153.2), 4E8 enhanced the ability of CD4+ T cells to secrete IFN-γ in MLR (4E8 of 10μg/mL group:IFN-γ secretion 2563±150.3 and 4E8 of 3.33 μg/mL group:IFN-γ secretion 1981±211.5) with statistically signifi-cant difference ( P<0.05) . In addition, the combined application of 4E8 with the anti-programmed death-1 ( PD-1) monoclonal anti-body nivolumab showed synergistic effects for increasing IFN-γ secretion in MLR assay ( 4E8 of 10 μg/mL group: IFN-γ secretion 3049±80.5 and 4E8 of 0.33μg/mL group:IFN-γsecretion 1957±321.3) as compared with 4E8 alone (10μg /mL group:IFN-γse-cretion 2563±150.3 and 0.33 μg/mL group:IFN-γ secretion 844±76.2) with statistically significant difference (P<0.05). Conclu-sion We successfully obtained a 4E8 clone of monoclonal antibody to human TIM-3 which may enhance the capacity of IFN-γsecre-tion from CD4+ T cells. The effect of enhancing IFN-γ secretion of CD4+T cells by TIM-3 monoclonal antibody was independent from blocking the binding of TIM-3 with Ptdser.
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Objective:To detect the expression levels of T cell immunoglobulin mucin-3 (Tim-3) in peripheral blood natural killer (NK) cells of patients with oral squamous cell carcinoma (OSCC) and to study their biological and clinical significance.Methods:The expression of Tim-3 in the CD3-CD56 + NK cells was examined in 72 patients with OSCC and 40 heathy controls (HC) by flow cytometry.The correlation of Tim-3 expression in NK cells with clinicopathological parameters was analyzed.Results:The proportion of CD3-CD56 + NK cells was significantly decreased in OSCC patients as compared with HC [(9.30 ± 2.52)％ vs (17.36 ± 3.15)％,P ＜ 0.001].The percentage of Tim-3 + CD3-CD56 + NK cells in OSCC patients was higher than that in HC [(14.35 ± 6.35) ％ vs (1.78 ± 0.86) ％,P ＜ 0.001].High percentage of Tim-3 + CD3-CD56 + NK cells was associated with cell differentiation,lymphatic metastasis and pathological stage of OSCC (P ＜ 0.01).Conclusion:Lower proportion of NK cells and higher level of Tim-3 in peripheral blood NK cells may play a role in the development of OSCC.
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Objective To study the expression of T cell immunoglobulin mucin-3(Tim-3)in hepatocellular carcinoma(HCC)cell line and its influence on the oncobiological behavior of HCC cells.Methods The expression of Tim-3 mRNA and protein in human normal liver cell line L02 and HCC cell line HepG2 and SMMC7721 was assessed by FQ-PCR and Western blot.The siRNA Tim-3 fragments were designed to silence the Tim-3 gene expression in HepG2 cel1.HepG2 cells were transfected with siRNA by using LipofectamineTM 2000.The expression of Tim-3 protein was detected after transfection by Western blot to screen the effective siR-NA fragment.The proliferation and migration potential of hepG2 cells was evaluated after Tim-3 gene silence by the cell growth curve MTT assay and the wound healing assay.Results Both expressions of Tim-3 mRNA and protein in human HCC cell line HepG2 and SMMC7721 were significantly higher than those in normal liver cell line L02(P<0.05).After siRNA transfection,the protein expression of Tim-3 in experimental group was significantly lower than that of the control group.Compared with control group,the proliferative activity and the migration ability were decreased significantly.Conclusion Expressions of Tim-3 mRNA and protein are increased in HCC cell line.Tim-3 expression promotes HCC cell proliferation and migration.