The Expression And Function Of Tim3 And Its Ligand Galectin-9 In Patients With Acute Myeloid Leukemia

Objective: To dynamic analyze the expression and the role of costimulatory molecules Tim3 on natural killer cells(NK cells) and NK cell subsets as well as its ligands galectin-9 expression on blast cells in patients with acute myeloid leukemia(AML) before and after chemotherapy. Consistently explore the clinical significance of Tim3 on NK cells and whether Tim3/Galectin-9 pathway involved in immune escape in AML.Methods: Clinical data of patients diagnosed with AML by bone marrow MICM classification were collected. Anticoagulated peripheral blood before and after treatment was taken from each candidate. The heparin anticoagulation peripheral blood from healthy volunteers was gathered as normal control. We used the methods of immune fluorescence labeling and flow cytometry to dynamic analyze expression of Tim3 on NK cells and NK cell subsets. Secondly detected secretion of IFN-γ on NK cells. Lastly, galetin-9 expressions on blast cells in AML patients were detected by real-time quantify PCR.Results:(1)The proportion of NK cells on lymphocytes in AML patients before chemotherapy was(6.46±6.09)%, which was significantly lower than the proportion of normal controls [(13.53±6.43)%](P<0.001). The levels of Tim3 expression on NK cells in AML patients before chemotherapy was(45.45±20.66)%, which was significantly lower than the levels of normal controls [(59.38±16.11)%](P<0.001).(2)further CD3-CD56+NK cells were divided into two subgroups:CD56dim subsets and CD56 bright subsets. The proportion of CD56 dim subsets on CD56+NK cells was(93.74±3.16)%, which was significantly higher than the proportion of CD56 bright subsets[(13.53±6.43)%] in healthy volunteers(P<0.001). The levels of Tim3 expression on CD56 dim subsets was(62.11±16.51)%, which was significantly higher than the levels of CD56 bright subsets[(35.49±13.88)%] in normal controls(P<0.001).(3)The proportion of CD56 dim subsets on CD56+NK cells in AML patients before chemotherapy was(76.88±20.77)%, which was significantly lower than the proportion of normal controls[(93.74±3.16)%](P=0.008). The levels of Tim3 expression on CD56 dim subsets was(46.57 ± 18.34)%, which was significantly lower than the levels of normal controls[(62.11±16.51)%](P=0.027).(4)The proportion of CD56 bright subsets on CD56+NK cells in AML patients before chemotherapy was(22.87±22.05)%, which was significantly higher than the proportion of normal controls[(5.86±3.01)%](P=0.011). The levels of Tim3 expression on CD56 bright subsets was(48.52±21.80)%, which was higher than the levels of normal controls[(35.49±13.88)%], but the difference was not statistically significant(P=0.076).(5)The proportion of NK cells on lymphocytes in AML patients achieved complete remission(CR) after chemotherapy was(10.37±10.03)%, which was significantly increased compared with the proportion before treatment [(6.46±6.09)%](P=0.032). The levels of Tim3 expression on NK cells in patients achieved CR after chemotherapy was(56.88±22.67)%, which was significantly were significantly increased compared with the proportion before treatment [(45.45±20.66)%](P=0.026). The proportions of NK cells and Tim3 on NK cells were significantly related with the number of chemotherapy and remission time of disease.(6)The proportion of CD56 dim subsets on CD56+NK cells in AML patients achieved CR after chemotherapy was(90.86±2.99)%, which was significantly increased compared with the proportion before treatment [(76.88±20.77)%](P=0.008). The levels of Tim3 expression on NK cells in patients achieved CR after chemotherapy was(61.17±18.96)%, which was significantly increased compared with the proportion before treatment [(46.57±18.34)%](P=0.033).(7)The proportion of CD56 bright subsets on CD56+NK cells in AML patients achieved CR after chemotherapy was(7.79 ±2.26)%, which was significantly reduced compared with the proportion before treatment [(22.87±22.05)%](P=0.007). The levels of Tim3 expression on CD56 bright subsets in patients achieved CR after chemotherapy was(38.03±15.12)%, which was decreased compared with the proportion before treatment [(48.52±21.80)%],but the difference was not statistically significant(P=0.114).(8)The secretion of IFN-γ in Tim3+NK cells was significantly increased than Tim3-NK cells(P<0.05).(9)Galectin-9 expression levels on blast cells in AML patients were higher than peripheral blood mononuclear cells in healthy candidates(2-△ △ CT:3.13±2.25 vs 0.86±0.35)(P<0.001). The levels of galectin-9 was significantly associated with chemotherapy response and NCCN risk groups(P≤0.05).Conclusion: Dynamic analysis of changes in the the proportions of NK cells as well as NK cell subsets and the level of expression Tim3 in AML patients before and after chemotherapy prompt that Tim3 may mediate very important function in the pathogenesis of AML. A significant reduction in the proportion of CD56 dimNK cells and downregulation of Tim3 on CD56 dimNK cells in newly diagnosed AML patients suggest that decrease of the function of mature NK cells inhibit NK cell cytotoxicity on tumor cells and secretion of cytokines, thus mediate immune escape of leukemic cells. In addition, galectin-9 expression levels were up-regulated and was associated with poor prognosis in AML patients. Prompt Tim3/galectin-9 pathway is involved in mechanism of occurrence and development in AML. It is expected to become a potential new target for immunotherapy of AML.