The Reasearch On Expression And Mechanism Of SLAMF7 In Large Granular T Lymphocytic Leukemia

ObjectiveTo explore expression and molecular mechanism of SLAMF7 on tumor large granular T lymphocytes in the peripheral blood of patients with newly diagnosed large granular lymphocytic leukemia(LGLL),and elucidate the relationship between SLAMF7 and large granular T lymphocytes(T-LGL),providing potential targets for LGLL diagnosis and treatment.Methods1.The SLAMF7 expression level of large granular lymphocytes in the peripheral blood from LGLL patients was determined by flow cytometry.2.The expression levels of perforin and granzyme B in LGL from patients were determined by flow cytometry.The correlation between SLAMF7 and perforin/granzyme B was analyzed by Pearson correlation analysis.3.The morphological characteristics of LGL were observed by Wright-Giemsa staining.4.Peripheral Blood Mononuclear Cells(PBMC)from healthy people was isolated by Ficoll density gradient centrifugation.After treating PBMC with different concentrations(0.25,0.5,1,2?g/mL)of phytohemagglutinin(PHA)for different times(6,24,48,72 h),the expression of SLAMF7 on CD8~+T cells was detected by flow cytometry. 5.The chronic activated lymphocytes model(CALM)was established by continuously stimulating human PBMC with low concentration of PHA(0.5 ?g/mL).The expression characteristics of SLAMF7 on CD4~+T and CD8~+T cells were analyzed by flow cytometry. 6.The changes of p65,p-p65,STAT3,p-STAT3,mTOR and p-mTOR protein levels in CD8~+T cells in CALM at 24 and 48 h were detected by immunoblotting.7.After the CALM was treated with IKK-16(2,4?M)and Rapamycin(10,20 nM) for 24 and 48 h,the expression of SLAMF7 on CD8~+T cells was detected by flow cytometry.8.After treatment with IKK-16(4?M)for 48 h,the expression levels of SLAMF7 and Granzyme B of CD8~+T cells in CALM were detected by flow cytometry.Results1.Compared with the healthy control group,the expression of SLAMF7 in T-LGL of patients with large granular T lymphocytic leukemia was significantly increased;similarly,SLAMF7 was strongly expressed in both normal NK cells and NK-LGL of large granular NK lymphocytic leukemia patients.2.The morphological characteristics of LGL are as follows:larger cell size,increased cytoplasm,large and irregular nucleus within more or less nucleoli,and the most characteristic feature is the appearance of azurophilic granules with different size in slightly basophilic cytoplasm.3.In LGL,the expression of SLAMF7 was positively correlated with the expression levels of Granzyme B and Perforin,the main components of azurophilic granules.4.Compared with the control group,after PHA(0.5,1,2?g/mL)treated PBMC at different times(24,48,72 h),the expression level of SLAMF7 on CD8~+T cells was significantly increased.5.In CALM:the expression of SLAMF7 on CD4~+T cells began to increase at 24 h and reached the peak at 48 h,then decreased after 72 h;SLAMF7 on CD8~+T cells increased from 24 h and reached the peak at 48 h,then remained unchanged at 72h.6.After the CALM was cultured for 24 and 48 h,the phosphorylation levels of NF-?B/STAT3/mTOR signaling pathway-related proteins(p65,STAT3,and mTOR) were increased in a time-dependent manner.7.The treatment of IKK-16(2,4?M)for 24 and 48 h significantly reduced the expression level of SLAMF7 on CD8~+T cells in CALM,and the effect was mostobvious when treatment of IKK-16(4??)for 48 h.8.IKK-16(4?M)treated the CALM for 48 h significantly reversed the upregulation of SLAMF7 and granzyme B expressions in CD8~+T cells.ConclusionIn patients with LGLL,there was a high expression level of SLAMF7 on neoplastic large granular T lymphocytes,which was related to the over-activation of NF-?B signal pathway.And the expression level of SLAMF7 was positively correlated with the levels of perforin and granzyme B in T-LGL.These results indicated that SLAMF7 might be recognized as a characteristic marker of T-LGL and as one of the potential targets for clinical diagnosis and treatment of LGLL.