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@#[Abstract] Objective: To explore the anti-tumor activity of MUC16-targeted chimeric antigen receptor modified NK-92 (CARNK-92) cells against ovarian cancer. Methods: The expression of MUC16 in surgically resected tumor tissues of 15 patients with ovarian cancer treated in the Department of Obstetrics and Gynecology of Qingyang Hospital of Traditional Chinese Medicine and 4 ovarian tumor cell lines was detected by Immunohistochemistry and Flow cytometry. MUC CAR sequence was synthesized by gene synthesis, and its lentivirus expression vector were constructed. CARNK-92 cells targeting MUC16 (MUC-BBz) were obtained by lentivirus infection. The expression of CD107a in MUC-BBz was detected by Flow cytometry. The activation of MUC-BBz cells and its cytotoxicity against SKOV3 target cells were characterized by the release of LDH assay. The xenograft nude mouse model of SKOV3 cells was established to verify the in vivo anti-tumor activity of MUC-BBz cells. Results: MUC16 was highly expressed in ovarian cancer tissues and human ovarian cancer cells. MUC-BBz was successfully constructed by infecting NK-92 cells with lentivirus, with a positive rate of (42.79±2.58)%. MUC-BBz could be specifically activated by MUC16 over-expressing tumor cells. After co-incubation of effector cells and target cells, the expression of CD107a on MUC-BBz was upregulated significantly (P<0.01), and the ability of MUC-BBz secreting cytokines IFN-γ and perforin also increased (all P<0.01). The LDH test indicated that with the increase of effector-target ratio, the cytotoxicity of MUC-BBz against 4 ovarian cancer cells (hey, COC1, SKOV3 and A2780) also significantly enhanced. The results of transplanted tumor model showed that transfusion of MUC-BBz could significantly inhibit the growth of SKOV3 xenograft in mice (P<0.01). Conclusion: The CARNK-92 cells can significantly inhibit the growth of ovarian cancer cells in vitro and in vivo, which provides an important basis for further evaluation of its clinical application.
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Objective To analyze the possibility of using intracellular cytokine staining (ICS) to evaluate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and to detect the changes in ADCC activity among patients with chronic HIV and/or HCV infection. Methods Flow cytometry was per-formed to determine the percentages of NK cells and the expression of NK cell receptors. ImageStreamX MarkⅡ system was used to identify the expression of CD3, CD56, CD16 and CD32 on CD56brightNK and CD56dimNK subsets. Degranulation process and cytokine production in NK cells were detected using an anti-gen-antibody complex model of P815/Ab in combination with ICS. Differences in NK cell-mediated ADCC were evaluated among patients infected with HIV and/or HIV and healthy subjects by flow cytometry. Re-sults The percentages of CD107a+and IFN-γ+NK cells were positively correlated with the decrease of mean fluorescence intensity (MFI) of CD16. ICS assay revealed a positive correlation between the secretion of CD107a and IFN-γ by NK cells. CD16 was highly expressed in CD56dimNK cells. The ADCC mediated by CD56dimNK cells was stronger than that mediated by CD56brightNK cells. The rate of target cell lysis detected by rapid fluorescence assay was positively correlated with the percentage of CD107a+/IFN-γ+NK cells meas-ured by ICS. NK cell-mediated ADCC was suppressed in patients with chronic HIV and/or HCV infection. Conclusion This study suggests that ICS assay could be used to evaluate NK cell-mediated ADCC. It also reveals that NK cell-mediated ADCC is suppressed in patients with chronic HIV and/or HCV infection.
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Objective:To explore the relationship between CD244 and the phenotype and function of CD56bright NK cells of patients with active pulmonary tuberculosis.Methods: PBMCs were isolated from peripheral blood by density gradient centrifugation.The expression of CD244,CD94,NKG2D on the CD56bright NK cells from the active pulmonary tuberculosis patients and healthy controls was detected by flow cytometry.And then analyzed the relationship of the expression of CD244 with Tim3,CD27,CD62L,CCR7,IFN-γ and CD107a in CD56bright NK cells by flow cytometry.Results: The expression of CD244 on the CD56bright NK cells showed no significant difference between the patients with active pulmonary tuberculosis and healthy controls without MTB antigen.The expression of CD244 was significantly increased on CD56bright NK cells of patients with tuberculosis stimulated with MTB antigen.The expression of CD94 and NKG2D on CD56bright NK cells showed no difference between patients and healthy controls.The proportion of Tim3+ cells in CD244+CD56bright NK cells was significantly higher than CD244-CD56bright NK cells.While the expression of CD62L and IFN-γ decreased significantly in CD244+CD56bright NK cells.The expression of CD107a on CD56bright NK cells was not significantly different between CD244+ cells and CD244-cells.Conclusion: The expression of CD244 on CD56bright NK cells in patients with active pulmonary tuberculosis increased significantly,maybe inhibit IFN-γ co-work with Tim3.CD244 has nothing to do with degranulation of CD56bright NK cells.
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Objective To investigate effect of interferon gemma on immune factor and CD69, CD107a in patients with delayed type hypersensitivity by interferon gamma.Methods 76 cases with delayed type hypersensitivity were selected and divided into 2 groups.38 cases in control group were treated conventional therapy, 38 cases in experiment group were treated by interferon gamma.Peripheral blood CD69, CD107a, immune factor, T cell subsets and the treatment efficiency were compared after the treatment.Results Compared with the control group, the serum CD69 and CD107a levels were lower in the experimental group (P<0.05), serum IgG and IgA levels were higher, the serum IgE level was lower (P<0.05), and the CD3 +,CD4 +,CD4 +/CD8 +level was higher, serum CD8 + was lower (P<0.05) .The effective rate of the treatment group was significantly higher than that of the control group (P<0.05) .Conclusion Interferon gamma has good clinical effect in the patients with delayed type hypersensitivity, and can effectively reduce the levels of CD69 and CD107a in the peripheral blood, and regulate the immune function of the body.
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Objective:To explore the changes of NK and NKT cells and the expression levels of their activated, inhibitory receptors in the peripheral blood of patients with newly diagnosed rheumatoid arthritis ( RA), and to reveal the potiential role of NK and NKT cells played in the pathogenesis of RA.Methods:32 patients with new onset RA and 15 healthy controls were recruited.Activated and inhibitory NK and NKT cells in peripheral blood were quantified by flow cytometry.The frequency of spontaneous and stimulated IFN-γ+NK and NKT cells and CD107aγ+NK cells were examined.Finally,the potential relationship between the frequency of NK and NKT cells subsets and clinical indexes were analyzed.Results:The frequencies of NK cells in peripheral blood in RA patients were sig-nificantly lower than those in the controls ( P=0.026 ).The frequencies of NKG2D+, NKP46+activated NK cells and NKG2C+, NKG2D+,NKP46+activated NKT cells in RA patients were significantly higher than those in the controls (P=0.011,P=0.010,and P<0.001,P=0.032,P=0.001,respectively),whereas the frequencies of KIR2DL3+,KIR3DL1+and NKG2A+inhibitory NK cells and KIR2DL3+,NKG2A+inhibitory NKT cells in RA patients were significantly lower (P=0.002,P=0.002,P=0.014,and P=0.027,P=0.002,respectively).Moreover, the frequencies of stimulated IFN-γ+NK cells and IFN-γ+NKT cells, spontaneous and stimulated CD107 aγ+NK cells in RA patients were significantly higher than that in the controls ( P=0.037, P=0.004 and P=0.001, P=0.001, respectively).Furthermore,the frequencies of NK cells,NKG2Aγ+and KIR2DL3γ+inhibitory NK cells were correlated significantly with the values of DAS28 in RA patients (r=0.357,P=0.045;r=0.399,P=0.024;r=0.468,P=0.021,respectively).Conclusion:Lower frequencies of NK cells, higher frequencies of activated NK cells and activated NKT cells, lower frequencies of inhibitory NK cells and inhibitory NKT cells, and higher NK cell activity may induce the autoimmune reaction involved in the pathogenesis of RA.
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Hemophagocytic lymphohistiocytosis (HLH) in different ethnicities has been described in the literature, but few cases in patients of Chinese descent have been reported. Here, we describe the case of a Chinese neonate presenting with HLH carrying novel, compound heterozygous mutations of the UNC13D gene, including [c.2295_2298delGCAG, p.Glu765Aspfs*27] in exon 23, c.-250C>T, c.1+30G>A, c.279C>T, c.888G>C, c.18+36A>G, c.20-48T>C, c.1977C>T, c.2296C>T, c.24-46C>T, c.26-9_26-8insC, c.2599A>G, c.28+48C>T and c.3198A>G, some of which have not been reported in the literature. Cytokine profile analyses were performed in this patient, and the results were consistent with our previous findings in HLH patients. Cytokine profile monitoring may be helpful in differentiating among various clinical phases of HLH.
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Humans , Infant, Newborn , Male , Asian People/genetics , Cytokines/blood , Heterozygote , Lymphohistiocytosis, Hemophagocytic/drug therapy , Membrane Proteins/genetics , MutationABSTRACT
Natural killer (NK) cells,a major cell type of the innate immunity,have a central role in infections,immunizations and tumors.The relationship between NK cells and hematological malignancies is increasingly pursued by domestic and foreign researchers.Exploring the relationship between function and activity of NK cells and malignant hematological disease is of great importance.