Diagnostic value of Flt3L and Gas6 in post-chemotherapy infection in patients with non-Hodgkin′s lymphoma / 中华检验医学杂志

Objective:To investigate the diagnostic value of Flt3 ligand (Flt3L) and growth arrest specific protein 6 (Gas6) in post-chemotherapy infection of patients with non-Hodgkin′s lymphoma.Methods:A total of 94 NHL patients admitted to Wuhan No.1 Hospital for chemotherapy from July 2019 to October 2020 were collected, and divided into infection group ( n=40) and non-infection group ( n=54). Fifty healthy subjects at the same period were selected as the control group to analyze the expression levels of Flt3L, Gas6 and general inflammatory indicators among the three groups. The relationship between Flt3L, Gas6 and general inflammatory indicators were analyzed. The predictive value of Flt3L and Gas6 for post-chemotherapy infection in NHL patients was analyzed by ROC curve. Kaplan-Meier curve was used to analyze the infection status of NHL chemotherapy patients with serum Flt3L and Gas6 levels. Results:The levels of serum Flt3L [807.80(215.10-1 232.00) pg/ml] and Gas6 [20.04(13.14-27.52) ng/ml] in the infection group were higher than those in the non-infection group and the control group, and the differences were statistically significant (all P<0.05);The levels of serum Flt3L[887.3(321.60-1 367.00) pg/ml] and Gas6[25.24(17.61-42.86) ng/ml] in the severely infection group were higher than those in the mildly infection group, and the differences were statistically significant (all P<0.05). serum Flt3L was negatively correlated with WBC and positively correlated with IL-6;serum Gas6 was negatively correlated with WBC.ROC curve analysis showed that the AUC and 95% CI of Flt3L and Gas6 were 0.814 (0.718-0.909) and 0.644 (0.523-0.765), respectively. The combined application of serum Flt3L with hsCRP, PCT and IL-6 significantly improved the diagnostic efficiency, and the area under the curve was 0.956, 0.923 and 0.865, respectively. Kaplan-Meier curve analysis showed that when serum Flt3L≥649.80 pg/ml and serum Gas6≥21.50 ng/ml in NHL chemotherapy patients, the infection rate was significantly increased ( P all<0.05). Conclusion:Serum Flt3L and Gas6 levels in post chemotherapy-infection of NHL patients were significantly increased and correlated with the severity of infection. Early detection of serum Flt3L levels is helpful to predict the diagnosis of infection in NHL patients after chemotherapy.

Development of a method for in vivo expansion, isolation and purification of mouse NK cells / 军事医学

Objective To stimulate the expansion of natural killer cells (NK) in vivo, to achieve high expression of Flt-3 ligand ( FL) in the mouse liver by hydrodynamic injection technology , to obtain high purity of NK for cell therapy and then to purify the spleen lymphocytes by MicroBeads sorting ( MACS ) technique.Methods C57BL/6 mice were repeatedly treated with hydrodynamic injection of FL expression plasmid .Then, surface markers of splenic NK were analyzed by flow cytometry and purified by MACS technique .Results Compared with mice treated with one hydrodynamic injection, the spleen of mice treated with two hydrodynamic injections showed significant variation, with the absolute num-ber of lymphocytes increased (6.02 ±1.15) times, the proportion of NK subsets increased (2.07 ±0.39) times, and the absolute number of NK subsets increased (12.49 ±2.39) times.Cell surface marker detection confirmed that NK activity and surface markers did not change significantly .NK with a purity of about (83.81 ±0.92)％ was obtained by the combination of two magnetic beads sorted with CD 5 ( Ly-1) MicroBeads Positive selection and EasySep TM Mouse NK Cell Isolation Kit negative selection .Conclusion NK can be expanded in the spleen of mice by hydrodynamic injection of FL plasmid without influence on the NK activity and surface markers.The combined use of CD5 (Ly-1) MicroBeads positive selection with EasySep TM Mouse NK Cell Isolation Kit negative selection can effectively remove T , B and other miscellaneous cells, thereby contributing to obtaining high-purity NK. This study provides good reference for the immunotherapy of tumor cells.

The inhibitory effects of the DNA vaccine co-expressing CEA tandem repeat epitopes and FL on the in vivo growth of heptocarcinomas in mice / 中国免疫学杂志

Objective:To observe inhibitory effrects of DNA vaccine co-expressing CEA tandem repeat epitopes and FL on cancer cells in mice.Methods:The encoding sequences for CEA tandem repeats and FL were inserted into plasmid pcDNA3.0 using gene recombinant technique.BALB/c mice were immunized intramuscularly with the co-expressing DNA vaccine.The survival time and tumor size were measured and specific CTL cytotoxicity was detected by ~(125)I-UdR release method.Results:Compared with that of the control,the survival time was prolonged (P＜0.01)and the tumors were significantly inhibited in the mice immunized with the vaccine peDNA-triCEA_(625-667)-sFL(P＜0.01).The splenic cells from mice immunized with the vaccine pcDNA-triCEA_(625-667)-sFL induced strongly cytotoxicity against tumor cells H22-CEA ~+(P＜0.01).Conclusion:The recombinant DNA vaccine co-expressing pcDNA-triCEA_(625-667)-sFL can suppress the growth of tumor expressing CEA in mice and enhance CTL response against CEA antigen.

Production of Recombinant Retroviruses Encoding Human Flt3 Ligand and IL-6 and Establishment of Genetically Modified OP9 Mouse Stromal Cells

Flt3 Ligand (FL) and IL-6 are multifunctional cytokines implicated in normal hematopoiesis and ex vivo expansion of hematopoietic stem cells. Retroviral vectors are useful for stable expression of genes in many cells. Here, we aimed to produce retroviral vectors directing expression of human FL and IL-6 genes. Recombinant retroviral vectors containing human genes for FL and IL-6 were constructed using a retroviral vector pLXSN. Recombinant retroviruses were produced from GP2-293 cells with the aid of pseudo-envelope protein gene VSV-G, and efficiently transduced to a mouse stromal cell line OP9. Genetically modified OP9 cells clearly showed expression of human FL or IL-6 gene at the mRNA level determined by RT-PCR. Based on the results from ELISA, human FL and IL-6 were detected in the cell culture medium of OP9/FL and OP9/IL-6 cells, respectively. As the recombinant human FL and IL-6 proteins are successfully produced and secreted to the culture medium, this system can be useful in future application such as ex vivo expansion of hematopoietic stem cells and differentiation of embryonic stem cells.

Strategies to Improve Dendritic Cell-based Immunotherapy against Cancer

Dendritic cells (DCs) play a pivotal role in T cell-mediated immunity and have been shown to induce strong antitumor immune responses in vitro and in vivo. Various approaches utilizing different vaccine cell formats, cell numbers, vaccination schedule, site of vaccination and maturation stages of DCs were investigated worldwide. While clinical trials have demonstrated the safety of such strategies, the clinical outcome was less than expected in most cases. This is due to in part host immunodeficiency imposed by tumors and immunoediting of tumor cells. To overcome these obstacles, new approaches to improve DC-mediated immunotherapeutic strategies are under investigation. First, functional enhancement of monocyte-derived DCs can be generated with using flt3-ligand (FL). Second, diverse antigenic determinants from heat shock-treated tumor cells may improve the immunogenicity of DC-based vaccines. Third, inclusion of ex vivo expanded NK/NKT cells in DC-based vaccines could be beneficial since the bidirectional interaction of these two cell types are known to enhance NK cell effector function and to induce DC maturation. Application of these approaches may induce a broadened antitumor immune response and thereby promote the elimination of tumor antigen-negative variant clones that had escaped immunosurveillance or undergone immunoediting. We are currently examining the feasibility of these immunotherapeutic approaches using a murine pancreatic cancer model system.

Effect of Flt-3 ligand on fusion vaccine of dendritic cells and induction of antitumor immunity in vitro / 医学研究生学报

Objective:To investigate the effect of flt-3 ligand on the preparation of the fusional vaccine with dendritic cells and colorectal cancer cells,and the anti-tumor immunological reaction of the vaccine. Methods: ①In the presence of Flt-3 ligand、GM-CSF、IL-4 and TNF,the maturation of dendritic cells was made from peripheral blood;The preparation of the fusional vaccine was made in the presence of PEG. ②The biological characteristics of fusion cells,including their morphological specialties, the expression of cell surface phenotypes, their growth curves, inducing specific cytotoxic T lymphocyte(CTL), their possibility to proliferate into a new carcinoma in nude mice etc, were tested to verify their safety when used as anti-tumor immune reactions. Results: ①Flt-3 ligand can stimulate the full maturation of dendritic cells. Dendritic cells and Lovo cells could be fused by PEG. ②The fusion cells exhibited typical biological characteristics. They could not proliferate into new carcinomas in nude mice, and can inhibit the proliferation of Lovo cells efficiently. Conclusion: Flt-3 ligand can enhance the maturation of dendritic cell. The fused cells had poor proliferation abilities and could not proliferate into new carcinomas in immunodeficient animals. The fusion vaccine could induce specific cytotoxic T lymphocytes in vitro and was safe as a vaccine.

Induction of Dendritic Cell and Cytokine Gene Expression by In situ Delivery of Flt3 Ligand Plasmid / 대한면역학회지

Dendritic cell (DC)s are protessional antigen presenting cells and they have been used for antitumor immunotherapy or cell vaccines. However therapy using DC is restricted because the number of DC available from tissue is very low. Flt3 ligand (FL) has been known as a hematopoietic growth factor that increases proliferation of hematopoietic stem cells and progenitor cells, and recently it showed inducibility of dendritic cell (DC)s and signiticant antitumor effects in vivo. Thus FL will be frequently used for DC induction and antitumor immunotherapy in future. Here we constructed FL plasmid and studied its in vivo effect. FL plasmids were made by cloning of partial FL cDNA into pcDNA3 plasmid, and gene expression and protein producibility of FL plasmid were confirmed in Renca cells transfected with FL plasmid. Mice were injected with FL plasmid (100ug/mouse) three times and 20 days later mouse spleens were harvested for staining and RT-PCR. There were lots of blastogenic cells in the spleen of mice treated with FL plasmid. FL plasmid also induced DEC205, IL-12 and GM-CSF gene expression in mouse splenocyte. All these data suggest FL plasmid may be used for induction of DC and antitumor therapy as DNA adjuvant.