Recombinant Salmonella Vaccine Enhances NK Cell Against Acute Myeloid Leukemia In Vitro And Establishment Of Human Acute Myeloblastic Leukemia Nude Mouse Model

BackgroundGreat interest has surrounded the activation of immune system as an oncolytic agent due to its ability to infect and induce death in a range of human malignancies whilst sparing normal cells, the innate immunity produced in the early primary stage of system development and the host immune response, and the most important function of which is to adjust the adaptive immune response, and not thought to develop immunological memory. However, evidences suggest that specific subsets of mouse NK cells can nevertheless develop long-lived and highly specific memory to a variety of antigens. Natural killer cells (NK cells) is a kind of large particles cell, different from T,B lymphocyte, distributing in the peripheral lymphoid organ and blood circulation system. Without the pre-stimulus and activation of antigen, It could play role in cytotoxic effects and secrete a variety of cell factors and chemotactic factors. NK cells expressed a series of activated receptors and suppressive receptors, both of which regulate the activation of NK cells. Normally, a combination of MHC-I molecule of the surface of target cells and suppressive receptors of the surface of NK cells conduction suppressive signals to inhibit the function of NK cells, and so target cells will not be under attack by NK cells. Pathologically, when this balance is broken, NK cells might be driving "killing" effect to the target cells. Research shows that inserting the immune activation gene can stimulate anti-tumour immune response, such as direct dissolve tumor cell function, suggesting exogenous DNA can be a stimulating factor of immune therapy and cytotoxic effects. So, whether tumor derived cells pre-treated by exogenous DNA could promote the innate or adaptive immune response to achieve the purpose of inducing tumor cell apoptosis? The secondary histocompatibility antigen gene HA-1specifically express hematopoietic cells. Cytotoxic T lymphocytes which can identify HA-1have the observable anti-leukemia effects, while the AML/ETO fusion gene is uniquely of acute myeloid leukemia-M2type, both plays an important role in the formation of leukemia.The salmonella typhimurium is a common aggressive intracellular bacteria, which significantly reduce poison to host through genetic engineering method, but still keep good invasiveness. It can directly submit immune antigen to immune cells to inducte specific immune response., and attenuated salmonella typhimurium as oral vaccine carrier is currently one of the hot topics in the study of vaccine immune. Our research aims to prove that after salmonella typhimurium was inserted the recombinant plasmid with immunogenic gene AML/ETO and HA-1and infected AML/ETO positive leukemia Kasumi-1cell, it could strengthen the killer effect of NK cells. At the same time, BALB/c nude mice immune system was restrained by the radiation or cyclophosphamide pretreatment, and then were inoculated with AML/ETO positive leukemia Kasumi-1cells to construct the low cost, high stability of the nude mice model, for further study on AML/ETO and HA-1recombinant salmonella vaccine enhances NK cell innate immunity against acute myeloid leukemia cell Kasumi-1.MethodsThe first part recombinant salmonella vaccine enhances NK cell innate immunity against acute myeloid leukemia cell Kasumi-11. HA-1and AML/ETO recombinant plasmid Constructing and reorganization of the construction of the plasmid salmonella vaccine building208bp AML/ETO cloning segment is inserted into the containing long segment (HA-1F) and short segment (HA-1M) plasmid respectively. Five restructuring plasmids containing AML/ETO, HA-1M, HA-1F, AML/ETO+HA-1M, AML/ETO+HA-1F fragment were transformed by electroporation into prepared salmonella competent, no-load as control group. Gene sequencing analysis confirmed gene correct connection with restructuring plasmid.2. acute myeloid leukemia M2type Kasumi-1cells culturing and recombinant salmonella vaccine infectingCells were cultured in RPMI1640medium supplemented with10%fetal bovine serum. Before infection with Salmonella, the Kasumi-1cells were washed and resuspended in fresh culture media without antibiotics. The Kasumi-1was seeded for1h at a concentration of2*105mL in cell culture bottles. Then, bacteria were added to mix with the Kasumi-1with the ratio of15bacteria per cell. The infection was allowed to proceed for2hours at37℃C cell incubator. The extracellular bacteria were killed by the addition of100mg/mL of gentamicin. After48h postinfection, the cells were detected ultrastructure by TEM to confirm successful infection.3. Culture of immature NK cell from peripheral blood monocytesHuman immature NK cell were cultured from peripheral blood monocytes. After adherence for1h, culture dishes were rinsed with HBSS and adherent cells were incubated overnight, and change into medium of NK92cells. After cultured for72h, cells were harvest and isolated by NK isolation kit. Cytotoxicity by NK cells after cocultured with Kasumi-1cells at effector-to-target (E:T) ratios ranging from20to5:1. Effector cells were incubated with5000target cells in a final volume of200μ1per well. Supernatants were harvested by centrifugated at3000rpm, which was detected by CytoTox96Non-Radioactive Cytotoxicity Assay.4. Cytotoxicity AssayCocultrue NK cells and Kasumi-1infected salmonella and harvest the supernatants. The equation is%Cytotoxicity=OD(Experimental-Effector Spontaneous-Target Spontaneous)/OD(Target Maximum-Target Spotaneous)×100. All procedures were according to the manufacturer’s recommendations.The second part the establishment of acute myeloid leukemia M2nude mouse model1. Establishment of nude mouse model of human acute myeloblastic Leukemia M2typeBALB/c mice were randomly divided into three groups by random draw method: One is cyclophosphamide group that nude mice were injected about8×105Kasumi-1cells through by caudal vein after intraperitoneal injection of cyclophosphamide2d; One is irradiation group that nude mice were pretreated by X-ray irradiation all over the body; Another is non-pretreated group and injected the Kasumi-1leukemia cells directly. In addition, three normal BALB/c mice as normal controls.2. The percentage of AML/ETO fusion gene positive cells of model mouse bone marrow by FISHCollect executed mouse bone marrow cells at each time point, grille piece2h in fluorescence in situ hybridization instrument at59℃, add the diluted DNA probe, coverslip and Rubber Cement, Fengpian denaturation for75℃2min, and then37℃overnight. Thrown off the coverslip carefully, wash and dry naturally at room temperature, add DAPI/Antifade to dye20min away from light, finally observed by fluorescence microscopy and image acquisition.3. RT-PCR detect leukemia cells on tumor loadWe found that Kasumi-1cells vaccined into nude mice for14d, then formed tumors by the above assay. Taken blood of orbital venous0.5ml, inoculated with tumor cells in nude mice for28days, total RNA was extracted, then purity and integrity of total RNA were identificated. The primers were designed:AML1-A:CTA CCG CAG CCA TGA AGA ACC; ETO-B:AGA GGA AGG CCC ATT GCT GAA, product size395bp. PCR conditions:95℃,30s;35cycles (94℃,30s;65℃, lmin;72℃,1min);72℃,3min. Gel imaging analysis system for imaging analysis.Statistical methodsThe results of experimental data with mean±standard deviation, significant differences (P<0.05), SPSS13.0statistical package for statistical analysis. Statistical methods using single-factor analysis of variance (One-Way ANOVA). Results The One Part Recombinant Salmonella vaccine enhanced innate immune response of NK cell for acute leukemia cells1. Builded recombinant plasmid carrying double geneConfirmed by agarose gel electrophoresis, PCR amplification of AML-ETO1and AML the-ETO2containing different restriction sites, PCR products were connected to plasmid of pcDNA3.1-HA-1H (HA-1M) and pEXP-cDNA3.1-the HA1-H-mycHIS-Neo-Jos (HA-1F) after purifying and digesting, then transformed the ligation products of pcDNA3.1-HA-1H-AML-ETO1(HA-1M) and pEXP-cDNA3.1-HA1-H-mycHIS-Neo-Jos-AML-ETO2into DH5a. The selection of monoclonal bacteria on the Petri dish was identified by PCR, the positive clones were sent to sequencing, and sequencing results shown that the AML-ETO gene had been correctly connected to the appropriate plasmid, and the sequence comparing with the original sequence was exactly the same.2. Builded and identified of recombinant vaccine strainsTransformed control group and five plasmid of pUHD-HA-AML1-ETO, pcDN A3.1-HA-1M,pEXP-cDN A3.1-HA1-H-mycHIS-Neo-Jos,pcDN A3.1-HA-1M-A MLthe-ETO1, pEXP-cDNA3.1-HA1-H-mycHIS-Neo-Jos-AML-ET02into Salmonella SL7207Respectively, PCR electrophoresis and DNA sequencing were confirmed that the conversion successed.3. TEM inspection of recombinant Salmonella typhimurium vaccine successfully infect Kasumi-1cellsInfected control group Salmonella SL7207and five recombinant vaccine bacteria ofSL7207/pUHD-HA-AML1-ETO,SL7207/pcDNA3.1-HA-1M,SL7207/pEXP-cDN A3.1-HA1-H-mycHIS-neo-Jos,SL7207/pcDN A3.1-HA-1M-AML-ETO1SL7207/pE XP-cDNA3.1-HA1-H-mycHIS-Neo-Jos-AML-ET02into acute myeloid leukemia Kasumi-1cells2days, then collected cells, selected non-infection Kasumi-1cells as the control group, TEM detection of recombinant vaccine SL7207/pEXP-cDNA3.1-HA1-H-mycHIS-Neo-Jos-AML-ETO2for the experimental group, the results shown that Salmonella vaccine infected cells appeared cytoplasmic vesicles, whereas the control group did not.4. Determination of cytotoxicityAs effector cells:target cells for8:1,16:1,32:1were co-incubated NK cells and infected Salmonella Kasumi-1, and centrifuged to obtain50μl supernatant, then mixed with50μl reaction liquid(SubstrateMix) of CytoTox96Non-Radioactive Cytotoxieity Assay, determined releasing of lactate dehydrogenase in the mixture. Formula for a percentage of the Cytotoxicity:cytotoxicity percentage=(OD experiment releasc—ODcfficient spontaneous release—OD target spontaneous release)/(ODtarget maximum release ODtarget spontaneous release)×100%. One-Way ANOVA statistical method analysised, when the titer of8:1, compared with the control group (P<0.05), attenuated Salmonella typhimurium carrying the AML/the ETO gene and/or HA-1gene induced DNA oral vaccine can pass specific tumor antigens to antigen presenting cells, activated killing effect of NK cell, especially attenuated Salmonella infection recombinant Salmonella typhimurium carrying the AML/ETO gene and HA-IM gene infected Kasumi-1cell, and had stronger killing effect as NK cells (P<0.05); when the title of16:1, recombinant Salmonella vaccine infection group at least carrying one HA-1gene, NK lethal higher than the infection of the AML-ETO single gene group and control group (P<0.05), the vaccines infection group of SL7207/pcDNA3.1-HA-1M-AML-ETO1was best (P<0.05); when the title of32:1, recombinant Salmonella vaccine infection carrying double gene, NK lethality higher than single gene and the control group (P<0.05), while the lethal infection group of SL7207/pcDNA3.1-HA-1M-AML-ETO1vaccine was the highest(P<0.05); when focus on the SL7207/pcDNA3.1-HA-1M-AML-ETOl vaccine infection group, we found that NK cell efficiency target ratio of32:1, NK cells best. In summary, NK cells with recombinant Salmonella vaccine of SL7207/pcDNA3.1-HA-1M-of AML the-ETO1infected Kasumi-1cells, as the title of32:1, the NK cell had the highest lethality.The Two Part Established nude mouse model of acute myeloid leukemia M21. Introduction of pathogenesisNude mice inoculated with Kasumi-1cells14d appeared leukemia, nude mice less energy and less movement, abdominal distension, subcutaneous palpable nodules of varying sizes, weight loss with the tumor increased, after42days, part of the mice appeared instability step and cornering or turning in circles.2. Leukemia in nude mice of various organs of the AML/ETO gene mRNA levelNormal control group of nude mice mononuclear cells of peripheral blood not founded Kasumi-1cells unique integration of AML/the ETO gene. Cyclophosphamide group, radiation group, no pretreatment group showed mRNA expression of integration gene AML/ETO, but Electrophoretic bands of the pretreatment group was weaker than the cyclophosphamide group, irradiated group.3. Positive cells appeared in Model of bone marrow AML/the ETO geneMouse cells was significantly less than human tumor cells, while only have DAPI nuclear staining. Normal control group of nude mice bone marrow cells not founded inKasumi-1cells-specific fusion gene-AML/ETO. After Kasumi-1vaccined cells4weeks, three groups of experiments founded hybridization signal of the integration gene of AML/ETO in the bone marrow mononuclear cells.Conclusions1. We successfully constructed six recombinant Salmonella vaccine with AML/ETO and/or HA-1gene;2. This experiment founded that compared with control group, recombinant Salmonella vaccine with simple gene and double gene infected Kasumi-1cells was the better killing effect of NK cells;3. This experiment founded that, in all groups recombinant Salmonella vaccine with AML/ETO and HA-1M infected Kasumi-1cells was the best killing effect of NK cells;4. We successfully constructed human acute myeloblastic Leukemia M2nude mouse model;5. Both Two pretreatment groups and unpretreatment group are successfully constructed human acute myeloblastic Leukemia M2nude mouse model;6. We successfully constructed human acute myeloblastic Leukemia M2nude mouse model, which lay the foundation for the further study of the innate immune response and anti-leukemia effect of restructured salmonella vaccine with AML/ETO and HA-1M in the body of leukiemia nude mouse model.