| Aim:To identify the killing effect of double negative T cells on myeloid tumor cells by in vitro killing test,and detect the expression of NKG2 D receptor on the double negative T cells and NKG2 D ligand on leukemic cells,and compare the killing efficiency of double negative T cells with different expression levels.Test the peripheral blood of the patients who were injected with double negative T cells and observe the changes of the double negative T cells in the patients.Methods:This study is divided into two parts: first,in vitro culture and killing experiments: the double negative T cells from normal human peripheral blood were extracted and amplified,and their purity was detected by flow cytometry.After that,the killing experiments of cryopreserved myeloid tumor primary cells and cell lines were carried out.Second,the infusion safety and body changes: After the infusion of cultured double negative cells to patients ready for the experiment,blood count,peripheral blood cells,finding abnormal biochemical and vital signs were detected regularly to assess efficacy and safety.Also,detect the number and percentage of double negative cells and the tumor cells in the peripheral blood of patients.The flow cytology data were plotted and analyzed by Flowjo7.6.1 software.The measured data obeying the normal distribution are expressed in the mean ± standard deviation,and the non‐normal distribution data are expressed in the median(range).The continuous measurement data were analyzed by single sample t test or nonparametric test.Using Graph Pad Prism 5 and R 3.2.2 statistical software for statistical image and data processing,and p<0.05 means statistically significant.Result:1.In vitro culture and killing experiment The extracted mononuclear cells were cultured after the omission of CD4 or CD8 positive T cells.The purity of the double negative cells was over 90%,and the cell multiplication rate was 483 times on the 17 day.For the cell lines: When the target ratio was 4:1,the killing efficiency of DNT to cell line OCI‐AML3 cells was 30.94 + 1.25%,and 50.3 + 1.40% for killing 411 cell lines,and DNT are sensitive to killing both two cell lines,compared with the control group(p<0.05).For primary cells: The killing efficiency of DNT cells to 6 AML patients are different.When the target ratio is 4:1,the killing efficiency is 18.1(4.7‐38.7)%.Although the killing efficiency is higher than the control group,there is no statistical significance(p=0.069).With the increase of effect target ratio,the killing efficiency is obviously improved.The killing efficiency of the two groups with the effect target ratio of 8:1 and 16:1 were significantly higher than that of the control group(p=0.033,0.00).2.Evaluation of the safety of infusion of double negative T cells and the change of the cell in the human body1 of the 4 patients underwent DNT cell infusion treatment(25%)had high fever,which was considered CRS.After the treatment of IL‐6(4mg/kg) antibody,the patient’s temperature was controlled.5 days after the infusion of DNT,another patient suffered scattered red rash,visible blood eosinophilia,which was considered allergic reactions.The symptoms disappeared after the treatment of cetirizine.All the 4 patients didn’t experience a GVHD or c GVHD,no damage to liver and kidney function or symptoms of central nervous system,immune globulin infusion of normal,smooth,no chills,respiratory distress,tachycardia and other symptoms,only 1 people have mild irritability symptoms;2‐3 weeks after infusion of neutrophil and platelet and hemoglobin were restored to the level before treatment,no obvious damage to the hematopoietic function.DNT cell infusion in patients after 6h in peripheral blood were detected in very low proportion,the number of DNT cells after 48 h began to increase 5 days after the infusion of the increase in the number of rapid pace;after the second infusion cells continued to increase the number of fifth days,the number decreased slightly,seventh days began to increase rapidly again,while maintaining a certain level in 28 days.The increase of the absolute value of DNT cells in peripheral blood after infusion was statistically significant(p<0.05).4 cases of patients with 3 cases of hematologic relapse,the first infusion of DNT cells,the proportion of primitive cells have different degrees of decline;long‐term follow‐up showed that the recurrence of the original disease by DNT cells after treatment,4 patients in 3 patients after the treatment of DNT in February,June,October died of primary disease in 1 patients,since 5 months is still living.Conclusion1.For the mononuclear cells without CD4/CD8 from healthy people,using the patent technology of professor L Zhan(Name: amplification of double negative T international patent number: PCT/CA2006/001870,US9018004;China patent No.: 200680043116.7)make the culture practical,and the amplification effect is good,and purity of training is extremely high;DNT cell cytotoxicity on myeloid tumor cell lines significantly;DNT cells in patients with AML of the original cell killing efficiency differences,but the overall effect is good,with statistical significance;it is suggested that DNT cells may through the NKG2 D ligand,DNAM‐1 and leukemia cell surface combined with the corresponding play killing effect.2.DNT cell infusion in treatment of human myeloid tumor is safe,no obvious Miss damage normal tissue,the incidence rate of CRS is low,there is no obvious complications;it can enter the body to continue proliferation in vivo in infusion within 28 days to maintain a certain level;DNT cell therapy for myeloid neoplasms the need to further Research on screening suitable patients and increase the dose to improve the curative effect. |
PDF Full Text Request