| Objectives:Burkitt’s lymphoma（BL）is the most common non-Hodgkin’s lymphoma in children,which is highly invasive and rapidly progressive.Short-cycle high-dose intensified multi-drug chemotherapy is commonly used for clinical treatment,with a cure rate of 80-90%,but there is still a 10-20%recurrence or death rate.Currently,rituximab（CD20 antibody）can be used to treat 95%of relapsed or chemotherapy-resistant patients with good results.However,there are still around 5%of patients who do not respond well due to the lack of CD20 expression on B cell surfaces,leading to low 3-year survival rates and poor prognosis.Therefore,effective treatment options are urgently needed for these patients,and in recent years,exosome-based immunotherapy has become a promising approach for tumor immunotherapy.Studies have shown that natural killer cell-derived exosomes（NK Exo）can be used to kill melanoma and ovarian cancer cells.Our previous research also found that NK Exo can be used for immunotherapy of pediatric neuroblastoma.However,there have been no reports on using NK Exo for immunotherapy of BL.Therefore,this study will explore this for the first time.In order to obtain more efficient activation of NK Exo for killing BL,this study will use genetic engineering technology to modify K562 cells to make them more strongly stimulate and activate NK cells,thereby secreting more highly active exosomes.Based on this,we will first use DNA recombinant technology to construct a membrane-binding interleukin 21（mb IL-21）lentiviral vector,and then transfected and screened the modified K562cells（mb IL-21 K562 cells）.After X-ray irradiation,we will co-culture them with peripheral blood mononuclear cells（PBMC）and induce rapid activation and expansion of NK cells under the stimulation of interleukin 2（IL-2）and interleukin 15（IL-15）,in order to obtain more NK cells with higher cytotoxicity.Then,we will use a laboratory-developed exosome isolation and extraction method to extract and isolate the above cultured NK cell supernatant,thereby obtaining a large number of highly efficient activated NK Exo,which will be used for immunotherapy of BL cell lines（Raji cells）and rituximab-resistant cell lines（ARH-77）.The goal is to discover a new immunotherapy method for relapsed/chemotherapy-resistant BL,providing new research ideas and theoretical basis for BL immunotherapy.Methods:The mb IL-21 lentiviral plasmid was constructed by homologous recombination method.After transfection with Polyethylenimine（PEI）into HEK293T cells,the virus was collected and used to infect K562 cells.Positive mb IL-21K562 cell lines were screened using G418 and confirmed by Western Blot（WB）to express IL-21.After X-ray irradiation at a dose of 100 Gy,the mb IL-21 K562 cells were co-cultured with PBMCs at a ratio of 1:1 for 14 days,along with soluble cytokines IL-2 and IL-15 to induce differentiation of PBMCs into NK cells.Flow cytometry was used to detect the proportion of NK cells（CD3-CD56+CD16+）,and the number of NK cells was calculated using Trypan blue staining.Activated NK Exo were obtained from the supernatant of high-capacity NK cell culture using Polyethylene Glycol（PEG）precipitation.The membrane structure and particle size of NK Exo were confirmed by transmission electron microscopy,and the size distribution and concentration were determined using nanoparticle tracking analysis.The expression of CD63,CD81,perforin,granzyme A,and granzyme B proteins in NK Exo was detected by WB.Co-culture of activated NK Exo with Raji and ARH-77cells was performed,and flow cytometry was used to detect apoptosis and cell cycle changes of Raji and ARH-77 cells at different time points.Results:Successfully obtained K562 modified cell line（mb IL-21 K562 cell line）,including:Successfully constructed mb IL-21-PCDH plasmid by homologous recombination method.PCR and sequencing results showed complete consistency with mb IL-21 design sequence;Transfected mb IL-21-PCDH lentiviral vector into HEK 293T cells and infected K562 cells with virus to obtain positive mb IL-21 K562cell line through G418 screening and cultivation.WB results showed that IL-21protein level in mb IL-21 K562 cells was significantly higher than that in untransfected K562 cells.After stimulation of mb IL-21 K562 cells and induction and cultivation with IL-2 and IL-15 for 14 days,NK cells with a purity of 90.4%could be obtained.Flow cytometry results showed that with the extension of co-culture time between X-ray irradiated mb IL-21 K562 cells and PBMC,the number of NK cells（CD3-CD16+CD56+cells）increased continuously,and on the 14th day of co-culture,the proportion of NK cells reached 90.4%,which was higher than that in the stimulated group of untransfected mb IL-21 K562 cells.Using the laboratory-developed exosome isolation and extraction method,a large number of activated NK Exo could be extracted and isolated from the above cultured NK cell supernatant.Transmission electron microscopy showed that NK Exo had typical double-layer membrane structure with a diameter of about 100-200 nm;Nanoparticle Tracking Analysis（NTA）showed that the size range of NK Exo was 70-200 nm,with a peak diameter distribution of about 131.3 diameter/nm and a concentration of 1-3.8×1011particles/ml.WB results detected the expression of CD63 and CD81,as well as perforin,granzyme A,and granzyme B proteins.Activated NK Exo could induce apoptosis in Raji and ARH-77 cells and arrest them in G1/S phase.Specifically,after treatment with NK Exo（3μg/μl）for 6 h and 24 h,Annexin V-FITC and PI double staining were used for flow cytometry analysis.The results showed that compared with the group without NK Exo,co-culture of NK Exo with Raji cells for 6 h induced cell apoptosis,while less apoptosis was observed in ARH-77 cells.However,after extending the co-culture time to 24 h,both Raji and ARH-77 cells underwent significant apoptosis,with an apoptosis rate of 79.6%and 82%,respectively.In addition,PI single-staining flow cytometry results showed that after treatment with NK Exo（3μg/μl）for 24 h,the proportion of cells in G1 phase was significantly higher than that in S and G2 phases,indicating that both cells were affected by activated NK Exo and their cell cycle was arrested in G1/S phase.Conclusions:Firstly,successfully obtained K562 modified cell line（mb IL-21K562 cell line）which can be used for significant amplification of NK cells in vitro.After X-ray irradiation,this cell line can stimulate rapid activation and expansion of NK cells,and after induction and co-culture with IL-2 and IL-15 for 14 days,highly efficient,high-purity and activated NK cells could be obtained.Secondly,through the laboratory-developed exosome isolation and extraction method,a large number of activated NK Exo could be obtained from the high-capacity NK cell culture supernatant.The extracted NK Exo had a high concentration,typical Exo double-layer membrane structure and expression of CD63 and CD81 exosome markers.In addition,NK Exo also expressed tumor-killing substances of NK cells:perforin,granzyme A and granzyme B,indicating that it had strong tumoricidal toxicity.Thirdly,the isolated activated NK Exo can significantly induce apoptosis of Raji and ARH-77 cells.Lastly,activated NK Exo obtained by stimulating and cultivating mb IL-21 K562 cell line may have potential immunotherapy effects on BL（including relapsed/chemotherapy-resistant BL）. |
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