The Effects Of Borojo On Regulating The Anti-tumor Activity Of Human Vγ9Vδ2-T Cells And Its Underlying Mechanisms

Objective:At present,the existing in vitro amplification system has been unable to meet the needs of the development of immune cell therapy.How to improve the quantity and quality of anti-tumor immune cells in vitro by optimizing conventional amplification methods has become a hot issue in the current immunotherapy research.The aim of this study was to study the effect of Borojo on the activity of human peripheral blood mononuclear cell(PBMC)and the antitumor activity of expanded Vγ9Vδ2-T cells in vitro,and to explore its possible mechanism.Methods:PBMC was isolated from venous blood of healthy volunteers by density gradient method.PBMC was treated with different concentrations of Borojo,and then the cytotoxicity of Borojo was detected by CCK-8 method.The effects of Borojo combined with IL-2 on PBMC were studied by CCK-8 method.CD3~+T lymphocytes were enriched from fresh PBMC by Immune Magnetic Beads negative selection method.The purity of CD3~+T lymphocytes was detected by flow cytometry,and the percentage of CD4~+T and CD8~+T lymphocytes was detected too.CD3~+T lymphocytes were stimulated by adding CD3/CD28 Dynabeads,meanwhile CD3~+T lymphocytes were stimulated by adding different concentrations of Borojo and the percentage of CD4~+T and CD8~+T lymphocytes were detected by flow cytometry 72 hours later.In addition,in order to detect the immune regulatory effect of Borojo on human Vγ9Vδ2-T cells,Pamidronate(PAM)and IL-2 were used to amplify Vγ9Vδ2-T cells in PBMC in vitro,and different concentrations of Borojo were used to interfere with the in vitro amplification of Vγ9Vδ2-T cells.The percentage of Vγ9Vδ2-T cells and the absolute number of cells were detected by flow cytometry at different time during amplification;In order to evaluate the anti-tumor effect of Borojo on Vγ9Vδ2-T cells,we cultured Vγ9Vδ2-T cells treated different concentrations of Borojo with K562labeled with CFSE for 6 hours according to 10:1ratio of effect to target,then stained with PI,and detected the percentage of PI~+in CFSE labeled K562 cells by flow cytometry,that is,the efficiency of killing K562 by Vγ9Vδ2-T cells.In order to study the anti-tumor mechanism of Borojo on Vγ9Vδ2-T cells,we will detect the phenotypic features and cytokine secretion of Vγ9Vδ2-T cells after incubation.Results:Borojo has no toxicity to human immune cells,and can significantly promote cell proliferation at the concentration of 10μg/mL;The PBMC was treated with10ug/ml Borojo combined with 50U/mL IL-2.on the 9th day,the level of cell proliferation was significantly different between IL-2 treatment group and 10μg/mL Borojo combined with IL-2 treatment group.CD3~+T lymphocytes were isolated by magnetic bead negative selection,and the purity of CD3~+T lymphocytes was up to 99%.CD3/CD28 stimulation combined with different concentrations of Borojo was used to treat the CD3~+T lymphocytes.We found that the percentage of CD8~+T lymphocytes was increased and the percentage of CD4~+T lymphocytes was relatively decreased in the group with Borojo,and the sum of the two was almost the same.There was no significant difference in the ratio of CD4~+T and CD8~+T lymphocytes treated with different concentrations of Borojo.PAM combined with IL-2 can be used to amplify Vγ9Vδ2-T cells in vitro.The process of culturing Vγ9Vδ2-T cells with different concentrations of Borojo showed that,although the percentage of Vγ9Vδ2-T cells was not different,the absolute number of cells had no significant difference in the first 20days of the amplification process,but the rate of decrease of the group cultured with100μg/mLBorojo was slower than that of the control group,which was significantly different from that of the control group.After 30 days of treatment of Vγ9Vδ2-T cells withμg/mL Borojo,the mortality of K562 was detected after incubation of Vγ9Vδ2-T cells with K562 for 6 hours.it was found that the killing rate of Vγ9Vδ2-T cells without Borojo on K562 was 13.43%1.8%,while that of Vγ9Vδ2-T cells with Borojo on K562 was 31.18%3.6%,indicating that the death rate of K562 target cells treated with Borojo had increased nearly18%.The surface and intracellular molecular markers of Vγ9Vδ2-T cells after 30 days of amplification were treated with Borojo We found that Borojo had no significant effect on the surface molecules TRAIL,FasL,NKG2D and intracellular dissolved particulate matter of Vγ9Vδ2-T cells,but the expression of Granzyme B increased and Fas decreased.After purified and co-cultured with K562cells in a ratio of 1:1 for 12h after the amplification of Vγ9Vδ2-T cells treated with Borojo,the results showed that compared with PBS control group,we found that there were significant differences in the levels of cytokines secreted by the treated group Vγ9Vδ2-T cells into the cell culture supernatant,such as INF-γ,TNF-α,Granulin,Granulase B,perforin and Granulysin.Conclusion:Borojo has no cytotoxicity to human PBMC,and can promote the proliferation of PBMC in vitro in a certain concentration range:Borojo has a tendency to promote CD3~+Tlymphocytes to differentiate into CD8~+T lymphocytes;With the concentration of 100μg/mL Borojo,Vγ9Vδ2-T cells can be cultured in vitro for a longer time,and their antitumor activity can be enhanced by secreting higher levels of INF-γ,TNF-α,GranzymeA,GranzymeB,perforin and Granulysin and decreasing Fas expression.