Effect Of Artocarpus Lingnanensis Lectin On Lymphocytes Subsets In Vitro And Vivo And Its Anti-hepatoma Effect

OBJECTIVE:Adoptive tumor immunotherapy is a major branch of tumor immunotherapy,mainly by inducing activation and expansion of immune effector cells from patients themselves or from donors,and infused anti-tumor effector cells to patients enhance the anti-tumor immunity.How to effectively activate and expand the required immune effector cells.The preliminary study of the ALL(Artocarpus lingnanensis lectin)independently isolated and purified in our laboratory found that ALL has a strong activation and expansion effect on human peripheral blood lymphocytes in vitro,but the cell survival time in vitro is not long,restricting the cells adoptive reinfusion therapy.IL-2(Interleukin-2)is an immunostimulatory cytokine and a key factor for maintaining the survival of lymphocytes in vitro.Therefore,how to using ALL and IL-2 combined application in vitro to the expansion of lymphocytes will likely solve the problem of short survival time in lymphocyte expansion.At the same time,it has not been reported whether ALL directly affects the proportion of lymphocytes and the body’s anti-hepatoma function.Therefore,this study intends to explore the effect of ALL and IL-2 combined application on the activation and expansion efficiency of human peripheral blood and mice spleen lymphocytes,as well as the proportion of different lymphocyte subsets in vitro,to explore the lethality of lymphocytes on liver cancer cells after ALL stimulation;to explore the in vivo conditions,the effect of ALL treatment on the ratio of different immune subset cells and tumor growth in the mice liver cancer model.METHODS:1.Exploring the optimum concentration of ALL.The freshly separated and purified ALL was dissolved to the required concentration by the research,and human peripheral blood and mice spleen lymphocytes were separated by erythrocyte lysis.Set different concentrations of ALL and IL-2 to stimulate mice spleen lymphocytes.The CCK8 method was used to explore and select the optimal concentration of ALL and IL-2 to amplified mice spleen lymph.2.ALL combined with IL-2 stimulates lymphocyte proliferation.CCK8 method and CFSE method were used to detect the proliferation of human peripheral blood and mice spleen lymphocytes stimulated by ALL combined with IL-2.Flow cytometry was used to detect the apoptosis rate of lymphocytes after stimulation.IL-2 was established as control.3.ALL combined with IL-2 stimulates lymphocyte activation and subpopulation changes.Flow cytometry was used to detect the expression of T lymphocyte activation markers CD69 and CD25,CD3~+T cells,B220~+B cells,CD49b~+NK cells,CD11C~+DC cells(CD3~+CD45RA~-CCR7~+)Tcm cells,(CD3~+CD45RA~-CCR7~-)Tem cells and(CD3~+CD45RA~+CCR7~+CD62L~+CD95~+CD122~+)Tscm cell ratio changes after ALL combined with IL-2 stimulated lymphocytes.;IL-2 was established as control.4.Killing effect of ALL activated lymphocytes on mice hepatocellular carcinoma cells.Collect mice Hepa1-6 hepatocellular carcinoma cells(target cells)and activated spleen lymphocytes(effector cells)stimulated by ALL combined with IL-2,which was called activated lymphocytes for short in following.CCK8method and LDH method were used to detect the effect cells proliferation and killing ability on target cells.Effects of ALL activated lymphocytes on the apoptosis rate of Hepa1-6 cells after co-culture for 72 hours with a flow cytometry detection target ratio of 20:1,10:1,5:1,1:1.IL-2 activated cells and Hepa1-6 cells were established as control.5.Cytokine secretion from activated lymphocyte.Collect the supernatant of activated lymphocytes and Hepa1-6 cells co-cultured,and elisa method were used to detect the secretion levels of IL-2,TNF-αand Gr B in the target-to-effect ratios of 20:1,10:1,1:1 for 24 to 96 hours culture supernatant.IL-2 activated cells and Hepa1-6 cells were established as control.6.Effects of ALL on tumor growth and immune system in mice liver cancer model.The mice liver cancer model was established in C57/BL6 mice.ALL was injected intravenously at day 0,2,4,6,and 8 days,to evaluate the antitumor effect of ALL.After ALL treatment,the proportion of T cell,Tscm cell,Tcm cell,Tem cell,B cell,NK cell,macrophage and DC cell subsets in peripheral blood,spleen and tumor infiltrating lymphocytes of mice liver cancer model were analyzed.Elisa method was used to detect the secretion levels of serum IL-2,TNF-αand Gr B in liver cancer model mice.PBS was established as control.RESULTS:1.Effects of ALL on lymphocyte proliferation and antitumor effects and different immune cell subsets in vitro.1.1 The optimal concentrations of ALL to stimulate mouse lymphocytes are 10μg/ml.CCK8 results showed that after ALL stimulated mice spleen lymphocytes,the cells aggregated into colony-like growth,and the cells proliferated faster.The best concentration of ALL was 10μg/ml 34.19%±1.51,was higher than other ALL(2.5μg/ml,5μg/ml and 7.5μg/ml)stimulation group(p<0.05).The 10ug/ml ALL was selected to be for subsequent experiments.IL-2 can maintain the growth of mice lymphocytes,and 12.5ng/ml,25ng/ml,and 37.5ng/ml have similar proliferation effects,and the difference is not statistically significant.The12.5ng/ml IL-2 was selected to be for subsequent experiments.1.2 ALL combined with IL-2 effectively promotes lymphocyte proliferation.The results of CCK8 showed that the relative proliferation rates of the splenic lymphocytes of the ALL+IL-2 and IL-2 group after 96 hours were 98.98%±6.12and 41.61%±2.78,respectively(P<0.001).CFSE results showed that after 72hours of stimulating human peripheral blood and mice splenic lymphocytes in the ALL+IL-2 and IL-2 groups,the green fluorescence showed a diminishing phenomenon,indicating that the lymphocytes were dividing and proliferating.It was 69.83%±3.17 and 55.73%±1.25(P=0.002);the ratio of cell proliferation in ALL+IL-2 and IL-2 groups of lymphocytes was 74.20%±2.50 and 61.43%±6.39(P=0.032),respectively.Flow cytometry detected the apoptosis rate of splenic lymphocytes stimulated by different concentrations of ALL+IL-2 groups for 72hours.The results showed that the apoptosis rates of the ALL group was19.31%±0.97;and the apoptosis rates of the IL-2 group and the blank control group were 28.85%±0.93 and 30.32%±0.85;which proved that ALL combined with IL-2 can promote cell proliferation and reduced apoptosis.1.3 ALL combined with IL-2 effectively promotes T lymphocyte activation.Flow cytometry analysis showed that the T-cell early activation marker CD69in the ALL+IL-2 group was 62.5%±2.45 after 24h stimulated,which was significantly higher than IL-2 group 11.13%±0.21,and continued to be high in subsequent cultures.The expression of CD25,a late activation marker of T cells,increased significantly after 48-72h of culture.The expression of CD25 in ALL+IL-2 group was 50.46%±4.38 after 72h on stimulated,the IL-2 group was15.33%±0.71,It shows that ALL combined with IL-2 effectively promotes T lymphocyte activation,and the expression of CD69 and CD25 is significantly higher compared with IL-2 group(P<0.05).1.4 The effect of ALL combined with IL-2 stimulation on different lymphocyte subsets.Flow cytometry showed that in the ALL+IL-2 group stimulated human peripheral blood lymphocytes for 72h.The results showed that the proportion of T lymphocytes,DC cells and TSCM cells in the ALL group was higher than IL-2group(P<0.01),the proportion of B lymphocytes and NK cells in the ALL+IL-2group was lower than IL-2 group(P<0.01).After IL-2 and ALL stimulated mice spleen lymphocytes for 72h,the results showed that the proportion of B lymphocytes,DC cells,Tscm cells and Tcm cells in the ALL+IL-2 group was higher(P<0.01),and the proportion of T lymphocytes,NK cells and Tem cells in the ALL group was lower(P<0.01).1.5 The antihepatoma activity of lymphocytes enhancement by ALL+IL-2 activity ALL+IL-2 activited lymphocytes were co-cultured with Hepa1-6 cells for 24-96h,and the results of CCK8 showed that the growth of Hepa1-6 cells was obviously inhibition,and with the increase of lymphocyte effect target ratio,the growth inhibition of Hepa1-6 cells gradually increased(P<0.05),the supernatant lactate dehydrogenase(LDH)activity was obvious increased,the LDH activity was much higher than the other groups when the effective target ratio was 10:1and 20:1(P<0.05).Flow cytometry was used to detect the apoptosis rate of Hepa1-6 cells in groups with different target-efficiency ratios after lymphocytes activated by ALL+IL-2 and Hepa1-6 cells were co-cultured for 72h.The results showed that ALL activated lymphocytes can promote hepatocellular carcinoma cell apoptosis in different effect-target ratio groups(P<0.05).Among them,the effect-target ratio of 10:1 group and the 20:1 group the apoptosis rate of cells is the highest.Elisa method was used to detect IL-2,TNF-αand Gr B in the supernatant of ALL+IL-2 activated lymphocytes and mice liver cancer cells in each target group(E:T=1:1,E:T=10:1 and E:T=20:1)secretion levels,the results show that as the effect-target ratio increases,the cytokine secretion level also increases,and the difference between each effect-target ratio is statistically different(P<0.05),and the level of cytokines was higher than IL-2 activated lymphocyte co-culture group.2.Effects of ALL on liver cancer growth and different immune cell subtypes.2.1 ALL could inhibits the growth of tumors transplanted in the mice.The tumor volume growth of tumor-bearing mice in the 12.5μg/g ALL and25μg/g ALL treatment groups was significantly inhibited,while the tumor growth of the mice in the PBS group continued to grow(P<0.05),but there was no statistically significant difference between different dose ALL treatment groups.2.2 Effect of ALL on different immune cell subtypes in peripheral blood,spleen and tumor infiltrating lymphocytes of liver cancer mice.2.2.1 ALL significantly increased the ratio of T cells and Tem cells in peripheral blood.The proportion of T lymphocytes,NK cells and Tem cells in the peripheral blood of mice in the ALL high and low dose groups were increased,but the proportion of B lymphocytes,MΦcells and Tcm cells was significantly reduced(P<0.001),while in the ALL high dose groups the proportion of DC cells showed an upward trend(P<0.001).2.2.2 ALL increases the proportion of B,DC and Tem cells in the spleen.The proportion of B lymphocytes,DC cells and Tem cells in the spleen tissue of mice in the ALL high and low dose groups all increased,while the proportion of T lymphocytes,NK cells and MΦcells all showed a decreasing trend.2.2.3 ALL increases the proportion of T,B and Tem cells in tumor infiltrating lymphocytesThe ratio of specific immune T lymphocytes,B lymphocytes and Tem cells in tumor infiltrating lymphocytes in mice of ALL high and low dose groups all increased,while the proportion of non-specific immune cells NK,MΦcells and DC cells were all showed decreasing trend.2.3 ALL induced mice bearing liver cancer to release IL-2,TNF-αand Gr BElisa method was used to detect the secretion levels of related cytokines in the serum of tumor mice after ALL treatment.The concentration of IL-2 in the ALL treatment group was significantly higher than PBS group(P<0.001),and the concentration of IL-2 in the ALL low-dose group were highest,which is statistically significant compared with the high-dose group(P<0.001);the concentration of TNF-αin the high and low dose ALL group was significantly higher than PBS group(P<0.01);the concentration of Gr B in the ALL low-dose group was obvious higher than PBS group and high-dose group(P<0.001).CONCLUSION:This study verified that ALL combined with IL-2 has activation and proliferation effects on lymphocytes in vitro,and can enhance the antitumor effect of lymphocytes.The use of ALL treatment can inhibit the growth of tumors in mice liver cancer models.The mechanism may be mainly through change the inbibition of T,Tem,B and DC cells in peripheral blood,change the proportion of cell subpopulations,and at the same time promote the secretion of related cytokines to achieve anti-tumor effects.