The Effect Of All On The Proliferation Of T Lymphocytes And The Expression Of Immune Checkpoint Protein

PurposeAffinity chromatography was used to separate and purify Artocarpus lingnanensis lectin（ALL）,to study the proliferation effect of ALL on T lymphocytes,and to further detect the level of immune checkpoint protein expression on CD4 and CD8 T lymphocytes stimulated by ALL.To study whether ALL can promote the proliferation of T lymphocytes by inhibiting immune checkpoints related pathways.MethodsSeparation,purification and analysis of biological activity of ALL.1.Affinity chromatography to separate and purify the ALL at different time and originGrind 60g Artocarpus lingnanensis seeds produced form June in Guangxi,August in Guangxi and June in Hainan into powder,extracted and filtered with PBS,and the protein was fractionated and precipitated by ammonium sulfate.After dialysis,the precipitated protein was applied to a Gal-sepharose 6B affinity chromatography column.After eluting the contaminated protein with PBS,it was replaced with a PBS buffer containing 0.2Mol/L Gal to continue the elution.The elution peak liquid was collected,and the PBS was dialyzed to no Gal.Freeze the dialysate into a dry powder form,which is the purified Artocarpus lingnanensis lectin,and put it at-80℃ for later use.2.Electrophoresis to detect ALL purity and molecular weightDissolve the lyophilized ALL powder with an appropriate amount of PBS,use BCA to determine the protein concentration of the ALL solutions in the three production areas,and calculate the ALL yields in the three production areas.The molecular weight and purity of ALL were analyzed by SDS-PAGE and non-denaturing PAGE.3.Hemagglutination test verifies the biological activity of ALLU-type blood coagulation test was used to detect the coagulation titer of ALL in Guangxi in June,Guangxi in August,and Hainan in June.The effect of ALL on the proliferation and activity of T lymphocytes1.CCK8 and cell count test the effect of ALL on the proliferation of T lymphocytes.The red blood cell lysate method was used to separate the peripheral blood lymphocytes of healthy subjects.The lymphocytes were adjusted to 1.0×10⁶/ml in the RPMI1640 cell culture medium containing 12.5ng/ml IL-2,and the lymphocytes were co-cultured with ALL.CCK8 and cell morphology were used to detect the effect of ALL on lymphocyte proliferation.2.Flow cytometry to detect the effect of ALL on the expression of CD3 T cells.The ALL was co-cultured with T lymphocytes,and flow cytometry was used to detect the effect of ALL on the expression of CD3 T cells.3.Flow cytometry to verify the effect of ALL on the apoptosis of T lymphocytes.Flow cytometry was used to determine the effect of ALL on lymphocyte apoptosis,and to detect the toxic effect of ALL on lymphocytes.4.ELISA technology detects the level of cell cytokine released by ALL stimulated lymphocytes.The ELISA technique was used to detect the levels of cytokines IL-2 and GZMB released by CD4 T cells and CD8 T cells in the cell culture supernatant.The effect of ALL on CD4 and CD8 T immune checkpoint protein expression.Lymphocytes were separated from the blood of healthy subjects by the red blood cell lysate method,and CD4 T and CD8 T cells were screened by CD4 and CD8 immunomagnetic bead positive selection technology.The cells were adjusted to 1.0×10⁶/ml with 12.5ng/ml IL-2 RPMI1640,seed the cells into a six-well plate,add different concentrations of ALL to culture for 24-96h,flow cytometry to detect CD4 T and CD8 T cell surface immune checkpoints PD-1,CTLA-4,TIM-3,The change trend of LAG-3 protein expression.ResultsALL separation and purification and activity analysis.1.Electrophoresis showed that the purified ALL was obtained by affinity chromatography.SDS-PAGE electrophoresis showed that the ALL produced from June in Guangxi,August in Guangxi,and June in Hainan showed two bands with molecular weights of 10KDa and 15KDa,respectively.Non-denaturing PAGE electrophoresis showed a band without obvious miscellaneous bands.The molecular weight was about 60KDa,and the purity was qualified.It is suggested that ALL is a tripolymer composed of two subunits.2.ALL has high hemagglutination activity.The yields of all isolated and purified from Guangxi in June,Guangxi in August,and Hainan in June were 850ug/g,770ug/g,and 820ug/g,respectively.The blood coagulation test showed that the coagulation activity of 2mg/ml and1mg/ml ALL in Guangxi was 2¹²in June,the coagulation activity of 2mg/ml and 1mg/ml ALL in Guangxi was 2¹²in August,and the coagulation activity of2mg/ml and 1mg/ml in Hainan was 2¹²in June.The blood clotting activity was2¹²and 2¹¹,respectively.Therefore,we believe that ALL purified by affinity chromatography has higher hemagglutination activity.ALL has proliferation and activation effects on lymphocytes1.ALL stimulates the colony growth of lymphocytes.Morphological observations showed that lymphocytes grew well after being stimulated by different concentrations of ALL in different places of origin.The cells in the ALL-stimulated group grew in colony style with massive proliferation,and the combination of high-concentration ALL and IL-2 had a better effect.2.ALL stimulates lymphocyte proliferation.After adding ALL-stimulated human lymphocytes to the cell culture medium containing 12.5ng/ml IL-2,the non-stimulated group was compared with the ALL-stimulated group.The number of cells in the ALL-stimulated group began to increase at 48h,increased significantly at 72h,and continued to increase at 96h.The results of CCK8 showed that adding 12.5μg/ml ALL produced in June in Guangxi,August in Guangxi,and June in Hainan,the maximum proliferation rates of lymphocytes were 80.13%±3.83,79.43%±1.04,76.3%±0.70,respectively;Adding 25μg/ml ALL produced in June in Guangxi,August in Guangxi,and June in Hainan to the cell culture medium,the maximum proliferation rate of lymphocytes was 99.03%±3.00,88.3%±4.68,94.03%±1.80,respectively.25μg/ml ALL has a better effect on cell stimulation than 12.5μg/ml ALL.There is a statistically significant difference between the low-concentration ALL stimulation group and the high-concentration ALL stimulation group（P<0.05）.Among them,June in Guangxi and June in Hainan ALL had better effect than the August ALL in Guangxi,and the difference was statistically significant（P<0.05）.Therefore,we will use the ALL produced in June in Guangxi for follow-up experiments.3.ALL can promotes CD3 T cell expression.Flow cytometry detection of CD3 T cell expression showed that ALL began to show effects after 48h.There was no significant difference between the 24h and 48h groups.The 72h,96h 12.5ug/ml ALL group and the 25ug/ml ALL group compared with the control group,the difference was statistically significant（P<0.005）.The expression levels of 96h control group,12.5μg/ml ALL group and 25μg/ml ALL group were 45.93%±1.09,52.5%±0.88,51.96%±1.48,respectively.4.ALL has no obvious toxicity to lymphocytesThe results of flow cytometry detection of lymphocyte apoptosis showed that the lowest apoptotic rates in the control group,12.5μg/ml ALL group,and25μg/ml ALL group were 15.82%±1.31,13.6%±3.07,13.96%±2.04,respectively;There was no statistically significant difference between the pairwise comparisons（P>0.05）.ALL showed no obvious toxicity to lymphocytes.5.ALL improves the release of T lymphocyte activity factorsELISA analysis showed that the release of cytokines IL-2 and GZMB in the ALL stimulation group increased significantly compared with the control group,regardless of the CD4 T or CD8 T subgroups.In CD4 T cell subsets,the levels of IL-2 and GZMB in the control group were 120±0.57pg/ml and9.66±2.51pg/ml,respectively;the levels of IL-2 and GZMB in the 12.5μg/ml ALL group were 471±7.2 pg/ml and 28.63±2.88pg/ml,respectively;the levels of IL-2 and GZMB in the 25μg/ml ALL group were 500±11.13pg/ml and40.86±3.44pg/ml,respectively.In CD8 T cell subsets,the levels of IL-2 and GZMB in the control group were 8.11±0.45 and 9.20±2.55 pg/ml,respectively;the levels of IL-2 and GZMB in the 12.5μg/ml ALL group were 459.66±7.50pg/ml,respectively,48.1±1.90pg/ml;the levels of IL-2 and GZMB in the25μg/ml ALL group were 494.66±7.37pg/ml and 49.13±2.15pg/ml,respectively.Compared with the control group,the levels of IL-2 and GZMB secretion level in the ALL group were significantly different（P<0.001）.ALL has no obvious effect on CD4 T and CD8 T cell immune checkpoints protein expression1.ALL has no significant effect on the expression of CD4 T cell immune checkpoint protein.Flow cytometry showed that after ALL stimulated CD4 T cells for 24-96h,the proportions of PD-1,CTLA-4,TIM-3,and LAG-3 in the control group were 18.2%±7.53,0.57%±0.43,33.13%±14.81,respectively,0.93%±0.15.The proportions of PD-1,CTLA-4,TIM-3,and LAG-3 in the 12.5μg/ml ALL group were 17.93%±7.41,0.50%±0.42,32.73%±14.1,1.04%±0.34,respectively The proportions of PD-1,CTLA-4,TIM-3,and LAG-3 in the 25μg/ml ALL group were 17.33%±6.98,0.48%±0.38,32.46%±13.71,0.99%±0.31,respectively.ALL has no significant difference in the expression of CD4 T cell immune checkpoint protein.It can be seen from the results that the expression levels of the four proteins in CD4 T cells are significantly different.TIM-3 and PD-1 are expressed in higher levels in T cells,but ALL has an effect on the four immune checkpoint proteins in CD4 T cells.The amount of expression has no obvious influence,the difference was not statistically significant.2.ALL has no significant effect on the expression of CD8 T cell immune checkpoint protein.Among the CD8 T cell subsets,flow cytometry showed that after ALL stimulated CD8 T for 24-96h,the proportions of PD-1,CTLA-4,TIM-3,and LAG-3 in the control group were 8%±0.36,1.8%±0.18,70%±6.23,0.26%±0.13,respectively.The proportions of PD-1,CTLA-4,TIM-3,and LAG-3 in the12.5μg/ml ALL group were 6.96%±0.41,1.64%±0.14,74.7%±12.56,0.25%±0.10,respectively.The proportions of PD-1,CTLA-4,TIM-3,and LAG-3 in the 25μg/ml ALL group were 6.91%±0.50,1.66%±0.12,75%±12.73,0.35%±0.075,respectively.By further testing the effect of ALL on the expression of PD-1,CTLA-4,TIM-3,and LAG-3 in the T cell subsets,the results showed that among the CD8 T cell subsets,the ALL stimulated group was compared with the control group.There was no statistically significant difference in the expression of immune checkpoints（P>0.05）.It sμggests that among the T cell subsets,ALL may not promote the proliferation of T lymphocytes by inhibiting the pathway of these four immune checkpoint proteins.Conclusion1.Purified ALL can be obtained by affinity chromatography with high recovery rate and hemagglutination activity.2.ALL has activation and proliferation effects on T cells.The isolated three areas of ALL can promote the growth of T cells,and the effect is better when produced in June in Guangxi and June in Hainan.3.Under the condition of RPMI1640 cell culture medium containing12.5ng/ml IL-2,25μg/ml ALL has more obvious effect on the proliferation of T lymphocytes than 12.5μg/ml ALL.4.Flow cytometry detection of apoptosis results showed that 12.5μg/ml ALL and 25μg/ml ALL had no obvious toxicity to lymphocytes.5.In CD4 T and CD8 T cells activated by ALL,the release of cytokines IL-2 and GZMB increased,and the difference was statistically significant（P<0.001）,indicating that the subset of T cells activated by ALL has stronger cells The ability of factor secretion can better kill tumor cells and play an anti-tumor effect.6.In CD4 T and CD8 T cells activated by ALL,the expression of immune checkpoints was not significantly affected,sμggesting that in the T cell subsets,ALL may not promote T lymphocytes by inhibiting the pathways of these four immune checkpoint proteins.