Effect Of Cryopreservation On T Cells And Induction Of Activation Of GPC3 Antigen Epitope Peptide On T Cells

Objectives: 1.To establish a culture method for rapid expansion of T lymphocytes in vitro.2.To evaluate the effect the state of T cells before cryopresevation on proliferation,phenotype and function after thaw.3.To establish GPC3 specific T cells for enhancing the tumor killing ability of adoptive immunotherapy in hepatocellular carcinoma(HCC)patients.Methods: 1.We isolated peripheral blood mononuclear cells(PBMCs)from HCC patients,a ALyS based training technology for the experimental group: ALyS505 N culture medium,adding IL-2 induced with traditional culture method;control group: application of 1640 culture medium,adding IFN-gamma +IL-2+IL-1 alpha;two groups of cell morphology,cell number,phenotype and killing efficiency were observed at different time point.2.PBMCs were isolated from HCC patient,and the T cells were frozen during cell culture at 0,4,8,12 days.After 2 months preservation the cells were thawed.After a period of re-culture,cell activity were detected by trypan blue,cell proliferation were evaluated by MTS assay,cell phenotype were detected by flow cytometry,IFN-gamma and IL-10 were detected by ELISPOT.3.We isolated umbilical cord blood PBMCs,attaching to the wall for 60-90 minutes,taking attachment cells to prepare DC,using HepG2 freeze-thaw antigen and GPC3 epitope antigen to load DC,taking TNF-alpha to promote DC maturation,and then collect DC after eight days.The morphology of the cells was observed by phase contrast microscopy,and the expression of DC,CD80,CD86 and CD83 on the surface antigen were detected by flow cytometry.We co-culture eighth days mature DC with suspension cell from the same umbilical cord blood for 7 days,collecting cells and then flow cytometry was used to detect T cell subsets,Th cell polarization and CCR4,CCR5,CCR6,CXCR3 and CXCR4 chemokines.Results: 1.After 18 days,the total number of cells in the experiment group was significantly higher than that in the control group(2-2.8)times(P<0.05),and the proportion of living cells in the two groups was higher than 95%.The ratio of CD25+ in the two groups was up to peak at 7 days.Compared to the control group,it was significantly higher than that in the experiment group(P<0.05)at 7,14 days.During 18 days culture,the proportion of CD3+,CD3+CD8+,CD3+CD56+ cells was increased,CD8+ cell ratio was(58.80 + 11.26)% in the experimental group at 18 days,significantly higher than the control group(40.35 + 4.98)%(P<0.05).The killing efficiency of T lymphocyte induced by SMMC-7721 in the experimental group was significantly higher than that in the control group,and it was 1.6-2.6 times higher than that of the control group(P<0.05).2.The cells in each group were recovered after 2 months of cryopreservation,and they maintained cell morphology.The survival rate of cryopreserved cells in each group was >90%.MTS method detected the proliferation ability for the re-culture cells.We found 4 days(F/T group),8 days(F/T group)and 12 days(F/T)group were significantly increased after 24 hours re-culture,and 0 days(F/T)increased after 72 hours.Compared to fresh group,CD3+,CD3+CD4+ and CD3+CD8+ cells showed no significant change on 4 days(F/T),8 days group(F/T)and 12 days(F/T)group.However,composed of CD3+CD4+ group and CD3+CD8+ cell was changes on the 0 day(F/T)group.The proportion of NKT cells was significantly decreased on 0 days(F/T),and 8 days(F/T)group;the proportion of Treg cells was significantly increased on 0 days(F/T)and 4 days(F/T)group.The results of ELISPOT showed that cryopreservation did not affect the ability of re-culture T cells to secrete IFN-gamma,but IL-10 in the T cells decreased significantly after 8 days(F/T)and 12 days(F/T).3.It was observed that the DC with typical morphology could be induced by different sensitization methods.The expression levels of DC surface antigen CD83,CD80 at 7th days.Compared to T cells without DC,CD8+T was increased on T cell co-culture with DC loaded by different and there was no significantly difference between the different loading way.Compared to T cells without DC,Th1 cell percentage was significantly increased in GPC3 epitope peptide sensitized DC induced T cells(DC-GPC3144-152-T group and DC-GPC3298-306-T group),while the proportion of Th2 cells was significantly decreased.The cell group without DC,sensitized and non sensitized DC induced T cells(DC-T group and DC-GPC3144-152-T group,DC-GPC3298-306-T group,DC-HepG2-T group)can improve the expression of CCR6 and CXCR3.And the T cells induced by DC(DC-T group)compared to the different way to sensitized(DC-GPC3144-152-T group and DC-GPC3298-306-T group,DC-HepG2-T group)were induced by increased expression of CCR6 sensitive DC induced T cells induced by GPC3298-306(DCGPC3298-306-T group)increased the expression of CCR5.Conclusions: Using ALyS culture technique can effectively expand T lymphocytes in HCC patients,and increasing the number of T cells.After 8 and 12 days of culture,T cells were frozen,which could be thawed to the same condition as the fresh culture.GPC3 epitope antigen peptide can effectively sensitizing DC.When cultured with DC,CD8+T lymphocyte subsets can be increased,and the expression of CCR6,CXCR3 and CCR6 can be effectively improved.