The Proliferation Of Human Umbilical Cord Blood T Cells By Using Cycloastragalus Combined With Interleukin IL-7 And IL-15 In Vitro

Objective:With the progress of modern medicine,the clinical treatment of cancer has been developed from surgery,radiotherapy,chemotherapy to biotherapy such as molecularly targeted therapy and immune cell therapy.The immune cell therapy,especially the T cell receptor engineered-T cell（TCR-T）therapy and chimeric antigen receptor engineered T-cell（CAR-T）therapy,is a promising new cancer treatment due to their good clinical outcome.These cell therapies are genetically engineered in vitro to enhance the T cell antigen recognition and cytolytic T-cell cytotoxicity.The reinfusion T-cell quality is the key to ensure the good clinical outcome of immunotherapy.Therefore,improvement of T cell isolation,activation and amplification in vitro to obtain more T cells with high activity is an important part of the T cell-based immunotherapy.Cycloastragenol（CAG）is a major saponins component of astragalus membranaceus,and has immunomodulatory,anti-inflammatory and antioxidant effects.It is also a traditional Chinese medicine as a telomerase activator.In this study we cultured T cells from the source of umbilical cord blood with different concentrations of the CAG combined with cytokines and detected T-cell proliferation and phenotypes of T-cell subsets by multicolor flow cytometry,observing the effect of CAG with different concentrations on T cell amplification in vitro.This study optimized the best condition of T-cell cultivation with CAG in vitro and provided an alternative T-cell cultivation method for immunological therapy.Methods:1.Theoretical study:We review the published research papers about T cell biofunctions and the T cell-based immune cell therapy.Considering the modern pharmacological study of CAG,we investigated the effect of CAG on T cell amplification and cell phenotype in vitro.2.Experimental study:2.1 Optimized Culture conditions of T cells in vitro:Sorted T lymphocytes from peripheral blood or umbilical cord blood were activated by anti-CD3/CD28-conjugated magnetic beads in the 1:2,1:1,2:1 or 3:1 bead/T-cell ratio and then cultured with RPMI 1640medium containing 5ng/ml IL-7 and 5ng/ml IL-15 for 7 days.T cells were counted and the phenotype of subsets were assessed at day 7 after stimulation by flow cytometry.The activation conditions of T cells from peripheral blood and umbilical cord blood were optimized.T cells derived from peripheral blood or umbilical cord blood were activated by anti-CD3/CD28-conjugated magnetic beads in a 1:1bead/T-cell ratio and then cultured in complete RPMI 1640 culture medium with（1）recombinant human（rh）IL-2 at 50 IU/ml;（2）rhIL-7 at 5 ng/ml;（3）rhIL-15 at 5ng/ml;（4）rhIL-7 at 5 ng/ml and rhIL-15 at 5 ng/ml each for 7 days.T cells were counted and phenotypes were detected by flow cytometry.The optimal conditions for T cell culture from peripheral blood and umbilical cord blood were obtained.Activated T cells were cryopreserved at different T cell densities（1E7/ml,2E6/ml and5E5/ml）in the 1ml cryopreservation reagent to investigate the optimal cryopreserved density of T cells.Peripheral blood-derived or cord blood-derived T cells were cryopreserved at the same cryopreservation density of 2E6/ml in different T cell cryopreservation volumes（1ml,0.5ml,0.2ml）to investigate the optimal cryopreservation volume of T cells.2.2 Different concentrations of CAG combined with cytokines in vitro expansion of umbilical cord blood-derived T-cells.Cord blood-derived T cells stimulated by anti-CD3/CD28 antibody-conjugated magnetic beads in a ratio of 3:1 bead/cell were cultured in RPMI 1640 complete medium with 5ng/ml IL-7 and 5 ng/ml IL-15 combining CAG at different concentrations of 0,0.04,0.2,1,5μM for 11 days.T cell expansion and cell phenotypes were detected by flow cytometry and statistic analysis was performed using GraphPad Prism 6.0.Results:2.1 The optimal activation conditions of T cells from different sources were different.When the peripheral blood derived T cells were stimulated by anti-CD3/CD28--conjugated magnetic beads in a ratio of 1:2 bead/cell,the cumulative proliferation ratio of T cells cultured continuously for 7 days was the highest,suggesting that the optimal ratio of bead/T cell was 1:2.The umbilical cord blood-derived T-cells were activated in a ratio of 3:1 bead/T cell,the cumulative proliferation ratio of T cells cultured continuously for 7 days was the highest,suggesting that the optimal magnetic bead activation ratio of cord blood-derived T cells was 3:1.In T cell culture experiments with different cytokines,T cells from peripheral blood or umbilical cord blood were cultured with cytokines IL-7 and IL-15for 7 days,which not only promoted T cell proliferation,but also maintained a higher ratio of T_SCM cell,better than the other culture conditions.T cell cryopreservation conditions experiment:the optimal cryopreservation density of T cells derived from peripheral blood was 1E7/ml,and the optimal cryopreservation volume was 1ml.The optimal cryopreservation density of T cells derived from umbilical cord blood was 1E7/ml,and the optimal cryopreservation volume was 0.5ml.2.2 After activation,0.2、1μM of the CAG combined with cytokine IL-7 and IL-15 combination group had a significantly higher effect on promoting T cell amplification than IL–7 group or IL-15group,and can promote the CD197⁺CD45RA⁺phenotypic expression of CD8⁺T cells,which maintain the high percentages of CD8⁺T_SCM subset.The difference between the 5μM CAG group and the DMSO control group was not significant,suggesting that high concentration of CAG did not promote T cell proliferation.Conclusion:low concentration of CAG combined with IL-7 and IL-15 can significantly improve the amplification ability of T cells and maintain a high proportion of T_SCM subsets in CD8⁺T cells.