The Studies On Apoptosis Of Tumor Cell Induced By CD4～+T Cell From CIKs In Vitro

Objects: To study the apoptosis-inducing activity of CD4~+T cells in CIKs and investigate the underlying mechanism. Methods: After large scale of amplification of CIKs in vitro, CD4~+T cells subset was isolated by magnetic beads separationcolumns. Cytotoxicity of purified CD4~+ T, CD4~-T cells subset in CIKs against raji cells were measured by using LDH-release assay. Apoptotic rates of raji cells cocultured with CD4~+CIK after 4 hours and 24 hours were evaluated by AnnexinV staining and expression of Fas was compared by flowcytometer as well. The influence of cell-cell contact and Fas/FasL interaction on apoptotic rates of raji cells was studied by transwell plate and antibody blocking experiment .The cytokines such as IL-2, IFN-γ, TNF-a secreted in supernatant was detected by ELISA. The expression of CD40L in CD4~+CIK was compared with CD4~+PBMC by RT-PCR, flowcytometry and ELISA. Results: The proportions of CD3~+ cells> CD3~+CD8~+ cells , CD3~+CD4~+ cells , CD3~+CD56~+ cells in CIKs increased significantly comparedwith those of PBMC. Purity of enriched CD4~+T cells reached 96.2%. Few raji cellswere lysed by CD4~+CIK after 4h cocultured. But the apoptotic rates of raji was observed and elevated with the increasing of effector cells after 24h cocultured Flowcytometry showed that Fas on raji cells was increased after 24h cocultured. The apoptotic rates decreased obviously after cocultured with anti-FasL blocking antibody. The cytokines ,such asIL-2, IFN-γ, in supernatant increased and there was no change about TNF-a. Comparing with PBMC, significant increase in mRNA andprotein expression of CD40L in CD4+CIK was observed(P<0.01)ConcIusion: (1)CD4+CIK were the effectors capable of inducing apoptosis in tumor cells through cell-cell-contact. (2)CD4+CIK induce apoptosis of tumor cells mainly through Fas/FasL pathway.(3)Comparing with PBMC, significant increase in mRNA and protein expression of CD40L was observed in CD4+CIK(i><0.01). (4) CD4+CIK can increase the expression of Fas and. induce apoptosis of tumor cell through the interaction of CD40/CD40L.Part II :The effect of CD40/CD40L in apoptosis of tumor cellinduced by CD4+T cells in CIKsObjects: To investigate the effect of CD40L in apoptosis induced by CD4+CIK. Methods: To construct plasmid carried sCD40L gene pIRES2-EGFP-sCD40L. CHO cells was transfected with plasmid pIRES2-EGFP-sCD40L(CHO-sCD40L) and control plasmid pIRES2-EGFP(CHO-pIRES2) separately. After transfected, SCD40L secreted by CHO-sCD40L was evaluated by ELISA. Then they were cocultured with T47D cells to observe the expression of Fas on T47D.The apoptotic rates of T47D cell ware observed after cocultured with the anti-Fas activating antibody(CHll).Results: We constructed plasmid carried sCD40L successfully. CHO cells was transfected with plasmid pIRES2-EGFP-sCD40L successfully and the transfected rate reached about 20%. SCD40L secreted by CHO-sCD40L increased significantly .After cocultured with CHO-sCD40L, the expression of Fas on T47D increased significantly. The apoptotic rates of T47D cell increased cocultured with the anti-Fas activating antibody(CH-ll).Conclusion: Theinteraction of CD40/CD40L can increase the expression of functional Fas andinduce apoptosis on tumor cell.