Research On CCL21-mediated Antitumor Response In Vitro

During the cancer initiation and progression, immunosurveillance and antitumor response play very important roles. However, there are many mechanisms for the tumor to evade the surveillance: lacking the necessary components to stimulate host immune response; producing immune negative modulators for an negative microenvironment for response; inducing specific T cells apoptosis through Fas ligand; apoptotic cell-mediated immune tolerance. How to improve the tumor immunogenicity and consequent antitumor response are goals for tumor immunotherapy. CC Chemokine Ligand 21, CCL21/6Ckine, is a potent chemoattractant for lymphocytes and mapped to human chromosome 9p13.CCL21 was constitutively expressed by high endothelium venules of lymph nodes, Peyer's patches and stromal cells in the T cell zone of secondary lymphoid organs.Its receptor CCR7 is expressed on naive T cells, B cells, dendritic cells and some cancer cells. Present researches demonstrated that the combination of lymphatic CCL21 and CCR7 induces cells'migration to secondary lymphoid tissue.Therefore,CCL21 is closely related with lymphocytes homing and lymphatic metastasis. In the tumor progression, the reaction between CCL21 and CCR7 facilitates the antitumor response and the tumor metastasis to CCL21-binding lymph node. Our previous study found that intratumoral injection of CCL21 blocked tumor progression, and attracted more infiltrated T cell in tumor.【Objective】Based on previous theories,we designed tumor-derived CCL21 to investigate the effects of tumor-derived CCL21 on CCR7+ tumor cells and immune cells ,and the subsequent antitumor response.Part I Construction and expression of human CCL21 plasmid 1 Construction of human CCL21The full sequence of CCL21 cDNA was amplified by reverse transcription PCR from a cDNA template derived from human spleen and subsequently cloned into red fluorescent protein eukaryotic expression vector pDsRed2-N1, with additional nucleotides for Kozak sequence, EcoRⅠand BamHⅠrestriction sites. The construction of recombinant plasmid was identified with digestion and sequencing.2. Transfection and expression of CCL21 plasmidRecombinant plasmid was transfected into MCF. 12 hours later, the intracellular expression of red fluorescence was observed with inverted fluorescence microscope. The supernatant was harvested and detected with fluorescence spectrophotometer and westernblot. The results indicated that CCL21-RFP was secreted into the supernatantan and its concentration peaked at 72 hours after transfection.After transfected with CCL21, G418 resisted MCF-7 clones were selected The stable transfected MCF-7 cells was named as MCF-7-CCL21.PartⅡThe MCF-7-CCL21 prompts MCF-7 their immunogenicity1. Groups:1)Untransfected MCF-7 control:MCF-72)Vector-transfected MCF-7 control:MCF-7-vector3)CCL21-transfected MCF-7:MCF-7-CCL214)The mixture of MCF-7-vector and MCF-7:MCF-7-vector cocultured with MCF-7 at 1:105)The mixture of MCF-7-CCL21 and MCF-7:MCF-7-CCL21 cocultured with MCF-7 at 1:102.Methods1) HLA classⅠexpression was detected with indirect immunofluorescence FCM Intracelluar TAP-1 expression was checked with westernblot . 2) FasL mRNA in tumor cells was detected with Real-time PCR. ELISA was used for TGF-βdetection in supernatantant.3) Mitomycin-C-induced tumor apoptosis was analyzed with FCM.4) PI staining was performed to detect PBMC proliferation induced by apoptotic cells using FCM.3.Results1) Compared with the controls, MCF-7-CCL21 up regulated its HLA-I and intracellular TAP-1 expression,which induced PBMC proliferation. Synthesization of FasL and the secretion of TGF-βwere decreased.The data suggested that CCL21 enhanced MCF-7 immunogenicity.2) After exposur to Mitomycin-C, more MCF-7-CCL21 cells underwent apoptosis, which mediated more powerful cytotoxicity of PBMC.3) Tumor-derived CCL21 also up-regulated MCF-7 immunogenicity.PartⅢEffects of CCL21 on DC antigen presentation and apoptosis1. Groups1)Blank: DC stimulated with saline2)DC /MCF-7:DC stimulated with MCF-73)DC /MCF-7-vector:DC stimulated with MCF-7-vector4)DC /MCF-7-CCL21:DC stimulated with MCF-7-CCL212. Methods1)DC preparation:PBMCs were isolated and were subsequently allowed to adhere in culture flasks for 7 h. The adherent cells were cultured for 6 d with granulocyte/macrophage colony-stimulating factor (50 ng/mL) or IL-4 (50 ng/mL) and then used as immature DCs.2) DC stimulation: 5×10~6 DCs cocultured with 5×10~6 Mitomycin-C-treated tumor cells for 2 d. 3) Isolated tumor-derived DC with different density of Ficoll-Hypaque①Chemotasis test was performed for DC migration.②FITC-dextran capture test was used to detect DC endocytosis③Real-time PCR was done for CD80/CD86 mRNA expression of DC④TAP-1 expression was detected by westernblot.⑤DC apoptosis was determined by Annexin-V staining⑥westernblot was used to detect Caspase-3 and Bcl-2 in DC⑦Nuclear factor-κB (NF-κB) activity in DC was detected with a NF-κB-luciferase reporter vector4) DC-activated lymphocytes'effects on MCF-7①Lymphocytes activation:The stimulated DC cocultured with lymphocytes for 5 d,at the ratio of 1:10. Then,the nonadheresent cells were harvested as activated effector lymphocytes. 1×10~6 tumor cells were labeled with CFSE, added to 5×107 activated lymphocytes,and cocultured for 48 h at 37℃②Lymphocytes cytotoxic effects was determined by PI and CFSE-double staining③FCM analyzed the percent perforin-producing CD8+ T cells④The concentrations of IFN-γ,TNF-αand IL-10 were detected with ELISA3. Result1)Comparing with the controls,CCL21-transfected MCF-7 facilitated DC migration, endocytosis, intracellular TAP-1 and CD80/CD86 levels, which suggested CCL21-transfected MCF-7 prompts DC antigen presentation.2) CCL21-transfected MCF-7 induced the reduction of DC apoptosis and expression of Caspase-3 , but up-regulated the expression of Bcl-2.3) CCL-21-transfected MCF-7 stimulated DC to induce lymphocytes to transform perforin-producing CD8+ T cell,and stronger cytotoxicity to MCF-7.The secretions of IFN-γ, TNF-αwere enhanced,opposite to IL-10.All data above demonstrated that CCL21-associated DC could elicit specific T cells against MCF-7,enhance the antitumor effects. 【Conclusion】Tumor-derived CCL21 improves tumor immunogenicity, enhanced mitomycin-C-induced apoptosis.Tumor-derived CCL21 facilitates DC antigen presentation,which induces effective antitumor effects of specific T cell and the suppression of negative immune modulators.