CTL诱导及杀伤实验

一、抗原肽诱导CTL制备1.PBMCs were separated from the whole blood of HLA-A2+ healthy controls. PBMCs (2 x106/ml) were cultured with each of the HLAA* 0201 refolding peptides at a concentration of 10 uM in RPMI 1640 medium containing 10% FCS and 20 U/ml recombinant human IL-2 (rhIL-2) in 24-well culture plate. Half of the medium was changed at day 4 with supplementation of rhIL-2 at 20 U/ml. At day 7, cells were harvested and tested for the presence of peptide-specific CD8+ T cells by an IFN-γrelease ELISPOT assay.---------------Zhou M, Xu D, Li X, Li H, Shan M, Tang J, Wang M, Wang FS, Zhu X, Tao H et al: Screening and identification of severe acute respiratorysyndrome-associated coronavirus-specific CTL epitopes. J Immunol2006,177(4):2138-2145.2. PBMCs were separated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation from HLAA*0201 normal donors and HCC patients. DCs were generated from peripheral blood monocytes as described by Romani. Briefly, PBMCs were seeded into six-well culture plates containing 3 ml RPMI-1640 and 10% FCS at5–10x106/well. Plates were incubated in a 37o C incubator for 2 h, then the non-adherent cells were removed and the adherent cells were cultured at 37o C in RPMI-1640 supplemented with 10% FCS, 1000 U/ml human recombinant GM-CSF and 500 U/ml human recombinant IL-4. All T cell stimulation was with day 7 DCs. As described below, DCs were matured with lipopolysaccharide (LPS) on day 6 and HCA587 antigen was added for effective processing at this time.Day 6 DCs were resuspended in RPMI-1640 with 1000 U/ml GM-CSF, 500 U/ml IL-4 and 10 ng/ml LPS and incubated at 37 o C. After 24 h cultivation the DCs were collected, washed and pulsed with 10 m g/ml HCA587 peptide for 3 h at 37 o C. The DCs were washed twice for the following stimulation.CD8+T cells were isolated by positive selection with CD8-Dynal immunomagnetic beads according to the manufacturer’s instructions.After washing, CD8+T cells were co-cultured with HCA587peptide-loaded DCs in 2 ml RPMI-1640medium supplemented with 10% AB serum, recombinant human IL-2(10 ng/ml) and IL-6 (500 U/ml). Seven days later, the cultured T cells were restimulated with freshly prepared peptide-pulsed DCs and cultured for another 7 days. After four consecutive rounds of stimulation, cultures were tested for the presence of HCA587-specific CTLs.-----------Li B, Wang Y, Chen J, Wu H, Chen W: Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587. Clin Exp Immunol2005, 140(2):310-319.3.Generation of CTLs in PBMCs from healthy donors：Dendritic cells (DCs) have the unique capacity of activating naive T cells and initiating primary T-cell response. Antigen-specific T-cell responses from peripheral blood mononuclear cells (PBMCs) can be elicited with antigenic peptide-pulsed autologous DCs. We obtained PBMCs from the buffy coat of heparinized whole blood samples of healthy donors by density gradient centrifugation on the Histopaque 1077. These cells were resuspended in serum-free RPMI 1640 and allowed to adhere to six well plates at a final concentration of 1 x 107cells/3 ml/ well. After 2 h of incubation at 37°C, non-adherent cells were gently removed with warm medium by gently pipetting. The non-adherent cells (effector lymphocytes) were cryopreserved in FCS supplemented by 10% DMSO. The resultant adherent cells containing DCs were cultured in medium supplemented with 800 U/ml GMCSF and 1,000 U/ml IL-4 in 37°C/5% CO2. Every 2 days, one-half of the medium was replaced by fresh medium containing a double concentration of GM-CSF and IL-4 as indicated above. On day 5, 10 ng/ml of recombinant human tumor necrosis factor a (TNF-a) was added to the medium to induce phenotypic and functional maturation of DCs. After 48 h, DCs were pulsed with 20 ug/ml peptide in the presence of 3 ug/ml b2- microglobulin at 37°C for 3 h and irradiated at 30 Gy before use. The thawed 2 x106 of non-adherent effector lymphocytes were cocultured with 2 x105 peptidepulsed irradiated autologous DCs in a 24-well plate in the presence of 10 ng/ml recombinant human interleukin-7 (IL-7). After 7 days, lymphocytes were restimulated with peptide-pulsed autologousPBMCs in medium containing 10 ng/ml IL-7 and 20 U/ml IL-2. About 20 U/ml of IL-2 was added 24 h later at regular intervals, 2 days after each restimulation. Lymphocytes were restimulated each week in the same manner. On the seventh day, after the three rounds of restimulation, cells were harvested and tested by ELISPOT assay.--------------An altered peptide ligand for nave cytotoxic T lymphocyte epitope of TRP-2(180–188) enhanced immunogenicity.Cancer Immunol Immunother (2007) 56:319–329 第三军医大吴玉章4.Generation of CTLs in healthy donorsDendritic cells are characterized by the unique capacity to activate naive T cells and initiate primary T-cell response. Antigen-specific T-cell responses from peripheral blood mononuclear cells (PBMCs) can be elicited with antigenic peptide- or protein-pulsed autologous DCs. Here, PBMCs were isolated from whole blood of 11 healthy HLA-A2.1_ volunteer donors by Ficoll/Hypaque density gradient centrifugation. Human peripheral blood monocyte-derived DCs were generated as previously described by us. On day 5 of culture, 10 ng/mL recombinant human tumor necrosis factor was added to the medium to induce phenotypic and functional maturation. Then, 48 hours later, DCs were pulsed with 20 ug/mL peptide in the presence of 3 ug/mL B2-microglobulin at 37°C for 3 hours and irradiated at 30 Gy before use. Peripheral blood lymphocytes (PBLs, 2 x106) were cocultured with 2 x 105 peptide-pulsed irradiated autologous DCs in a 24-well plate in the presence of 10 ng/mL recombinant human interleukin-7. The next day, recombinant human IL-10 was added to the culture medium, to give a final concentration of 10 ng/mL. After 7 days, lymphocytes were restimulated with peptide-pulsed irradiated autologous DCs in medium containing 10 ng/mL IL-7 and 10 ng/mL IL-10, and then supplemented with 20 IU/mL IL-2 24 hours later. Lymphocytes were restimulated each week in the same manner. At 7 days after the fourth round of restimulation, cells were harvested and tested by ELISPOT assay, cytotoxicity assay, and tetramer staining. CD8T lymphocytes were purified by CD4_ cell–negative depletion using human CD4microbeads.---------Wang B, Chen H, Jiang X, Zhang M, Wan T, Li N, Zhou X, Wu Y, Yang F, Yu Y et al: Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein.Blood2004, 104(1):200-206.第二军医大曹雪涛5. 抗原肽诱导CTL制备：密度梯度离心法分离经EDAT抗凝的静脉血中的外周血单个核细胞（peripheral blood mononuclear cells，PBMC)，分离得到的PBMC种于6孔细胞培养板，在含10%FCS的RPMI1640培养基中，37℃5%CO2孵育1.5h。