Amplification Ex Vivo And Cytocidal Activity Of Leukemia Tumor-associated Antigen-specific Cytotoxic T Lymphocytes

Objective This study was aimed to explore the feasibility of generating leukemia tumor associated antigens-specific cytotoxic T lymphocytes(TAA-CTLs) ex vivo and to evaluate cytotoxicity of TAA-CTLs.Methods Peripheral blood mononuclear cells were enrichment by density gradient centrifugtion. TAA-CTLs were generated by stimulation of PBMC with peptide-pulsed DC at an effector-to-target ration of 10:1. Immunophenotype of TAA-CTLs were analyzed by flow cytometer. Cytotoxicity assay was used to evaluate the cytotoxic activity of TAA-CTLs against peptide-pulsed autologous target cells(PHA-Blasts).Results1)Peripheral blood mononuclear cells were treated with GM-CSF, IL-4 combined effects of three days, the cells develop into irregular shape and gathered into a group, some cells from adherent state to the suspended state. Cultured for 7 days after induction of maturation, microscope, most of the cells into a state of suspension, the cells became larger, irregular and there is a lot of burr-like protrusions. Flow cytometry showed that mature mature DC cell surface expression of HLA-DR and CD86 was significantly increased.2) TAA-CTLs expanded from volunteer showed a mean expansion of 3.81±1.61. Phenotyping of the TAA-CTLs were predominantly CD3+(97.22±0.71)% with varying content of CD4+(41.47±27.08)% and CD8+(56.40±11.15)% T cells. It also contain few nature killer cells(0.50±0.31)% and rare residual B cells(0.14±0.20)%. Thesubpopulations of TAA-CTLs and CTLs were not statistically significant differences in the proportion(P>0.05).3) The intracellular cytokines were detected after stimulation with peptide by Flow cytometry. The secretion rates of IFN-γ and TNF- ɑin CD8+TAA- CTLs were(27.67±2.21) % and(34.2±0.71)%, while the rates were(21.6±2.55) % and(9.97±3.44)% in CD4+TAA-CTLs. Compared with the CD8+TAA-CTLs group, the secretion rates of IFN-γ and TNF- ɑwere(1.36±0.04) % and(5.58±0.03)% in CD8+CTLs, the rates of IFN-γ and TNF- ɑwere(0.91±0.06)% and(1.60±0.07)% in CD4+CTLs. The secretions of IFN-γ and TNF- in CTLs were both significantly lower ɑthan that in TAA-CTLs(p<0.01).4) The specific killing efficiency of the TAA-CTLs against TAA-pulesd target cells were(77.00±1.00)%,(67.40±3.60)%,(60.55±2.45)% and(26.85±5.25) %, which were under the effector-to-target ratio of 40:1, 20:1, 10:1 and 5:1. And there was negligible lysis of TAA-CTLs for PHA-blast(P<0.01).We have shown that antigen-specific cytotoxic T lymphocytes can be successfully induced and generated ex vivo from the healthy volunteer peripheral blood and be capable of killling efficiency specificlly.Conclusions:We have shown that antigen-specific cytotoxic T lymphocytes can be successfully induced and generated ex vivo from the healthy volunteer peripheral blood and be capable of killling efficiency specificlly.