PE-Cyanine 5
Excitation: 496nm, Emission: 667nm
The present study aimed to establish a novel method for efficiently inducing cytotoxic T lymphocytes (CTLs) in vitro, in order to develop an immune-based therapy for suppressing and killing ovarian cancer cells with a high safety and efficacy. Peripheral blood mononuclear cells (PBMCs) were stimulated with CpG oligodeoxynucleotide (CpGODN) and ginsenoside Rg1, which were united as an immune adjuvant, and human epidermal growth factor receptor 2 (HER2/neu) antigen peptide, in order to establish a specific CTL culture system in vitro. Chromosome karyotype analysis, growth curve construction and flow cytometric analysis of immune phenotypes, including cluster of differentiation (CD)3, CD4 and CD8, were performed to characterize the stimulated PBMCs in vitro. Subsequently, SKOV3 ovarian cancer cells were treated with the specific CTL culture system in vitro, and MTT assays were performed to test the inhibitory and lethal effects of the CTLs on SKOV3 cells. The number of CTLs was significantly increased from day 7 of stimulation with the specific mixture (CpGODN, ginsenoside Rg1 and HER2/neu) (P<0.01), and plateaued on day 19. Following activation, the number of CD3+, CD3+CD4+ and CD3+CD8+ cells was significantly increased (P<0.01). The lymphocyte karyotype did not change following exposure to antigen. After treatment with the specific CTL system, the number of SKOV3 cells in the experimental group was significantly reduced compared with that in the control group (P<0.01). The results of the present study suggested that two novel immune adjuvants, CpGODN and ginsenoside Rg1, could be combined with the HER2/neu antigen peptide to establish a specific CTL culture system in vitro. This system demonstrated a high antigen specificity, safety and proliferative ability, and exerted significant lethal and inhibitory effects on SKOV3 cells in vitro.
The gammadelta T cell receptor for antigen (TCR) comprises the clonotypic TCRgammadelta, the CD3 (CD3gammaepsilon and/or CD3deltaepsilon), and the zetazeta dimers. gammadelta T cells do not develop in CD3gamma-deficient mice, whereas human patients lacking CD3gamma have abundant peripheral blood gammadelta T cells expressing high gammadelta TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human gammadelta TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR-specific antibodies. The gammadelta TCR isolated in digitonin from primary and cultured human gammadelta T cells includes CD3delta, with a TCRgammadeltaCD3epsilon(2)deltagammazeta(2) stoichiometry. In CD3gamma-deficient patients, this may allow substitution of CD3gamma by the CD3delta chain and thereby support gammadelta T cell development. In contrast, the mouse gammadelta TCR does not incorporate CD3delta and has a TCRgammadeltaCD3epsilon(2)gamma(2)zeta(2) stoichiometry. CD3gamma-deficient mice exhibit a block in gammadelta T cell development. A human, but not a mouse, CD3delta transgene rescues gammadelta T cell development in mice lacking both mouse CD3delta and CD3gamma chains. This suggests important structural and/or functional differences between human and mouse CD3delta chains during gammadelta T cell development. Collectively, our results indicate that the different gammadelta T cell phenotypes between CD3gamma-deficient humans and mice can be explained by differences in their gammadelta TCR composition.