Cytotoxic T lymphocyte
a cytotoxic, t lymphocyte technology, applied in the field of t lymphocytes, can solve the problems of insufficient preparation of cells in a necessary amount for immunization, troublesome induction of a ctl utilizing the antigen peptide, and high labor intensity, and achieve excellent effect, excellent effect, and excellent effect of specific recognition
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example 1
Identification of HLA-A2402-Restricted CTL Epitope (Antigen Peptide) Using HLA-A2402 Transgenic Mice
(1) Materials and Method
HHDA2402±β2m− / − mice
[0127] A DNA construct (HHDA2402) containing an HLA-A2402 leader sequence, human β2 microglobulin, HLA-A2402 α1 and α2 domains, an H-2Db α3 transmembrane domain, and a cytoplasm domain was constructed. The resulting construct was cloned into an expression vector pcDNA 3.1 (manufactured by Invitrogen Corporation). The HHDA2402 construct (4 kb SalI-NotI fragment) was injected into fertilized eggs of C57BL / 6 mice, to give HHDA2402-expressing mice. Then, the HHDA2404-expressing mice were bred with β2m− / − mice (manufactured by The Jackson Laboratory). The resulting HHDA2402±β2m± were bred with β32m− / − mice to give HHDA 2402±β2m− / − mice (referred to hereinafter as “HHDA2402 mice”).
(2) Cell Strain
[0128] TAP transporter-deficient strain T2 (J. Immunol., 167, p.2529-2537 (2001)) was transfected with an HLA-A2402 cDNA to prepare T2A24 strain. ...
example 2
Preparation of HLA-2402 Antigen Peptide-Specific Human Cytotoxic T Lymphocytes (CTLs) Using CD4-Positive PHA Blast Cells into which mRNA was Introduced
(1) Preparation of mRNA
[0144] MAGE-A4 plasmid and SAGE plasmid were linearized. The resulting products and T7 polymerase (trade name: mMESSAGE mMACHINE T7 Kit, manufactured by Ambion, Inc.) were used, to conduct in vitro transfer according to a manufacturer's manual. Thereafter, the resulting product was polyadenylated with poly A polymerase (trade name: Poly(A) Tailing Kit, manufactured by Ambion, Inc.) according to a manufacture's manual. The resulting RNA was stored at −80° C. until use.
(2) Preparation of CD4-Positive Phytohemagglutinin (PHA) Blast Cells
[0145] By using positive selection using MACS CD4 microbeads (manufactured by Miltenyi Biotec), fresh CD4-positive cells were separated from PBMC. The resulting CD4-positive cells were seeded on a 24-well plate (manufactured by Coming Incorporated) at a cell density of from 1 ...
example 3
SAGE715-723-Specific CD8+ T Cells are Induced from A2402-Positive Healthy Normal Humans with High Probability
In Vitro Induction of Human CTLs Using CD8− PBMC Pulsed with a Peptide
[0166] The number 1×107 of CD8-negative PBMC were pulsed with 10 μM peptide by incubation at room temperature for 1 hour and at 37° C. in 5% CO2 for 1 hour, in 200 μl RPMI1640 medium containing 25 mM Hepes, 10 wt % inactivated human AB-positive serum, 2 mM L-glutamine, 100 U / milliliter penicillin, and 100 μg / milliliter streptomycin. The resulting cells were used as antigen-presenting cells. The number 5×105 of CD8+ T cells that were separated were then stimulated by incubation for from 10 to 12 days with 1×106 cells of the peptide-pulsed CD8− PBMC. On Days 1, 4 and 7, half amount of the medium was exchanged with fresh one, and human IL-2 (20 IU / milliliter) and IL-7 (50 ng / milliliter) were added thereto. Culturing for induction was conducted in 200 μl RPMI1640 medium (containing 25 mM Hepes, 10 wt % inact...
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