| Project/Area Number |
11670849
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Dermatology
|
|---|
| Research Institution | Keio University |
|---|
Principal Investigator |
SAKURAI Toshiharu (2000) School of Medicine, Keio University Instructor, 医学部, 助手 (20101933)
池田 英之 (1999) 慶應義塾大学, 医学部, 講師 (40301494)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Yuriko School of Medicine, Keio University Instructor, 医学部, 助手 (40255435)
FUJITA Tomonobu School of Medicine, Keio University Instructor, 医学部, 助手 (20199334)
KAWAKAMI Yutaka School of Medicine, Keio University Professor, 医学部, 教授 (50161287)
TOMITA Masato School of Medicine, Keio University Instructor, 医学部, 助手 (90296608)
桜井 敏晴 慶應義塾大学, 医学部, 助手 (20101933)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥3,000,000 (Direct Cost: ¥3,000,000)
|
|---|
| Keywords | HLA tetramer / HLA-A2 / HLA-A24 / Tumor infiltration lymphocyte (TIL) |
|---|
| Research Abstract |
The purpose of this project is to construct the soluble class I MHC-peptide complex ("tetramers") and to visualize the melanoma antigen-specific cytotoxic lymphocytes (CTL) using the produced tetramers.
1. We have produced HLA-A2/gp100_<(209-217)>, HLA-A2/MART-1_<(27-35)>, and HLA-A24/EBV-P tetramers. The β2-microglobulin (β2m) and soluble HLA-A2 and A24 heavy chains linked at its carboxyl terminus to a BirA substrate peptide were expressed separately in Escherichia coli. The expressed HLA-A2 or A24-BirA substrate peptide and β2m subunits were refolded together in vitro in the presence of synthetic antigenic peptides, corresponding to the HLA-A2-restricted melanoma-immunogenic epitopes of gp100_<(209-217)> (ITDQVPFSV) and MART-1_<(27-35)> (AAGIGILTV), and HLA-A24 restricted viral-immunogenic epitope of EBV-P (TYGPVFMCL). Folded materials were then subjected to enzymatic biotinylation with BirA enzyme. The HLA-A2 or A24/peptide complexes were purified first on a get filtration column and
… More
subsequently on a Mono Q ion exchange column. Tetrameric complexes of the biotinylated HLA-A2 or A24/peptide were finally produced by mixing the purified biotinylated heterodimers with Streptavidine-phycoerythrin (PE) conjugates at a molar ratio of 4 : 1.
2. The specific binding of the HLA-A2/gp100_<(209-217)> and HLA-A2/MART-1_<(27-35)> tetramers were assessed by staining the relevant peptide-specific tumor-infiltrating T-lymphocytes (TILs), TIL1520 and TIL1235 respectively, and analyzing them on flow cytometry. The HLA-A2/gp100_<(209-217)> tetramer specifically stained more than 90% of TIL1520 specific for the gp100_<(209-217)> peptide, but, did not stain TIL1370 specific for irrelevant gp100_<(154-162)> (G154 : KTWGQYWQV) peptide. In addition, using HLA-A24/EBV-P tetramer, we have analyzed EBV-P specific CTL that was generated by in vitro stimulation with the peptide from peripheral blood mononuclear cells (PBMCs) of HLA-A24 healthy donor and the CTL were found to stain with the tetramer.
These results indicate that the produced peptide-MHC tetramers can be used to specifically bind to antigen-specific CTL restricted by both HLA-A2 and HLA-A24, and possibly to enrich specific CTL among a heterogeneous population. Less
|
