Targeted cloning of cytotoxic T cells specific for minor histocompatibility antigens restricted by HLA class I molecules of interest.
BACKGROUND
T cells specific for minor histocompatibility antigens (mHAgs) play a major role in both graft-versus-host disease and the graft-versus-tumor effect after HLA-identical bone marrow transplantation (BMT). However, characterization of individual T-cell responses to mHAgs is difficult and has generally involved extensive screening of T-cell clones isolated from bulk T-cell cultures generated from BMT recipients. In this report, we describe a new approach that permits both direct visualization of CD8 T-cell responses to mHAgs and cloning of T cells reacting with mHAgs presented by individual HLA alleles of interest.


RESULTS
Panels of Epstein-Barr virus-transformed B-cell lines (B-LCL) expressing retrovirally transduced HLA cDNA were used as stimulator cells in an enzyme-linked immunospot (ELISPOT) assay to identify CD8 T cells reacting with mHAgs in cultures generated from postBMT recipient peripheral blood. T cells specific for mHAgs presented by selected HLA alleles could then be captured and cloned using an interferon-gamma secretion assay and magnetic bead selection. A majority of T-cell clones thus isolated exhibited cytolytic activity against the same HLA-transfected B-cell lines used for the ELISPOT assay.


CONCLUSIONS
The ELISPOT assay was useful for identification of the HLA alleles presenting mHAgs recognized by individual T-cell lines. This approach for isolating mHAgs-specific CD8 T-cell clones should assist in characterizing responses restricted by HLA alleles of interest, which are common in a certain ethnic group.
Akatsuka
Yoshiki
Y
Division of Immunology, Aichi Cancer Center, Nagoya, Aichi, Japan. yakatsuk@aichi-cc.jp.
Kondo
Eisei
E
Taji
Hirohumi
H
Morishima
Yasuo
Y
Yazaki
Makoto
M
Obata
Yuichi
Y
Kodera
Yoshihisa
Y
Riddell
Stanley R
SR
Takahashi
Toshitada
T
D000483:Alleles; D016026:Bone Marrow Transplantation; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D015395:Histocompatibility Antigens Class I / Q000032:analysis* / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000378:metabolism; D015778:Minor Histocompatibility Antigens / Q000032:analysis* / Q000235:genetics; D013194:Staining and Labeling; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry* / Q000166:cytology* / Q000502:physiology
D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D015778:Minor Histocompatibility Antigens; D007371:Interferon-gamma