[The characteristics of anti-B-cell malignance cytotoxic T lymphocytic clones induced by lymphoma relative peptides].
OBJECTIVE
To analyze if the cytotoxic T-cell (CTLs) are peptide-special and HLA restrict, and what is the sequence characteristic of TCRbeta genes.


METHODS
Using an antigen-specific T-cell expansion system in vitro, the peripheral blood mononuclear cells (PBMCs) from healthy HLA-A0201 positive donor were stimulated by PBMCs and T2 cells loading the IgHV1-QLVQSGAEV nonapeptide or IgHV3-QLVQSGAEV, B-lymphoma-related nonapeptides, as antigen presenting cells (APCs) once a week for four weeks so as to obtain peptide-specific CTLs. PBMCs from non-HLA-A * 0201 positive donors were used as controls. The immunophenotypes of the CTLs (CD3, CD4, or CD8) were identified by flow cytometry. The proportions of CD8 and peptide/tetramer double positive cells were assayed by using peptide/ HLA tetramer method, The IFN-gamma-releasing capacity of the CTLs incubated together with different target cells was assayed by ELISA. The changes of lymphocyte clones were analyzed TCR beta genes were identified by RT-PCR and spectral type method and then sequenced.


RESULTS
After four times stimulation, the CD4/CD8 ratio of the cultured cell decreased obviously from 1.43 to 0.10 (P < 0.05), showing a proliferation of CD8(+) CTLs. The frequency of CD8 and IgHV1-QLVQSGAEV/ HLA-A * 0201 tetramer double positive CTLs was 49.83%, significantly higher than that before stimulation (0.04%). The IFN-gamma secretion detected by ELISA indicated that these CTLs were capable of recognizing the target cells in a peptide-specific and MHC-restricted way. Spectral type method showed that the TCRbeta repertoires were skewed in only a few TCR families.


CONCLUSIONS
The peptides derived from IgHV succeeds in generation of peptide-specific CTLs in vitro in a clonality manner. These CTLs are capable of recognizing the target cells in a peptide-specific and MHC-restricted way, Understanding the function of these CTLs and the molecular structures of these TCR identification-related antigen peptides help discover the interaction between the B-cell and T-cell clones.
Guo
Xiao-ling
XL
Department of Hematology, Peking University First Hospital, Beijing 100034, China.
Zhu
Ping
P
Zhu
Xia
X
Liu
Jing
J
Ou
Yuan
Y
Du
Jin-wei
JW
D016516:CD4-CD8 Ratio; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D019674:Genes, T-Cell Receptor beta; D006801:Humans; D016393:Lymphoma, B-Cell / Q000276:immunology / Q000378:metabolism*; D008297:Male; D010455:Peptides / Q000494:pharmacology; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis* / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000378:metabolism; D014157:Transcription Factors / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; C485514:ZBTB7A protein, human