Purification of Ag-specific T lymphocytes after direct peripheral blood mononuclear cell stimulation followed by CD25 selection.: I.: Application to CD4+ or CD8+ cytomegalovirus phosphoprotein pp65 epitope determination

被引:24
作者
Gallot, G
Vivien, R
Ibisch, C
Lulé, J
Davrinche, C
Gaschet, J
Vié, H
机构
[1] Inst Biol, INSERM U463, F-44035 Nantes, France
[2] INSERM U395, Toulouse, France
关键词
D O I
10.4049/jimmunol.167.8.4196
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
The two main constraints that currently limit a broader usage of T cell therapy against viruses are the delay required to obtain specific T cells and the safety of the selection procedure. In the present work we developed a generally applicable strategy that eliminates the need for APC for timing reasons, and the need for infectious viral strains for safety concerns. As a model, we used the selection of T lymphocytes specific for the immunodominant CMV phosphoprotein pp65. PBMC from healthy seropositive donors were first depleted of IL-2R alpha -chain CD25(+) cells and were then stimulated for 24-96 h with previously defined peptide Ags or with autologous PBMC infected with a canarypox viral vector encoding the total pp65 protein (ALVAC-pp65). Subsequent immunomagnetic purification of newly CD25-expressing cells allowed efficient recovery of T lymphocytes specific for the initial stimuli, i.e., for the already known immunodominant epitope corresponding to the peptides used as a model or for newly defined epitopes corresponding to peptides encoded by the transfected pp65 protein. Importantly, we demonstrated that direct PBMC stimulation allowed recovery not only of CD8(+) memory T lymphocytes, but also of the CD4(+) memory T cells, which are known to be crucial to ensure persistence of adoptively transferred immune memory. Finally, our analysis of pp65-specific T cells led to the identification of several new helper and cytotoxic epitopes. This work thus demonstrates the feasibility of isolating memory T lymphocytes specific for a clinically relevant protein without the need to prepare APC, to use infectious viral strains, or to identify immunodominant epitopes.
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收藏
页码:4196 / 4206
页数:11
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