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

The two main constraints that currently limit a broader usage of T cell the rapy 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 g enerally applicable strategy that eliminates the need for APC for timing re asons, and the need for infectious viral strains for safety concerns. As a model, we used the selection of T lymphocytes specific for the immunodomina nt CMV phosphoprotein pp65. PBMC from healthy seropositive donors were firs t 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 infect ed with a canarypox viral vector encoding the total pp65 protein (ALVAC-pp6 5). Subsequent immunomagnetic purification of newly CD25-expressing cells a llowed efficient recovery of T lymphocytes specific for the initial stimuli , i.e., for the already known immunodominant epitope corresponding to the p eptides used as a model or for newly defined epitopes corresponding to pept ides encoded by the transfected pp65 protein. Importantly, we demonstrated that direct PBMC stimulation allowed recovery not only of CD8(+) memory T l ymphocytes, but also of the CD4(+) memory T cells, which are known to be cr ucial to ensure persistence of adoptively transferred immune memory. Finall y, our analysis of pp65-specific T cells led to the identification of sever al new helper and cytotoxic epitopes. This work thus demonstrates the feasi bility 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.