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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 Determination¹

Géraldine Gallot^*, Régine Vivien^*, Catherine Ibisch^*, Jaqueline Lulé, Christian Davrinche, Joëlle Gaschet^* and Henri Vié^2,*

^* Institut National de la Santé et de la Recherche Médicale Unité 463, Nantes, France; and Institut National de la Santé et de la Recherche Médicale Unité 395, Toulouse, France

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 -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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