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
G. Gallot et al., 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, J IMMUNOL, 167(8), 2001, pp. 4196-4206
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.