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 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.
Gallot
G
G
Institut National de la Santé et de la Recherche Médicale Unité 463, Nantes, France.
Vivien
R
R
Ibisch
C
C
Lulé
J
J
Davrinche
C
C
Gaschet
J
J
Vié
H
H
D000328:Adult; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000276:immunology*; D015704:CD4 Antigens / Q000276:immunology; D016827:CD8 Antigens / Q000276:immunology; D028121:Canarypox virus / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells; D003601:Cytotoxicity Tests, Immunologic; D000939:Epitopes; D005822:Genetic Vectors; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D007156:Immunologic Memory; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology*; D015375:Receptors, Interleukin-2 / Q000302:isolation & purification; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000276:immunology*
D000956:Antigens, Viral; D015704:CD4 Antigens; D016827:CD8 Antigens; D000939:Epitopes; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D010750:Phosphoproteins; D015375:Receptors, Interleukin-2; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa