Expansion of human cytomegalovirus-specific T lymphocytes from unfractionated peripheral blood mononuclear cells with artificial antigen-presenting cells.
BACKGROUND
The aim of this study was to find a simple and feasible method for ex vivo expansion of human cytomegalovirus (CMV)-specific cytotoxic T cells from unfractionated peripheral blood mononuclear cells (PBMNCs).


METHODS
Unfractionated PBMNCs from three HLA-A*0201-CMV-seropositive donors were stimulated with CMVpp65(495-503) peptide-loaded HLA-A*0201-immunoglobulin fusion protein (HLA-A2-Ig) based artificial antigen-presenting cells (aAPCs) on Day 1. Once a week the CMV-specific T cells were harvested and restimulated with fresh aAPCs. T-cell cultures were maintained for 28 days and then analyzed.


RESULTS
With aAPCs and starting with 1x10(7) freshly isolated PBMNCs that were less than 0.1 percent CMV-specific, more than 1x10(7) T cells with a CMV-specific frequency greater than 93 percent in all donors tested were generated. Expanded CD8+ cytotoxic T lymphocytes were functionally active and showed antigen-specific secretion of interferon-gamma and cytotoxic activity. No alloreactivity against unpulsed HLA-A*0201-positive cells was detected.


CONCLUSIONS
Herein is reported the successful in vitro expansion of CMV-specific cytotoxic CD8+ T cells from unfractionated PBMNCs of healthy CMV-seropositive blood donors by the use of HLA-A2-Ig-based aAPCs. This study demonstrates that more than 1x10(7) CMV-specific T cells can be generated from approximately 1x10(7) unfractionated PBMNCs within 1 month under highly reproducible conditions.
Paine
Ananta
A
Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
Oelke
Mathias
M
Blasczyk
Rainer
R
Eiz-Vesper
Britta
B
D000938:Antigen-Presenting Cells; D000956:Antigens, Viral / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018929:Cell Culture Techniques / Q000379:methods; D003587:Cytomegalovirus / Q000276:immunology*; D015789:HLA-A2 Antigen; D006801:Humans; D007136:Immunoglobulins; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008213:Lymphocyte Activation; D010455:Peptides / Q000276:immunology; D011993:Recombinant Fusion Proteins; D037182:T-Cell Antigen Receptor Specificity
D000956:Antigens, Viral; D015789:HLA-A2 Antigen; D007136:Immunoglobulins; D010455:Peptides; D011993:Recombinant Fusion Proteins