Isolation and expansion of human cytomegalovirus- specific cytotoxic T lymphocytes using interferon-gamma secretion assay.
OBJECTIVE
The aim of this study was to isolate and expand donor-derived human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs) for adoptive transfer of 10(7) cells per m(2) of body surface area to restore protective immunity after stem cell transplantation.


METHODS
A new strategy to generate HCMV-specific CTLs using the interferon-gamma (IFN-gamma) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 and feeder cell stimulation, is described.


RESULTS
From 1 to 5 x 10(4) HCMV peptide-specific T lymphocytes (greater than 90% CD3(+)CD8(+)) were isolated from 1 to 2 x 10(8) peripheral blood mononuclear cells comparable to 50 to 100 mL of blood from HLA-A*0201 HCMV seropositive blood donors (n = 14) and expanded ex vivo after a median of 16 days (range 8-28 days; n = 13) to greater than 10(7)/m(2) HCMV peptide-specific CTLs using autologous (n = 2) or allogeneic (n = 11) feeder cell stimulation. In three experiments, expansion to 6 weeks was performed, achieving a median of 1.6 x 10(9) cells (range 6.1 x 10(8)-3.3 x 10(9)). Characterization of these HCMV-specific CTL lines revealed an average purity of 89.2% (range 66.2-99.3%) using HCMV pp65 peptide HLA-A*0201 tetramer staining (n = 14) and 89.4% (range 64.4-99.5%) by peptide-specific IFN-gamma secretion (n = 7). A median of 82.6% (range 76.0-88.0%) showed perforin secretion (n = 3) and 57.5% (range 22.2-80.7%) specific lysis of peptide-pulsed T2 cells (n = 5). A median of 52.2% (range 35.2-7.3%) revealed specific killing of HCMV-infected autologous, but not allogeneic, fibroblasts (n = 6).


CONCLUSIONS
IFN-gamma secretion assay allows development of a simple and rapid protocol with short expansion times for generation of greater than 10(7)/m(2) HCMV-specific CTLs for adoptive immunotherapy.
Bissinger
Alfred Lennart
AL
Medizinische Klinik II, Eberhard-Karls-Universität Tübingen, Tübingen, Germany.
Rauser
Georg
G
Hebart
Holger
H
Frank
Friederike
F
Jahn
Gerhard
G
Einsele
Hermann
H
D019264:Adoptive Transfer / Q000379:methods; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D007371:Interferon-gamma