Isolation and expansion of human adenovirus-specific CD4+ and CD8+ T cells according to IFN-gamma secretion for adjuvant immunotherapy.
OBJECTIVE
In patients with lymphopenia following allogeneic stem cell transplantation adenovirus (ADV) infection is associated with high morbidity and mortality despite aggressive antiviral drug therapy. Virus-specific T cells seem to be essential for virus elimination. The aim of this study was to isolate and expand donor-derived human ADV-specific T lymphocytes for adoptive transfer of sufficient cell numbers to restore protective immunity after allogeneic stem cell transplantation.


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


RESULTS
A mean number of 3.4 x 10(6) (+/-3 SD) ADV antigen-specific T lymphocytes were isolated from 0.1 to 2 x 10(9) mononuclear cells from peripheral blood (n=5) or leukapheresis products (n=6). Characterization of ADV-specific T cells after isolation revealed a mean purity of 85.1% (+/-12% SD) using antigen-specific intracellular cytokine staining. Isolated cells were expanded ex vivo for a median of 18 days (range 7-29 days; n=5) to greater than 10(8) total cells using IL-2 and autologous feeder cell stimulation. ADV-specific response to adenovirus antigen was confirmed in the generated T cell lines, using intracellular cytokine staining, IFN-gamma Elispot assay, and (3)H-thymidine uptake. Generated T-cell lines showed specific killing of ADV-infected B-LCL (n=4). Alloreactive proliferation of generated T-cell lines in mixed lymphocyte cultures was significantly reduced when compared to unmanipulated PBMCs.


CONCLUSIONS
Generation of adenovirus-specific T cells in a simple and rapid clinical-grade protocol was established, using IFN-gamma secretion assay with short expansion times, leading to sufficient numbers of ADV-specific T cells that can be used for adoptive immunotherapy.
University Children's Hospital, Eberhard-Karls University, Tübingen, Germany. tsfeucht@med.uni-tuebingen.de
Tobias
T
Feuchtinger
Peter
P
Lang
Klaus
K
Hamprecht
Michael
M
Schumm
Johann
J
Greil
Gerhard
G
Jahn
Dietrich
D
Niethammer
Hermann
H
Einsele
D000257:Adenoviridae Infections / Q000517:prevention & control; D000260:Adenoviruses, Human / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured / Q000276:immunology; D002499:Centrifugation, Density Gradient; D002985:Clinical Protocols; D002999:Clone Cells / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D006801:Humans; D018189:Immunomagnetic Separation; D016219:Immunotherapy, Adoptive; D007371:Interferon-gamma / Q000378:metabolism*; D007937:Leukapheresis; D008213:Lymphocyte Activation; D033581:Stem Cell Transplantation; D014184:Transplantation, Homologous
D000956:Antigens, Viral; D007371:Interferon-gamma