A sealed and unbreached system for purification, stimulation, and expansion of cytomegalovirus-specific human CD4 and CD8 T lymphocytes.
BACKGROUND
Recent clinical trials have demonstrated the efficacy of adoptive cellular therapy with virus-specific lymphocytes in patients with defective cellular immune responses. Immunoreconstitution has become a challenge for cellular immunology and for transfusion medicine. In fact, both expertises are required to provide effective and safe cellular products. Because of in vitro manipulation, T-lymphocyte cultures are at risk of contamination even under good manufacturing procedure (GMP) conditions.


METHODS
To further improve the quality of these GMP cellular products, a procedure was designed for purification, stimulation, and expansion of antigen-specific CD4 and CD8 T-lymphocytes in a sealed, unbreached system. Leukopacks from the blood bank that fulfill the requirements of a GMP product were the starting material. Gradient separation and washing were performed in bags with sterile connecting devices on the bench-top, as well as addition of ingredients (antigen, interleukin-2) or transfer to larger bags.


RESULTS
The method is described in detail, and it is shown that increase in number of cytomegalovirus-specific CD4 or CD8 T-lymphocytes was similar to procedures based on open culture systems. Cell expansion after 4 weeks ranged from 800- to 2400-fold for CD4 lymphocytes and 300- to 900-fold for CD8 lymphocytes. Antigen specificity and loss of alloreactivity were demonstrated on the expanded cells with proliferation, intracytoplasmic interferon gamma-gamma staining, cytolytic activity, and pentamer binding.


CONCLUSIONS
This procedure can be applied to improve sterility under GMP conditions when T-cell lines are generated for adoptive immunotherapy and may increase biosafety for the staff when cell lines are generated from subjects infected with dangerous pathogens.
Li Pira
Giuseppina
G
Advanced Biotechnology Center, Genoa, Italy. lipira@cba.unige.it
Bottone
Laura
L
Ivaldi
Federico
F
Pelizzoli
Roberta
R
Risso
Marco
M
Tripodi
Gino
G
Manca
Fabrizio
F
D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D003470:Culture Media; D003587:Cytomegalovirus / Q000276:immunology*; D006801:Humans; D016219:Immunotherapy, Adoptive; D008213:Lymphocyte Activation
D003470:Culture Media