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  • Title: A sealed and unbreached system for purification, stimulation, and expansion of cytomegalovirus-specific human CD4 and CD8 T lymphocytes.
    Author: Li Pira G, Bottone L, Ivaldi F, Pelizzoli R, Risso M, Tripodi G, Manca F.
    Journal: Transfusion; 2006 Dec; 46(12):2053-62. PubMed ID: 17176316.
    Abstract:
    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. STUDY DESIGN AND 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. CONCLUSION: 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.
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