Positive selection and expansion of cytomegalovirus-specific CD4 and CD8 T cells in sealed systems: Potential applications for adoptive cellular immunoreconstitution

Administration of pathogen-specific T-cell lines can reconstitute the cellular immune function of immunocompromised patients. Selection and expansion of specific T cells for reinfusion pose unique challenges owing to the fact that good manufacturing procedures must be implemented. Cytokine secretion-based methods can identify and select specific T cells. We showed here that it is possible to combine this method with procedures for cell handling performed in a sealed, unbreached system from start to end. Peripheral blood mononuclear cells, obtained from blood samples and processed in a sealed system, were stimulated in Teflon bags with a library of selected CD4 and CD8 peptides derived from the immunodominant cytomegalovirus protein pp65. The stimulated T cells were labeled with reagents for interferon-γ surface capture and selected on a magnetic column using a sealed system connected to the Teflon bags. Elution and final expansion were also performed with an unbreached protocol with preservation of sterility even if the steps were run on the bench top. Expanded cells exhibited the appropriate functions. The use of this unbreached procedure proves that safety of cellular products generated in a good manufacturing procedures facility can be further improved. Similar sealed protocols can also be applied for T-cell therapies directed against tumor antigens.