Basic StudiesSimultaneous Isolation of CD8+ and CD4+ T Cells Specific for Multiple Viruses for Broad Antiviral Immune Reconstitution After Allogeneic Stem Cell TransplantationZandvliet, Maarten L.*; van Liempt, Ellis†; Jedema, Inge†; Kruithof, Simone†; Kester, Michel G.D.†; Guchelaar, Henk-Jan*; Falkenburg, J.H. Frederik†; Meij, Pauline*Author Information *Department of Clinical Pharmacy and Toxicology †Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands All authors have declared that there are no financial conflicts of interest with regard to this work. Reprints: Maarten L. Zandvliet, Department of Clinical Pharmacy and Toxicology, L0-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands (e-mail: email@example.com). Received May 9, 2010 Accepted January 2, 2011 Journal of Immunotherapy: April 2011 - Volume 34 - Issue 3 - p 307-319 doi: 10.1097/CJI.0b013e318213cb90 Buy Metrics Abstract Opportunistic viral infections can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation. Clinical studies have shown that adoptive transfer of donor-derived T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV), or human adenovirus (HAdV) can be a safe and effective treatment of infections with these major viral pathogens. The aim of this study was to develop a method for the simultaneous isolation of coordinated CD8+ and CD4+ memory T-cell responses against a broad repertoire of viral epitopes. To ensure that the method was applicable to a wide variety of virus-specific T cells that may differ in phenotypic and functional properties, we focused on T cells specific for the persistent viruses, CMV and EBV, and T cells specific for HAdV and influenza (FLU), which are not repetitively activated in vivo after initial viral clearance. Following in vitro activation, nearly all T cells specific for these viruses produced interferon γ (IFN-γ) and tumor necrosis factor α, and expressed CD137, whereas the populations varied in the production of interleukin-2, degranulation, and expression of phenotypic markers. Different kinetics of IFN-γ production were observed in CMV/EBV-specific T cells and HAdV/FLU-specific T cells. However, after the stimulation of peripheral blood from seropositive donors with viral protein-spanning peptide pools, the activated virus-specific CD8+ and CD4+ T cells could be simultaneously isolated by either IFN-γ-based or CD137-based enrichment. This study provides an efficient and widely applicable strategy for the isolation of virus-specific T cells, which may be used for the reconstitution of virus-specific immunity in allogeneic stem cell transplantation recipients. © 2011 Lippincott Williams & Wilkins, Inc.