title: Co-ordinated isolation of CD8(+) and CD4(+) T cells recognizing a broad repertoire of cytomegalovirus pp65 and IE1 epitopes for highly specific adoptive immunotherapy.
abstract: OBJECTIVE
  Adoptive transfer of cytomegalovirus (CMV)-specific memory T cells can be used for treatment of CMV reactivation after allogeneic stem cell transplantation. As co-ordinated CD8(+) and CD4(+) T cells specific for a broad repertoire of CMV epitopes may be most effective for adoptive immunotherapy, the aim of this study was to isolate these cells from peripheral blood of CMV seropositive donors, irrespective of their HLA type.
  
  
  METHODS
  Activation of CMV-specific CD8(+) and CD4(+) T cells was compared after stimulation of donor peripheral blood with minimal epitope peptides, pools of overlapping 15-mer peptides or full-length protein. Furthermore, the kinetics of interferon (IFN)-γ production after stimulation was analyzed to determine the optimal time-point for IFN-γ-based isolation of CMV-specific T cells. The specificity, phenotype and functionality of generated T-cell lines were analyzed.
  
  
  RESULTS
  CMV protein-spanning 15-mer peptide pools induced simultaneous activation of both CD8(+) and CD4(+) CMV-specific T cells, while full-length CMV protein only efficiently activated CD4(+) CMV-specific T cells. Isolation of IFN-γ-secreting cells at the peak of the IFN-γ response after 4-h stimulation with CMV pp65 and IE1 peptide pools resulted in efficient enrichment of CMV-specific T cells. The T-cell lines contained high frequencies of CD8(+) and CD4(+) T cells recognizing multiple CMV pp65 and IE1 epitopes, and produced IFN-γ and tumor necrosis factor (TNF)-α upon specific restimulation.
  
  
  CONCLUSIONS
  This study provides a feasible strategy for the rapid generation of clinical-grade CD8(+) and CD4(+) T-cell lines with high specificity for multiple CMV pp65 and IE1 epitopes, which may be used for effective adoptive immunotherapy.
authors.affiliation: Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, the Netherlands. m.l.zandvliet@lumc.nl
authors.forename: Maarten L
authors.initials: ML
authors.lastname: Zandvliet
authors.forename: Ellis
authors.initials: E
authors.lastname: van Liempt
authors.forename: Inge
authors.initials: I
authors.lastname: Jedema
authors.forename: Louise A
authors.initials: LA
authors.lastname: Veltrop-Duits
authors.forename: Roel
authors.initials: R
authors.lastname: Willemze
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: Guchelaar
authors.forename: J H Frederik
authors.initials: JH
authors.lastname: Falkenburg
authors.forename: Pauline
authors.initials: P
authors.lastname: Meij
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D002460:Cell Line; D003587:Cytomegalovirus / Q000472:pathogenicity / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000276:immunology* / Q000503:physiopathology / Q000628:therapy*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000276:immunology / Q000378:metabolism; D007156:Immunologic Memory; D007165:Immunosuppression Therapy / Q000009:adverse effects; D016219:Immunotherapy, Adoptive; D007371:Interferon-gamma / Q000378:metabolism; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D033581:Stem Cell Transplantation; D037182:T-Cell Antigen Receptor Specificity; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000276:immunology / Q000378:metabolism; D014775:Virus Activation
chemical_list: D018984:Epitopes, T-Lymphocyte; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D007371:Interferon-gamma