title: Simultaneous generation of multivirus-specific and regulatory T cells for adoptive immunotherapy.
abstract: Previous studies have established that adoptive immunotherapy with donor-derived virus-specific T cells can prevent/treat viral complications post-stem cell transplant and regulatory T cells show promise as inhibitors of graft-versus-host disease. On the basis of flow cytometric analysis of upregulation of activation markers after stimulation with viral peptide pools, we have developed a rapid and clinically applicable protocol for the simultaneous selection of virus-specific T cells (after stimulation with peptide pools for the immunodominant antigens of cytomegalovirus, Epstein-Barr virus, and adenovirus) and regulatory T cells using CD25 immunomagnetic selection. Using tetramer staining, we detected enrichment of CD8 T cells recognizing peptide epitopes from cytomegalovirus and Epstein-Barr virus antigens after CD25 selection in 6 of 7 donors. Enzyme-linked immunospot assays demonstrated the simultaneous presence of bivirus-specific or trivirus-specific cells in all evaluated donors, with a median 29-fold (6 to 168), 40-fold (1 to 247), and 16-fold (1 to 219) enrichment of cells secreting interferon-γ in response to cytomegalovirus pp65, adenovirus hexon, and Epstein-Barr virus lymphoblastoid cells compared with unmanipulated peripheral blood mononuclear cells from the same donors. Furthermore, the CD25-enriched cells lost alloreactivity in H-thymidine proliferation assays and showed highly effective (median, 98%) suppression of alloreactivity in all evaluated donors. In summary, we have developed a rapid, simple Good Manufacturing Practice compliant methodology for the simultaneous selection of T cells with multiple viral specificities and regulatory T cells. Adoptive transfer of T cells generated using this strategy may enable restoration of cellular immunity to viruses after allogeneic stem cell transplant with a low risk of graft-versus-host disease. Owing to the speed and simplicity of this methodology, this approach may significantly broaden the applicability of adoptive immunotherapy.
authors: Department of Molecular Immunology, Institute of Child Health, Great Ormond Street Hospital for Children NHS Trust, London, UK.
authors: Gertjan
authors: G
authors: Lugthart
authors: Sarah J
authors: SJ
authors: Albon
authors: Ida
authors: I
authors: Ricciardelli
authors: Michel G D
authors: MG
authors: Kester
authors: Pauline
authors: P
authors: Meij
authors: Arjan C
authors: AC
authors: Lankester
authors: Persis J
authors: PJ
authors: Amrolia
mesh_terms: D000256:Adenoviridae / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D036022:Capsid Proteins / Q000276:immunology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000276:immunology* / Q000472:pathogenicity; D003586:Cytomegalovirus Infections / Q000276:immunology* / Q000628:therapy; D003602:Cytotoxicity, Immunologic; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D007165:Immunosuppression Therapy; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007371:Interferon-gamma / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D007959:Lymphocyte Culture Test, Mixed; D010750:Phosphoproteins / Q000276:immunology; D037182:T-Cell Antigen Receptor Specificity; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D014763:Viral Matrix Proteins / Q000276:immunology
chemical_list: D036022:Capsid Proteins; D053645:Interleukin-2 Receptor alpha Subunit; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; C018315:hexon capsid protein, Adenovirus; D007371:Interferon-gamma