title: Cytomegalovirus-Specific T Cells Isolated by IFN-γ Secretion Assay Do Not Induce Significant Graft-Versus-Host Reactions In Vitro.
abstract: BACKGROUND
  Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the immunogenicity of virus-specific T cells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage.
  
  
  METHODS
  Cytomegalovirus-specific CTLs were isolated by the IFN-γ secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-γ, cytotoxic activity, IFN-γ and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay).
  
  
  RESULTS
  Cytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65 stimulation by secreting IFN-γ and killing virus peptide loaded autologous phytohemagglutinin (PHA) blasts. Compared with unselected peripheral blood mononuclear cells, CMV-CTLs induced significantly less severe cutaneous GvH tissue damage. This observation coincided with low levels of CD25 expression, as well as IFN-γ and granzyme B secretion after allogeneic antigen stimulation in both the mixed lymphocyte reaction and in the skin explant assay.
  
  
  CONCLUSIONS
  Cytomegalovirus-specific CTLs isolated by the IFN-γ secretion assay from HLA-unmatched healthy donors exhibited a high level of anti-CMV potency without inducing significant cutaneous GvH tissue damage in vitro. This finding provides novel evidence supporting the safe use of in vitro expanded CMV-CTLs as an antiviral therapy in transplant patients with refractory CMV infections.
authors.affiliation: 1 Alcyomics Ltd, Newcastle upon Tyne, United Kingdom. Haematological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom. 2 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. 3 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School (MHH), Hannover, Germany. 4 Haematological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Marsela
authors.initials: M
authors.lastname: Qesari
authors.forename: Anne
authors.initials: A
authors.lastname: Richter
authors.forename: Justyna
authors.initials: J
authors.lastname: Ogonek
authors.forename: Eva
authors.initials: E
authors.lastname: Mischak-Weissinger
authors.forename: Xiao-Nong
authors.initials: XN
authors.lastname: Wang
authors.forename: Anne M
authors.initials: AM
authors.lastname: Dickinson
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D003587:Cytomegalovirus / Q000276:immunology*; D006087:Graft vs Host Reaction; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D007371:Interferon-gamma; D053804:Granzymes