title: New tools to monitor the impact of viral infection on the alloreactive T-cell repertoire.
abstract: Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B*4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands. l.j.a.d_orsogna@lumc.nl
authors.forename: L J
authors.initials: LJ
authors.lastname: D'Orsogna
authors.forename: A L
authors.initials: AL
authors.lastname: Amir
authors.forename: Y M
authors.initials: YM
authors.lastname: Zoet
authors.forename: P M W
authors.initials: PM
authors.lastname: van der Meer-Prins
authors.forename: A R
authors.initials: AR
authors.lastname: van der Slik
authors.forename: M G D
authors.initials: MG
authors.lastname: Kester
authors.forename: M H M
authors.initials: MH
authors.lastname: Heemskerk
authors.forename: I I N
authors.initials: II
authors.lastname: Doxiadis
authors.forename: D L
authors.initials: DL
authors.lastname: Roelen
authors.forename: F H J
authors.initials: FH
authors.lastname: Claas
mesh_terms: D017951:Antigen Presentation; D000954:Antigens, Surface / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003429:Cross Reactions; D017931:DNA Primers / Q000737:chemistry; D020031:Epstein-Barr Virus Infections / Q000235:genetics / Q000276:immunology* / Q000821:virology; D006680:HLA Antigens / Q000276:immunology* / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000276:immunology / Q000472:pathogenicity*; D006801:Humans; D007156:Immunologic Memory; D010446:Peptide Fragments / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D000954:Antigens, Surface; D000956:Antigens, Viral; D017931:DNA Primers; D006680:HLA Antigens; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell