title: Development of an IFNγ ELISPOT for the analysis of the human T cell response against mumps virus.
abstract: In the last decade, mumps virus (MuV) causes outbreaks in highly vaccinated populations. Sub-optimal T cell immunity may play a role in the susceptibility to mumps in vaccinated individuals. T cell responses to mumps virus have been demonstrated, yet the quality of the MuV-specific T cell response has not been analyzed using single cell immunological techniques. Here we developed an IFNγ ELISPOT assay to assess MuV-specific T cell responses in peripheral blood mononuclear cells (PBMC) of healthy (vaccinated) donors and mumps patients. Various in vitro MuV-specific stimulation methods of PBMC were compared, using either live or inactivated MuV alone or MuV-infected autologous antigen presenting cells, i.e. Epstein Barr Virus-transformed B lymphoblastoid cell lines (EBV-BLCL) or (mitogen pre-activated) PBMC, for their ability to recall IFNγ-producing responder cells measured by ELISPOT. For the detection of MuV-specific T cell responses, direct exposure (24h) to live MuV was the preferred stimulation method when assay sensitivity and practical reasons were considered. Notably, flowcytometric confirmation of data revealed that primarily T cells and NK cells produce IFNγ upon live MuV stimulation. Depleting PBMC from CD56(+) NK cells prior to stimulation with live MuV led to the enumeration of MuV-specific T cell responses by ELISPOT. Our assay constitutes a tool to evaluate memory MuV-specific T cell responses in MuV vaccinated or infected persons. Furthermore, this study provides evidence that live MuV not only induces IFNγ production by T cells, but also by NK cells.
authors.affiliation: Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands. Electronic address: wanda.han@rivm.nl.
authors.forename: Wanda G H
authors.initials: WG
authors.lastname: Han
authors.affiliation: Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Maarten E
authors.initials: ME
authors.lastname: Emmelot
authors.affiliation: Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Haziz
authors.initials: H
authors.lastname: Jaadar
authors.affiliation: Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Hinke I
authors.initials: HI
authors.lastname: Ten Hulscher
authors.affiliation: Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Cécile A C M
authors.initials: CA
authors.lastname: van Els
authors.affiliation: Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Patricia
authors.initials: P
authors.lastname: Kaaijk
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000821:virology; D009109:Mumps virus / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D014709:Vero Cells
chemical_list: D007371:Interferon-gamma
keywords: IFNγ; Mumps virus; NK cells; Optimization; T cell ELISPOT