Development of an IFNγ ELISPOT for the analysis of the human T cell response against mumps virus
Wanda G H Han1, Maarten E Emmelot1, Haziz Jaadar1
1Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
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.
Insights
Mumps virus (MuV) outbreaks occur in vaccinated people due to weak T cell immunity. This study developed an assay to measure MuV-specific T cell responses, finding both T and NK cells produce IFNγ.
Area of Science:
- Immunology
- Virology
- Vaccinology
Background:
- Mumps virus (MuV) outbreaks persist in highly vaccinated populations, suggesting potential issues with vaccine-induced T cell immunity.
- Previous studies demonstrated T cell responses to MuV, but the quality of these responses, particularly at the single-cell level, remains under-analyzed.
Purpose of the Study:
- To develop and validate an Interferon-gamma (IFNγ) ELISPOT assay for assessing MuV-specific T cell responses in vaccinated and infected individuals.
- To compare different in vitro stimulation methods for optimal detection of MuV-specific T cell recall responses.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) from healthy vaccinated donors and mumps patients were stimulated with live or inactivated MuV, or MuV-infected antigen-presenting cells (APCs).
- IFNγ production was measured using ELISPOT assay.
- Flow cytometry was used to confirm cell types producing IFNγ, and NK cells were depleted to isolate T cell responses.
Main Results:
- Direct stimulation with live MuV for 24 hours was identified as the preferred method for assay sensitivity and practicality.
- Flow cytometry confirmed that both T cells and Natural Killer (NK) cells produce IFNγ upon live MuV stimulation.
- Depletion of CD56(+) NK cells enabled the specific enumeration of MuV-specific T cell responses by ELISPOT.
Conclusions:
- The developed IFNγ ELISPOT assay is a valuable tool for evaluating memory MuV-specific T cell responses in individuals vaccinated against or infected with mumps.
- Live MuV induces IFNγ production not only from T cells but also from NK cells, highlighting a broader immune response.
