Abstract
BackgroundMass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry.MethodsIn this study, we applied CyTOF to comprehensively characterize the circulating immune cell populations in elderly individuals both before and after administration of an investigational adjuvanted protein vaccine against respiratory syncytial virus (RSV) in a Phase 1a trial. Antigen-specific T cell responses to RSV by IFNγ ELISPOT had been observed in most but not all recipients in the highest dose cohort in this trial. Here, CyTOF was used to characterize the cellular response profile of ELISPOT responders and non-responders in this vaccine dose cohort.ResultsBoth CD4+ and CD8+ T cell antigen-specific IFNγ responses were observed. Principal components analysis revealed baseline differences between responders and non-responders, including differences in activated (HLA-DR+) CD4+ and CD8+ T cells, which were higher in non-responders versus responders. Using viSNE to analyze RSV-responsive CD4+ and CD8+ T cells, we also found increased expression of HLA-DR, CCR7, CD127 and CD69 in non-responders versus responders.ConclusionsHigh parameter CyTOF can help profile immune components associated with differential vaccine responsiveness.
Highlights
Mass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry
Viable cells were plated at 300,000 cells/well in quadruplicate in human IFNgamma enzyme-linked immune spot (ELISPOT) plates (Mabtech, USA) and stimulated with either medium containing 0.1% dimethyl sulfoxide (DMSO) or 2 μg/ mL of overlapping peptide pools of respiratory syncytial virus (RSV) F (JPT GmbH, Berlin, Germany). 30,000 viable cells/well were stimulated with Staphylococcus aureus enterotoxin B (SEB) as a positive control
T cell responses by IFNγ ELISPOT As previously reported, pre- and post-vaccination Peripheral blood mononuclear cells (PBMC) obtained from the clinical trial participants were tested for T cell responses by a qualified F-specific IFNγ ELISPOT assay, with a peak response at Day 8 post vaccination [21]
Summary
CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry. Aged adults have decreased immune responses compared to younger adults and are more prone to acute infections as well as reactivation of latent viruses. Waning adaptive immunity can be seen in adults as young as 50 years old [1]. Extensive research on immune senescence in the elderly has identified multiple pathways by which aging mechanisms adversely affect immune responses, T cell responses [2,3,4]. T cells in combination with neutralizing antibodies may have a key role in controlling respiratory viruses such as influenza and respiratory syncytial virus (RSV) that can cause more acute infections in the elderly versus healthy young adults. In an elderly adult population, T cell IFNγ responses to influenza could distinguish between those protected by vaccination and those who subsequently developed influenza illness [8].
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