Reduced Bordetella pertussis-specific CD4+ T-Cell Responses at Older Age

Abstract

Pertussis, a human-specific respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis (Bp), remains endemic with epidemic years despite high vaccination coverage. Whereas pertussis vaccines and natural infection with Bp confer immune protection, the duration of protection varies and is not lifelong. Recent evidence indicates a considerable underestimation of the pertussis burden among older adults. Whereas the impact of increasing age on Bp-specific humoral immunity has been demonstrated, little is known on immunosenescence of CD4⁺ T-cell responses in the context of Bp. Here, we aimed to address whether increasing age impacts responsiveness of the Bp-specific CD4⁺ T-cells in the memory pool following a clinically symptomatic pertussis infection in whole cell vaccine-primed pediatric and adult cases. Cytokine and proliferative responses and phenotypical profiles of CD4⁺ T cells specific for Bp antigens at an early and late convalescent timepoint were compared. Responses of various Th cytokines, including IFNγ, were significantly lower in older adults at early and late timepoints post diagnosis. In addition, we found lower frequencies of Bp-specific proliferated CD4⁺ T cells in older adults, in the absence of differences in replication profile. Phenotyping of Bp-specific CD4⁺ T cells suggested reduced expression of activation markers rather than increased expression of co-inhibitory markers. Altogether, our findings show that the magnitude and functionality of the Bp-specific memory CD4⁺ T-cell pool decrease at older age. Declined CD4⁺ T-cell responsiveness to Bp is suggested to contribute to the burden of pertussis in older adults.

Keywords: Bordetella pertussis; CD4+ T-cell cytokines; IFNγ ELISpot; aging; infection; proliferation.

FIGURE 1

Adults show lower frequencies of PT-specific IFNγ-producing cells and IFNγ levels in the early phase after clinical pertussis (SKI). PBMCs of youngsters (“Y”) and adults (“A”), from an early timepoint (‘E’; within 4 months after diagnosis), and late timepoint (“L”, 8–80 months) after clinical infection were stimulated with Bp proteins PT (left panels), FHA (middle panels) and PRN (right panels) for 5 days. (A) Numbers of IFNγ-spot forming cells per 10⁵ PBMCs measured by ELISpot. (B) IFNγ levels in culture supernatants by Luminex and expressed as [pg/ml]. Symbols show individual cases, while lines and bars indicate medians and interquartile ranges. Statistical significance was calculated with Mann Whitney U-test. *p < 0.05, **p < 0.01; # = significantly lower compared to the early phase.

FIGURE 2

Adults show lower levels of secreted Th-type cytokines after pertussis infection (SKI). Bar graphs show the concentration of IL-2, TNF-α, IL-5, and IL-13 in supernatants of PBMCs in vitro stimulated with Bp proteins PT (left panels), FHA (middle panels) and PRN (right panels) in youngsters (“Y”, white bars) and adults (“A”, dark bars) in early (“E”, open bars) and late (“L”, dotted bars) phase after clinical diagnosis. Dots show individual cases while bars indicate medians and interquartile range. Statistical significance was calculated with Mann Whitney U-test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Longitudinal follow up of older adults confirms lower cytokine production after Bp infection (Immfact). Bar graphs show the concentrations of IL-2, TNF-α, IL-5, and IL-13 in supernatants of PBMCs in vitro stimulated with Bp peptide pools Bp132, PT S1 (shown as PT), FHA and PRN, as indicated, in youngsters (“Y”, white bars) and older adults (“O”, dark bars) in early (“E”, open bars) and late (“L”, dotted bars) phase after clinical diagnosis. Dots show individual cases while bars indicate medians and interquartile range. Statistical significance was calculated with Mann Whitney U-test. *p < 0.05.

FIGURE 4

Frequencies of naïve and memory cells within the CD4⁺ T-cell population in longitudinal PBMC samples of youngsters (Y) and older adults (O) pre- and post in vitro culture. (A) Gating of naive and memory subsets based on CD45RO and CD27 expression. (B) Proportion of naïve/Tem/Tcm/Temra subsets within the CD4⁺ T-cell population at early and late phase prior to stimulation; (C) after 6-day antigenic stimulation in the early phase; and (D) after 6-day antigenic stimulation in the late phase. Stacked bars indicate median relative percentage of subsets per age group and timepoint. Statistical significance of proportional differences between CD4⁺ T-cell subsets of youngsters and older adults was calculated with Mann Whitney U-test. *p < 0.05.

FIGURE 5

Reduced proliferative response of Bp-specific CD4⁺ T cells at older age. PBMCs of youngsters (Y) and older adults (O) of early (E) and late (L) timepoints were labelled with a proliferation dye and subsequently assessed on sequential halving of fluorescent intensity after 6-days stimulation Bp peptide pools Bp132, PT S1 (shown as PT), FHA, and PRN. (A) Gating strategy of proliferating and non-proliferating CD4⁺ T cells as shown for Bp132 peptide pool-stimulated (left plot) and medium (unstimulated) (right plot) conditions. (B) Frequencies of proliferating CD4⁺ T cells post stimulation. (C) Replication index. Dots show background subtracted frequencies of individual cases, while bars represent medians and interquartile range youngsters (Y), white bars; older adults (O), dark bars; early (E) phase samples, open bars; late (L) phase samples, dotted bars, as indicated. Statistical significance was calculated with Mann Whitney U-test and Wilcoxon matched pairs signed-rank test. * = p < 0.05.

FIGURE 6

Minor age-related phenotypical differences of proliferated CD4⁺ T cells responding to Bp132 stimulation in youngsters and older adults in the early phase. (A) Cell density maps show phenotyping analysis by dimensionality reduced single cell data by viSNE show clustering of populations within the proliferating cells of youngsters and older adults of pooled flow cytometry datafiles pre-gated on proliferating CD4⁺ T cells and identified clusters indicated with letters a-g. (B) Dot-plot z-axis shows the location and intensities of each marker within the identified clusters. (C) Frequencies of identified viSNE clusters, based on their identifying marker expression as indicated, were compared on individual levels via FlowJo analysis. Bars represent median frequency of proliferated CD4⁺ T cells with interquartile range. Cluster proportions in youngsters and older adults as indicated identified by vISNE analyses. (D) Heatmap showing Arcsinh-transformed median expression of markers within each cluster of the viSNE plots of youngsters and older adults. (E) Frequencies of CD127^-FoxP3^-Helios⁺ proliferated CD4⁺ T cells in youngsters and older adults. Statistical significance was calculated with Mann Whitney U-test. * = p < 0.05, ** = p < 0.01.