Figure 5. Age-associated B cell responses to the influenza vaccine.

A. Number of samples and sampling timepoints across 2 flu seasons in the young and older adult cohorts that received the same seasonal vaccines. B-C. B/Washington and D-E. B/Phuket flu-specific total IgG antibody expression (B, D) in plasma compared to expression at baseline (Day 0) for both young (n=26) and older (n=42) adult cohorts in Flu Vax Year 1 and Mean percent inhibition of flu hemagglutinin (HA) antigen as determined by the HAI assay (C, E) for both young (n=21) and older (n=22) adult cohorts at days 0 and 7 in Flu Vax Year 1. P-values were calculated using Wilcoxon’s signed-rank test (paired) for the comparison between Day 0 and Day 7, and using the Wilcoxon rank-sum test for all other comparisons. F. Peripheral memory and antibody-secreting B cell population frequency changes in young (teal) and older adult (bronze) cohorts pre-vaccination (Day 0), and post-vaccination (Day 7). P-vals determined by Wilcoxon’s signed-rank test (paired). G. Enrichment plot for the top Hallmark pathway in CD27- effector B cells when comparing Day 7 transcriptome between young and older adults after gene set enrichment analysis. H. Sample level enrichment analysis scores for the Hallmark Reactive Oxygen Species Pathway at each timepoint for CD27- effector B cells in young and older adults. I. Volcano plot of DEGs for CD27- Effector B cells between the age cohorts at Day 7 post-vaccination. Highlighted genes are those previously shown to define a flu-specific effector B cell subset in a vaccinated adult cohort. Dark teal and bronze dots signify significantly different genes, and light-colored dots indicate nominal significance, while gray dots indicate no significance between age cohorts. J-K. Longitudinal expression of selected genes by CD27- effector B cell subset, averaged for each age cohort at each timepoint. L. Representative flow cytometry plot of CD19 and IgG protein expression on CD27- effector B cells in young and older adults at day 7 post-vaccination, based on flow cytometry analysis of 6 young and 6 older adult subjects that overlap with the scRNA data cohort. M. CLR-transformed frequency comparison of surface IgG+ CD27- effector B cells pre- and post-vaccination in young and older adults, as determined by flow cytometry. P-values were determined by Wilcoxon rank sum test with the alternative hypothesis ‘less’. N. Median surface CD19 protein expression comparison on CD27- effector B cells pre- and post-vaccination in young and older adults, as determined by flow cytometry. P-values were determined by Wilcoxon rank sum test with the alternative hypothesis ‘less’.