Estrogen upregulates NF-κB and TNF signaling in B cells to cause sex and age differences in antibody responses to seasonal influenza vaccination

Sex differences in the humoral immune responses to the seasonal quadrivalent influenza vaccine (QIV) in young adults (YA; 18-49yo) or high dose QIV in old adults (OA; 75+yo) were analyzed to evaluate how age-related changes in steroids impact sex differences in B cell responses. Among YAs, females had greater H3N2, but not H1N1, neutralizing antibody titers and greater proportions of hemagglutinin (HA)+CD19+ B cells and HA+ memory B cells than males through 28 days post-vaccination (DPV), which was not observed among OAs. Machine learning algorithms illustrated that baseline (0 DPV) steroids, including 17-hydroxyprogesterone, estrogens, and testosterone, as well as HA+CD19+ B cells and HA+ antibody-secreting B cells (ASCs) were major predictors of subsequent seroconversion at 28 DPV, particularly in YA. Single cell RNA sequencing demonstrated CD19+ B cells from YA females had greater transcriptional activity at 7 DPV than YA males, with upregulation of genes with estrogen-response elements (EREs) along NF-κB-mediated TNF signaling pathways in B cell subsets, including naïve, memory, and ASCs, which was mitigated in OA. Estradiol treatment of ASCs from YA females, but not males, increased the number and size of HA+IgG+ ASCs and expression of genes with EREs along the NF-κB-mediated TNF signaling pathway, which were inhibited by an estrogen receptor antagonist. Pharmacological inhibition of either NF-κB or TNF signaling blocked the ability of E2 to upregulate antibody secretion from YA female ASCs. This study provides mechanistic insights into estrogen mediated increases in influenza vaccine-induced antibody responses among reproductive-aged females as compared with age-matched males and suggests a role for estrogen signaling in the reduction of sex differences in vaccine-induced immunity with old age. Estrogenic activity in B cells causes greater influenza vaccine-induced immunity in young adult females than age-matched males, with these sex differences being mitigated in old adults.