Sex and age differences in antibody responses to seasonal influenza vaccination are mediated by estrogenic upregulation of NF-κB and TNF signaling in B cells

ABSTRACT Sex differences in the humoral immune responses to the seasonal quadrivalent influenza vaccine (QIV) in young adults (YA; 18–49 years old) or high-dose QIV in old adults (OA; 75+ years old) were analyzed to determine how age-related changes, including in steroids, impact sex differences in B cells. 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), that 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 seroconversion at 28 DPV, particularly in YA. Single-cell RNA sequencing demonstrated that 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, which was mitigated in OA. Estradiol treatment of ASCs from YA females, but not males, increased the number and size of HA+ IgG+ cells and expression of ERE genes along the NF-κB-mediated TNF signaling pathway,that was inhibited by an estrogen receptor antagonist. Pharmacological inhibition of either NF-κB or TNF signaling blocked the ability of E2 to upregulate antibody secretion in cells from YA females. This study provides mechanistic insights into estrogen-mediated increases in influenza vaccine-induced antibody responses among reproductive-aged females and suggests a role for estrogen signaling in the reduction of sex differences in vaccine-induced immunity with old age. IMPORTANCE Sex differences in influenza vaccine-induced immune responses become less pronounced with old age, which we hypothesize could be related to changes in circulating gonadal steroids. Our study shows that after receipt of the seasonal influenza vaccine, young adult females, who have elevated estrogenic activity, have more B cells that recognize influenza hemagglutinin; their B cells have greater activity along estrogen signaling and inflammatory pathways, and mount stronger antibody responses than young adult males, with these sex differences being mitigated in old adults. We identify estrogen as a key driver of sex differences in influenza immunity by showing that ex vivo estradiol increases antibody production by B cells through the estrogen receptor and engagement with NF-κB. These findings help explain the biological basis for sex differences in vaccine immunity and suggest that the hormonal environment, not just chronological age, shapes how well a person responds to vaccination.