The cohort consists of 1,000 healthy volunteers (500 men and 500 women) aged 20–69 y old equally distributed across five decades of life who were selected based on stringent inclusion and exclusion criteria (55 (link)). The study was approved by the Comité de Protection des Personnes—Ouest 6 and the Agence Nationale de Sécurité du Médicament and is sponsored by the Institut Pasteur (ID-RCB no. 2012-A00238-35). The study protocol was designed and conducted in accordance with the Declaration of Helsinki and good clinical practice as outlined in the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guidelines for Good Clinical Practice (https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6/E6_R1_Guideline.pdf), and all subjects gave informed consent. Stimulations were performed on 1 mL whole blood for 22 h using TruCulture tubes (61 (link)), and flow cytometry analyses were performed with an eight-color cytometry panel (62 (link)). Gene expression was performed using the Human Immunology v2 Gene Expression CodeSet, which contains 594 gene probes that encompass major immune pathways and functions, such as the TLR, Jak-STAT, and MAPK signaling pathways, cytokine–cytokine receptor interactions, apoptosis, and the complement and coagulation cascades. For each gene in each stimulated condition, a paired t test was used to compare expression levels in stimulated and nonstimulated states, controlling for FDR. Seven multiple regression models were built to estimate the effects of age and sex on gene expression. Structural equation modeling (44 ) was used to investigate the ways in which the different cell populations mediate the effects of age and sex on gene expression. DNA genotyping was performed using the HumanOmniExpress-24 BeadChip and the HumanExome-12 BeadChip (Illumina). After imputation using the 1,000 Genomes Project imputation reference panel (45 (link)), a final dataset of 5,265,361 SNPs was obtained. eQTLs mapping was performed with a linear mixed model implemented in GenABEL (63 (link)). Interaction effects between variables (genetics, sex, and age) on gene expression were estimated using ProbABEL v.0.4.5 (64 (link)).
Detailed information about the experimental methods and statistical analyses may be found in SI Materials and Methods.