Abstract Text: The Milieu Intérieur (MI) project aims to establish the boundaries of a healthy immune response and define the determinants of immune response variation, integrating the impact of hereditary and non-hereditary factors. Immunophenotyping of the 1000 healthy donor cohort by flow cytometry in the initial study in 2011 revealed seropositivity to cytomegalovirus, age, and sex as major determinants for the variability of immune cell counts and showed that genetic factors influence the expression levels of surface immune cell markers. Ten years later, 415 participants from the original cohort were recruited for a longitudinal assessment to compare individual ageing effects with population-level effects (MIv3). One of the main objectives is to define the changes in the initially measured immune phenotypes of peripheral blood cells and to study the contribution of genetic, epigenetic and environmental determinants on these modifications. To this end, we have developed two high-dimensional spectral flow cytometry panels that allow deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively) and standardized the protocol for sample handling, staining, acquisition and data analysis. This permits the reproducible quantification of roughly 200 immune cell phenotypes through standardized immunophenotyping at a single site. Repeatability testing of the two panels shows coefficients of variation (CVs) between 1.09% and 9.13% (cell proportions) and between 4.33% and 14.82% (absolute cell numbers). The reproducible results were with CVs in the range of 3.55 –13.30%. This highly standardized protocol was applied to MIv3 cohort and samples from patients with diverse autoimmune diseases.
