AUTHOR=Botafogo Vitor , Pérez-Andres Martín , Jara-Acevedo María , Bárcena Paloma , Grigore Georgiana , Hernández-Delgado Alejandro , Damasceno Daniela , Comans Suzanne , Blanco Elena , Romero Alfonso , Arriba-Méndez Sonia , Gastaca-Abasolo Irene , Pedreira Carlos Eduardo , van Gaans-van den Brink Jacqueline A. M. , Corbiere Véronique , Mascart Françoise , van Els Cécile A. C. M. , Barkoff Alex-Mikael , Mayado Andrea , van Dongen Jacques J. M. , Almeida Julia , Orfao Alberto 

TITLE=Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube

JOURNAL=Frontiers in Immunology

VOLUME=Volume 11 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.00166

DOI=10.3389/fimmu.2020.00166

ISSN=1664-3224

ABSTRACT=CD4+ T-cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow-cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models -monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM) and common variable immunodeficiency (CVID).  Overall, 130 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design-testing-evaluation-redesign, combined with in vitro functional studies, gene expression profiling and multicentric evaluation of manual vs automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0-89y) were used to establish reference values and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL (n=8), SM (n=7) and CVID (n=10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T-cells, Th1, Th17, TFH and Tregs from birth until 2y, followed by a decrease of naïve, Th2 and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4-tube allows fast, automated and reproducible identification of ≥89 subsets of CD4+ blood T-cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.