Frontiers in Immunology (Feb 2020)

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

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

DOI
https://doi.org/10.3389/fimmu.2020.00166
Journal volume & issue
Vol. 11

Abstract

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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, 145 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 0–89 years) 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, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, 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.

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