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, 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.
Highlights
The heterogeneous CD4+ T cells coordinate adaptive immune responses via secretion of cytokines and direct cell-to-cell contact [1]
All 12 markers mentioned above were stained together, and the profiles for the 10 CD4+ T-cell maturation-associated markers were compared via multivariate analysis (PCA) of single cells to identify the optimal combination of markers for maturation-related subsetting of blood CD4+ T cells (Figures 1E–R)
The preliminary age-related reference values set the basis for future immune monitoring in the clinical settings
Summary
The heterogeneous CD4+ T cells coordinate adaptive immune responses via secretion of cytokines and direct cell-to-cell contact [1]. CD4+ T cells comprise multiple functional subsets, including different subpopulations of Thelper (Th) cells [i.e., both classical Th cells, such as the Th1, Th2, and Th17 subsets [1, 3, 4], and non-classical Th1/Th17, Th22, Th9, or Th25 cells [1, 5]] each orchestrating different immune responses [2], regulatory T cells (Tregs) [1, 2], and follicular helper T (TFH) cells [1, 6] Since these multiple functionally distinct populations of CD4+ T cells play a critical role in coordinating immune responses, monitoring of their kinetics has become relevant in distinct conditions such as autoimmune and inflammatory diseases [7], allergy [8, 9], organ/tissue transplantation [10, 11], classical and novel targeted therapies (e.g., antitumor immunotherapy) [12], and vaccination [13,14,15]. Minimally invasive blood monitoring might still be possible to dissect the composition of the CD4+ T-cell compartment in clinical settings, if sufficient cells are acquired
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