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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 tw

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+H

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 tw, SCOPUS: ar.j, info:eu-repo/semantics/published

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. > 20 years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG1 (SAG1, HP6188, HP6001 and HP6186), IgG2 (SAG2, HP6014 and HP6002), IgG3 (SAG3, HP6095 and HP6050), IgG4 (SAG4), IgA1 (SAA1, H69-11.4 and B3506B4) and IgA2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues.

[Background]: Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated. [Objective]: We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments. [Methods]: B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n = 19) and peripheral blood (n = 215) from healthy donors aged 0 to 90 years by using flow cytometry and nephelometry, respectively. [Results]: IgH-switched MBCs expressing IgG, IgG, IgG, IgA, and IgA were already detected in cord blood and newborns at very low counts, whereas CD27IgMIgD MBCs only became detectable at 1 to 5 months and remained stable until 2 to 4 years, and IgD MBCs peaked at 2 to 4 years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG, IgG, and IgA) peaked later during childhood (2-4 years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG, IgG, and IgA) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11 months for IgM, IgG, IgG, IgG, IgA, and IgA; until 2 to 4 years for IgD; and until 5 to 9 years for IgG and decreasing thereafter. For most IgH isotypes (except IgD and IgG), maximum plasma levels were reached after PC and MBC counts peaked. [Conclusions]: PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of