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A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood
Published on: February 8, 2016
High-content cytometry and transcriptomic biomarker profiling of human B-cell activation
Christian Hennig1, Claudia Ilginus1, Kaan Boztug2
1Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hannover, Germany.
Insights
Researchers identified novel biomarkers for primary antibody deficiencies by studying B-cell differentiation kinetics. Complete absence of class-switched B cells is a promising diagnostic marker for activation-induced cytidine deaminase defects.
Area of Science:
- Immunology
- Cell Biology
Background:
- Primary antibody deficiencies are common but complex inborn immunodeficiencies.
- Understanding the cellular and molecular pathogenesis is crucial.
Purpose of the Study:
- To investigate CD40 ligand/IL-21-induced B-cell differentiation kinetics.
- To identify novel biomarker sets for primary antibody deficiency research.
Main Methods:
- Utilized high-content screening, including chip cytometry and RNA microarrays.
- Monitored B-cell activation, differentiation, and gene expression in vitro.
Main Results:
- Successfully tracked B-cell activation, proliferation, and immunoglobulin class-switching.
- Discovered new pathways in B-cell activation, like CXCL9/CXCL10 secretion.
- Identified a biomarker set that predicted AID and DNA repair defects in patients.
- Complete absence of class-switched B cells predicted AID deficiency.
Conclusions:
- The identified biomarkers can advance the study of B-cell activation.
- These biomarkers may elucidate the development of primary antibody deficiencies.
Background:
Primary antibody deficiencies represent the most prevalent, although very heterogeneous, group of inborn immunodeficiencies, with a puzzling complexity of cellular and molecular processes involved in disease pathogenesis.
Objective:
We aimed to study in detail the kinetics of CD40 ligand/IL-21-induced B-cell differentiation to define new biomarker sets for further research into primary antibody deficiencies.
Methods:
We applied high-content screening methods to monitor B-cell activation on the cellular (chip cytometry) and transcriptomic (RNA microarray) levels.
Results:
The complete activation process, including stepwise changes in protein and RNA expression patterns, entry into the cell cycle, proliferation and expression of activation-induced cytidine deaminase (AID), DNA repair enzymes, and post-class-switch expression of IgA and IgG, was successfully monitored during in vitro differentiation. We identified a number of unknown pathways engaged during B-cell activation, such as CXCL9/CXCL10 secretion by B cells. Finally, we evaluated a deduced set of biomarkers on a group of 18 patients with putative or proved intrinsic B-cell defects recruited from the European Society for Immunodeficiencies database and successfully predicted 2 AID defects and 1 DNA repair defect. Complete absence of class-switched B cells was a sensitive predictor of AID deficiency and should be further evaluated as a diagnostic biomarker.
Conclusion:
The biomarkers found in this study could be used to further study the complex process of B-cell activation and to understand conditions that lead to the development of primary antibody deficiencies.
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