High-content cytometry and transcriptomic biomarker profiling of human B-cell activation

J Allergy Clin Immunol. 2014 Jan;133(1):172-80.e1-10. doi: 10.1016/j.jaci.2013.06.047. Epub 2013 Sep 5.


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.

Keywords: AID; Activation-induced cytidine deaminase; B-cell immunology; CD40 ligand; CD40L; CSR; CVID; Class-switch recombination; Common variable immunodeficiency; MACS; Magnetic-activated cell sorting; PID; Primary immunodeficiency; chip cytometry; primary antibody deficiency.

MeSH terms

  • Adolescent
  • Adult
  • B-Lymphocytes / immunology*
  • Biomarkers / metabolism
  • Cell Differentiation
  • Cells, Cultured
  • Chemokine CXCL10 / genetics
  • Chemokine CXCL10 / metabolism
  • Chemokine CXCL9 / genetics
  • Chemokine CXCL9 / metabolism
  • Child
  • Female
  • Gene Expression Profiling
  • High-Throughput Screening Assays
  • Humans
  • Image Cytometry
  • Immunoglobulin Class Switching
  • Immunologic Deficiency Syndromes / diagnosis*
  • Immunologic Deficiency Syndromes / immunology
  • Infant, Newborn
  • Lymphocyte Activation / genetics*
  • Male
  • Microarray Analysis
  • Middle Aged
  • RNA, Messenger / analysis
  • Transcriptome / immunology
  • Young Adult


  • Biomarkers
  • Chemokine CXCL10
  • Chemokine CXCL9
  • RNA, Messenger