Presentation: Using Flow Cytometric Analysis of Protein Phosphorylation to Study B Cell Receptor Signaling in Tolerized Human B Cells (2007)

1418 Using Flow Cytometric Analysis of Protein Phosphorylation to Study B Cell Receptor Signaling in Tolerized Human B Cells

Purpose
How tolerance to self is established and maintained is a central questions in immunology. B cells recognized by the 9G4 anti-idiotype antibody (anti-VH4.34 heavy chain) provide an excellent model for studying human B cell tolerance. Although, 9G4+ B cells recognize self glycoproteins they are not deleted during development and can be readily detected in normal subjects. These 9G4+ B cells are excluded from germinal center (GC) reactions and post germinal center memory populations and remain functionally tolerant. In contrast, in Systemic Lupus Erythematosus (SLE) autoimmune patients 9G4+ B cells are no longer excluded from participating in germinal center reactions and can be detected in GC and memory populations by both flow cytometry and histology of tonsil biopsies. How this autoreactive 9G4+ B cell population is excluded from GC reactions in healthy subjects and why these regulatory mechanisms fail in SLE is still not clear. One appealing hypothesis to explain 9G4+ B cell tolerance is that constant exposure to self antigens results in altered B Cell receptor (BCR) signaling that is incapable of driving cell proliferation and differentiation. The purpose of these studies was to test the hypothesis that BCR signaling is impaired in autoreactive B cells.
Method

Recent advances in studying protein phosphorylation by flow cytometric analysis make it possible to simultaneously measure multiple signaling molecules and cell surface markers. Because 9G4+ B cells are a minor population and isolation of these cells may alter their phenotype, we used flow cytometry to directly measure signaling on a single cell level. Peripheral blood lymphocytes from 7 healthy donors were stimulated with either anti-IgM, anti-IgD, or anti-IgG antibodies. After fixation and permeabilization cells were stained for CD20, CD27, and IgM to differentiate naïve, IgM+CD27+ memory, and switched memory populations. Cells were also stained with 9G4 and antibodies specific for phosphorylated ERK, MAP Kinase p38, PLC-γ, or isotype control. Stimulation was expressed as mean fluorescence intensity over isotype control.
Results
A low level of basal phosphorylation of ERK and MAPKp38 but not PLC-γ was detectable in naïve B cells. After stimulation with anti-IgM or anti-IgD, phosphorylation of all three signaling molecules was increased in naïve B cells but not increased after the negative control anti-IgG stimulation. Although both 9G4+ and 9G4- showed increased phosphorylation after anti-IgM stimulation 9G4+ B cells reproducibly had lower (70-80%) amounts of phosphorylated ERK and PLC-γ.
Conclusion
While BCR signaling is detectable in 9G4+ B cells, the degree of phosphorylation is reduced, suggesting that exposure to self antigen can negatively alter BCR signaling. This impaired signaling is a potential regulatory mechanism for maintaining tolerance in autoreactive B cells. Studies are now underway to further dissect other signaling pathways and also evaluate signaling of 9G4+ B cells in SLE patients.

 S.A. Jenks, None; I. Sanz, None.