Detection of epsilon class switching and IgE synthesis in human B cells.
We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.
Pène
Jérôme
J
Institut National de la Santé et de la Recherche Médicale, Unite 454, Centre Hospitalier Universitaire Arnauld de Villeneuve, Montpellier, France.
Guilhot
Florence
F
Cognet
Isabelle
I
Guglielmi
Paul
P
Guay-Giroux
Angélique
A
Bonnefoy
Jean-Yves
JY
Elson
Greg C
GC
Yssel
Hans
H
Gauchat
Jean-François
JF
D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D019013:CD40 Antigens / Q000276:immunology; D023201:CD40 Ligand / Q000276:immunology; D002478:Cells, Cultured; D046508:Culture Techniques; D004270:DNA, Circular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005312:Fetal Blood / Q000166:cytology; D006801:Humans; D017578:Immunoglobulin Class Switching; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology*; D007139:Immunoglobulin epsilon-Chains / Q000235:genetics / Q000276:immunology*; D018189:Immunomagnetic Separation / Q000379:methods; D008213:Lymphocyte Activation; D014066:Palatine Tonsil / Q000166:cytology / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology
D019013:CD40 Antigens; D004270:DNA, Circular; D007139:Immunoglobulin epsilon-Chains; D023201:CD40 Ligand; D007073:Immunoglobulin E