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Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
Published on: August 14, 2010
A sensitive and efficient induction system for murine IgE. Single cell analysis at the clonal level
F Ledermann1, C Schlienger, K Wagner
1Research Department, CIBA-GEIGY, Ltd., Basel, Switzerland.
Insights
A new culture system using CB5.1 feeder cells enhances immunoglobulin E (IgE) expression analysis in single B cells. This method improves the detection of IgE-secreting B cells, crucial for understanding allergic responses.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Analyzing immunoglobulin E (IgE) expression in single B cells is challenging.
- Existing feeder cell systems have limitations in supporting IgE class switching.
Purpose of the Study:
- To develop and characterize a novel culture system for analyzing IgE expression by single murine B cells.
- To evaluate the efficacy of the CB5.1 stroma cell line as a feeder layer for IgE class switching.
Main Methods:
- Utilized a CB5.1 stroma cell line as a feeder layer for lipopolysaccharide (LPS)-stimulated B cells.
- Employed interleukin-4 (IL-4) to induce IgE class switching.
- Developed a highly specific sandwich-enzyme-linked immunosorbent assay (ELISA) for IgE detection.
Main Results:
- CB5.1 feeder cells significantly enhanced the frequency of IgE-secreting B cells (2.5-fold increase) compared to thymocytes.
- Optimal IgE switching occurred at lower IL-4 concentrations than IgG1 switching, differing from thymocyte cultures.
- The system allowed for the detection of IgE production at the single-cell level within B cell clones.
Conclusions:
- The CB5.1 cell line provides a superior and defined feeder system for studying IgE class switching in B cells.
- This novel system facilitates the analysis of IgE-producing cells at the single-cell level.
- The findings offer a valuable tool for immunological research, particularly in allergy and B cell differentiation studies.
Abstract:
A culture system is described which permits the analysis of IgE expression by single murine cells within clones of B cells. The system is based on the use of a CB5.1 stroma cell line as a feeder which optimally supports the IL-4-induced switch to IgE of LPS-stimulated B cells in culture. In this system 100 U/ml IL-4 induces the switch to IgE, in 3-5% of B cells and the switch frequency to IgG1 was as high as 2%. Five ng IgE or 12 ng IgG1 were produced per clone containing on average 13-15 PFC. The detection of single IgE secreting B cells was possible due to two newly developed, highly specific rat anti-mouse IgE antibodies used in a sandwich-ELISA. The frequency of IgE-secreting B cells was enhanced 2.5 times when the fibroblastoid CB5.1 cells rather than thymocytes were used as feeder cells. CB5.1 cells supported the differentiation of B cells to IgM-PFC almost as well as rat thymocytes (which have, to date, been used as the standard feeder layer) whereas the amount of secreted IgG1 was about 3 times lower than in thymocyte cultures. Optimal switching to IgE occurred at concentrations of IL-4 which were 10-fold lower than that required for IgG1 expression, a situation quite opposite to that observed in the rat thymocyte-supported culture system. In confirmation of established data the switch of B cells to IgE or IgG1 occurred randomly. The advantages of CB5.1 cells as feeder cells are (1) the use of a homogeneous and defined cell line, (2) their limited release of defined lymphokines (IL-6 and GM-CSF), and (3) the low degradation and consumption of cytokine factors. The combination of the CB5.1 cell line with a highly specific IgE ELISA assay made it possible to analyse the appearance of IgE producing cells within a developing B cell clone.
