Induction and assessment of class switch recombination in purified murine B cells.
Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.
Zaheen
Ahmad
A
Department of Immunology, University of Toronto.
Martin
Alberto
A
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000502:physiology*; D003564:Cytidine Deaminase / Q000096:biosynthesis / Q000276:immunology; D004790:Enzyme Induction; D017578:Immunoglobulin Class Switching / Q000235:genetics* / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000235:genetics* / Q000276:immunology; D051379:Mice; D011995:Recombination, Genetic / Q000276:immunology
D007132:Immunoglobulin Isotypes; D003564:Cytidine Deaminase