Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.
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 1, 2.
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 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. 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.
Step I: Isolation of splenic B cells via magnetic enrichment
Step II: CFSE cell staining
Step III: Cell stimulation with LPS
Step IV: Flow cytometric analysis of class switching
Representative Results
Following magnetic enrichment, the cell suspension should look like a homogenous population of indistinguishable small circular cells (Figure 1A). After 48-72 hours of stimulation, isolated clusters of enlarged proliferating cells will be clearly visible (Figure 1B). A large proportion of the non-proliferating cells will appear small and granular as they undergo apoptosis.
CSR can normally be detected at the highest levels between 72 and 96 hours after stimulation before most of the cells begin to die 7. At this stage, the cells should have undergone a number of cell divisions with switched cells appearing in the later daughter cell population. A representative flow cytometric plot of CFSE dilution and surface expression of IgG3 after 96 hours of LPS stimulation can be seen in Figure 2.
Table 1. Isotype switching cocktails. Note: Stimulant concentration values are recommendations only. Titrations may be necessary.
Desired Isotype | Stimulation Cocktail |
IgG1and IgE | LPS (25μg/mL) and IL-4 (10 ng/mL) |
IgG1and IgE | Anti-CD40 (10μg/mL) and IL-4 (10 ng/mL) |
IgG2a | LPS (25μg/mL) and IFN-γ(10 ng/mL) |
IgG2b and IgG3 | LPS (25μg/mL) |
IgA | LPS (25μg/mL), TGF-β(2 ng/mL) and IL-5 (1.5 ng/mL) |
Figure 1. Isolation and LPS stimulation of splenic B cells. (a) Magnetically enriched splenic B cells before LPS stimulation. (b) 48 hours after stimulation with 25 μg/mL of LPS. Proliferating cells are seen as clusters in a background of blasting and apoptotic cells.
Figure 2. Proliferation and class switch recombination after 96 hours. (a) CFSE dilution for LPS stimulated cells after 96 hours in culture. Each independent peak represents a dilution in CFSE fluorescence corresponding to an independent cell division. (b) IgG3 expression showing the emergence of an IgG3 positive population following several rounds of cell division.
The protocol outlined above provides a standard assay to analyze AID expression and function during the class switching of primary murine B cells. This protocol uses bacterial LPS to induce switching from IgM to IgG3, although modifications to the stimulation media can be made to induce switching to other isotypes (summarized in Table 1 8, 9).
We have noted that, for as yet unknown reasons, fetal calf serum can affect the level of class switching observed in vitro. It is crucial that the same lot number be used for all CSR experiments to ensure experimental consistency. It may also be beneficial to screen a panel of fetal calf serum sources to attain optimal in vitro switching rates. Mouse strains can also affect the degree of class switching seen in vitro 10. Depending on the assay in question, poorly switching murine models, such as the SJL mouse, must be sufficiently backcrossed to the C57BL/6 background to attain appreciable switching rates.
Step I may be omitted if a pure B cell culture is not desired. The stimulation of a mixture of splenic cells will still induce class switching, although it may result in a larger population of apoptotic non-B cells. Furthermore, subsequent flow cytometric analysis must include an established B cell marker (e.g. B220, CD19). The purification kit used in this protocol is supplied by StemCell Technologies (see reagents table). Alternate kits that use a negative enrichment process may also be used according to the manufacturer’s instructions.
CFSE fluorescence can be very intense and thus each experiment requires an appropriate compensation control for flow cytometric analysis. In addition, CFSE will slowly leak out of cells resulting in a reduction of overall fluorescence over time. For these reasons, it is recommended that all experiments include an unstimulated CFSE-stained cell population. Over a short period of time, these cells will remain static in culture and can thus be used as a compensation control for CFSE and to identify the non-proliferating cell population.
The authors have nothing to disclose.
We are grateful to the Martin laboratory for helpful discussions. This publication is supported by a grant from the Canadian Institute of Health Research (MOP-89783). A.M. is supported by a Canada Research Chair Tier II Award.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Phosphate Buffered Saline | Gibco | 14190 | ||
EasySep Mouse B Cell Enrichment Kit | Stemcell Technologies | 19754 | ||
Polystyrene tubes | Becton Dickinson Falcon | 352058 | ||
Bovine Serum Albumin | Sigma Aldrich | A7030 | ||
CellTrace CFSE Cell Proliferation Kit | Invitrogen | C34554 | ||
Bovine Calf Serum | HyClone | SH30072.03 | ||
RPMI 1640 Culture Medium | Gibco | 11875 | ||
Lipopolysaccharides from Escherichia coli | Sigma Aldrich | L6529 | ||
β-mercaptoethanol | Gibco | 21985 | ||
Fetal Bovine Serum | HyClone | SH30396.03 | ||
Normal Mouse Serum | Jackson ImmunoResearch | 011-000 | ||
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) | Becton Dickinson Pharmingen | 553141 | ||
Rat Anti-Mouse IgG3 | Becton Dickinson Pharmingen | 553401 |