Detection of True IgE-expressing Mouse B Lineage Cells

Summary

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Abstract

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

Introduction

During immunoglobulin heavy chain (IgH) class switch recombination (CSR) in mice and humans, the IgH μ constant region exons (Cμ) are deleted and replaced by one of several sets of downstream constant region exons (C_Hs) (e.g. Cγ, Cε, and Cα), resulting in a switch from the production of IgM to the production of other Ig classes (e.g. IgG, IgE, or IgA). CSR occurs within switch (S) regions, which are 1-10 kb sequences located 5’ to each set of C_Hs¹. The Activation-Induced Cytidine Deaminase (AID) enzyme initiates CSR via cytidine deamination activity.

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Protocol

NOTE: All experiments described here were in accordance with The Institutional Animal Care and Use Committee (IACUC) guidelines and approved by Animal Research Children’s Hospital (ARCH), Boston, Mass.

1. Preparation of Reagents

1. 1,000x IL-4 Stock: Dilute IL-4 cytokine to 20 µg/ml in H₂O or 0.1% bovine serum albumin (BSA). Dispense in 200 µl aliquots to 1.5 ml microcentrifuge tubes and store at -20 °C.
2. 1,000x anti-CD40: Obtain from the manufacturer at 1.0 mg/ml, store at 4 °C.
3. B cell stimulation media: To 425 ml of RPMI-1640 medium, add 75 ml of freshly thawed fetal calf serum (FCS), 10 ml of 1....

Results

This procedure has been successfully implemented to study CSR to IgE in mouse B cells. To demonstrate the efficiency in measuring CSR, we stimulated mouse splenic B cells with anti-CD40 and IL-4 as described previously¹¹. After five days of stimulation, cells were collected and processed using the protocol described above and stained with fluorescently-labeled IgM, IgG1, IgE, and B220 (CD45R) antibodies. Gated on the blasting lymphocytes (Figure 1), a clear population of IgE+ cells cannot be c.......

Discussion

Stimulation of mouse splenic B cells in culture with anti-CD40 and IL-4 will simulate T helper type 2 (T_H2) interactions, encouraging class switching to IgG1 and IgE⁵. B cells can be activated for CSR in the context of total splenocytes¹² or as purified splenic B cells¹¹. As noted in the protocol (step 2.3), B cell enrichment is optional, and is at the discretion of the experimenter to determine if it would beneficial. Positive magnetic separation of B cells using CD45R(B220)-l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-Mouse IgE FITC	BD Pharmingen	553415	clone R35-72
Rat Anti-Mouse IgG1 PE	BD Pharmingen	550083	clone A85-1
Methanol	BDH	BDH1135-4LP	Keep at -20 °C
Falcon Cell Strainer 40 µm Nylon	Corning	352340
Falcon Cell Strainer 70 µm Nylon	Corning	352350
Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5	eBioscience	45-4052-82	clone RA3-6B2
anti-Mouse/Rat CD40	eBioscience	16-0402-85	clone HM40-3
RPMI Medium 1640	Gibco	11875-093
HEPES (1M)	Gibco	15630-080
MEM-NEAA (100x)	Gibco	11140-050
Phosphate Buffered Saline (10x)	Lifetechnologies (Corning)	46-013-CM
MACS CD45R (B220) microbeads	Miltenyi Biotec	130-049-501
MACS purification column	Miltenyi Biotec	130-042-401
IL-4	PeproTech	214-14	Reconstitute in water or 0.1% BSA
Formalin Solution, Neutral Buffered, 10%	Sigma Aldrich	HT501128-4L
Trypsin-EDTA Solution (10x)	Sigma Aldrich	T4174-100 ml	5.0 g Trypsin, 2.0 g EDTA•4 Na per Liter of 0.9% NaCl
Penicillin-Streptomycin	Sigma Aldrich	P0781-100 ml	10,000 U Penicillin; 10 mg/ml Streptomycin (100x)
L-Glutamine 200 mM	Sigma Aldrich	G7513
2-mercaptoethanol (99%)	Sigma Aldrich	M6250-100 ml
Red cell lysis buffer	Sigma Aldrich	R7757
Rat Anti-Mouse IgM RPE/Cy7	SouthernBiotech	1140-17	clone 1B4B1
HyClone Fetal Calf Serum	Thermo	SH30910.03
B cell Stimulation media			B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1x MEM-NEAA, 2.0 mM L-glutamine,  1x Penicillin-Streptomycin (Penicillin: 100 U/ml, Streptomycin: 100 µg/ml), Beta-mercaptoethanol (7 µL/L), IL-4 (20 ng/ml), and anti-CD40 (1 µg/ml)