The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Summary

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

Introduction

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized prototypically by anti-nuclear antibody (ANA) production and glomerulonephritis. Numerous other sequelae, including dermal, cardio-pulmonary, and hepatic lesions are associated with disease in some individuals. Prevalence estimates in the US vary widely, from 150,000-1,500,000^1,2, with particularly high incidence in women and minorities³. Although the etiology of SLE has been difficult to discern, it is thought to arise from the interplay of various genetic and environmental factors, which culminate in systemic autoimmunity.

Numerous anima....

Protocol

Animal work was performed under specific pathogen-free conditions in accordance with guidelines set by the Association for Assessment and Accreditation of Laboratory Animal Care International and our Institutional Animal Care and Use Committee (IACUC).

NOTE: Incorporate mice expressing a congenic marker such as CD45.1 on either donor or recipient animals if possible, because this allows for the monitoring of donor graft efficiency and specific expansion of the donor CD4 T cell population. If considering the use of otherwise genetically modified mice as donor or recipients, ensure the strain is properly backcrossed to the B6 background, or t....

Results

Diseased mice develop splenomegaly in as little as 14 days, exhibiting spleens 2-3 times the size of healthy mice in terms of mass and cellularity (Figure 2).

Splenocytes are sequentially gated on light scatter (FSC-A by SSC-A), elimination of doublets (FSC-W or -H by FSC-A), viable cells (low staining of viability dye), and CD4⁺TCRβ⁺ (Figure 3A). Donor cells are distinguished from recipient cells based on CD45.1 and CD45.2 (Figur.......

Discussion

The bm12 inducible model is a relatively easy and efficient way to study the cellular and molecular processes of SLE. Chronic activation of adoptively transferred CD4 T cells directed against self antigens leads to the accumulation of Tfh, GC B cells, and plasma cells which can be measured by flow cytometry, as described here. Future studies using this model can quickly and easily interrogate the role of candidate genes and novel therapies in the autoimmune germinal center processes which resemble those occurring in pati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B6.SJL-Ptprca Pepcb/BoyJ	The Jackson Laboratory	001162	CD45.1+ BoyJ mouse strain
B6(C)-H2-Ab1bm12/KhEgJ	The Jackson Laboratory	001162	Bm12 mouse strain
FastDigest PsuI	Life Technologies	FD1554	Restriction digest enzyme for genotyping
1X RBC Lysis Buffer	eBioscience	00-4333-57
IMDM	GE Healthcare	SH30228.01
Plasma Separation Tube (PST)	BD	365974	Blood collection tube with Dipotassium EDTA
Serum Separation Tube (SST)	BD	365967	Blood collection tube with Clot activator / SST Gel
Ficoll	GE Healthcare	17-1440-02	High density cell separation solution
Lympholyte-M	Cedarlane	CL5030	High density cell separation solution
GL-7-biotin	eBioscience	13-5902-82
Streptavidin-BUV395	BD	564176
CD138-BV421	BioLegend	142508
CD4-BV510	BioLegend	100559
TCRβ-BV605	BD	562840
CD45.1-BV711	BioLegend	110739
CD45.2-FITC	BioLegend	109806
PD-1-PE	BioLegend	135206
CD19-PerCP	BioLegend	115532
Fas-PE-Cy7	BD	557653
CXCR5-APC	BioLegend	145506
Fixable Viability Dye ef780	eBioscience	65-0865-18
CD4-BV421	BioLegend	100443
1.2 ml FACS tube inserts, racked	USA Scientific	1412-1400
BD Falcon™ Round-Bottom Tubes	BD	352017