Characterization of the Cell Surface Phenotype and Regulatory Activity of B-Cell IgD Low (BD_L) Regulatory B Cells

B-cell IgD Low (BD_L) B cells have been shown to promote immunological tolerance by inducing proliferation of CD4⁺Foxp3⁺ T-regulatory cells (Treg) in a glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL, Tnfsf18)-dependent manner. BD_L cells constitute a small subset of splenic B lymphocytes that, in mice, are characterized by the B220⁺IgM^intCD21^intCD23⁺CD93^-IgD^low/- cell surface expression profile. In this chapter, we show the flow cytometry gating strategy developed to identify and purify BD_L. In addition, we describe an in vitro assay and two in vivo assays to assess BD_L regulatory activity by quantitating Treg expansion/proliferation and indicate how they can be used in mouse models of disease. Collectively, these methods are useful to track and quantitate BD_L and Treg numbers and assess their regulatory activity in inflammatory disease models.