Characterization and Activation of Fas Ligand-Producing Mouse B Cells and Their Killer Exosomes

B lymphocytes make several contributions to immune regulation including production of antibodies with regulatory properties, release of immune suppressive cytokines, and expression of death-inducing ligands. A role for Fas ligand (FasL)-expressing "killer" B cells in regulating T helper (T) cell survival and chronic inflammation has been demonstrated in animal models of schistosome worm and other infections, asthma, autoimmune arthritis, and type 1 diabetes. FasL B cells were also capable of inducing immune tolerance in a male-to-female transplantation model. Interestingly, populations of B cells found in the spleen and lungs of naïve mice constitutively expresses FasL and have potent killer function against T cells that is antigen-specific and FasL-dependent. Epstein-Barr virus-transformed human B cells constitutively express FasL and package it into exosomes that co-express MHC Class II molecules and have killer function against antigen-specific T cells. FasL exosomes with markers of B-cell lineage are abundant in the spleen of naïve mice. Killer B cells therefore represent a novel target for immune modulation in many disease settings. Our laboratory has published methods of characterizing FasL B cells and inducing their proliferation in vitro. This updated chapter will describe methods of identifying and expanding killer B cells from mice, detecting FasL expression in B cells, extracting FasL exosomes from spleen and culture supernatants, and performing functional killing assays against antigen-specific T cells.