Figure 5
From: Reduced Fas ligand-expressing splenic CD5+B lymphocytes in severe collagen-induced arthritis
Peptide-specific and Fas ligand-dependent killing of T hybridoma cells by purified B cells. Splenic CD19+ B cells were magnetic bead-purified from incomplete Freund's adjuvant/type II collagen (cII)-immunized human major histocompatibility complex (MHC) class II DR4 transgenic mice. Purified B cells (> 98% pure) were pulsed with peptides cII259-273 or HCgp39263-275 and then cultured with neutralizing antibodies against human MHC class II, mouse interleukin-10 (IL-10), or mouse Fas ligand. An isotype control antibody (rat IgG1) and an agonist anti-mouse CD4 antibody, used as a positive control for induction of T-cell apoptosis, were added to some wells as controls. One hour after the addition of antibodies, DR4-restricted, gp39-specific, T hybridoma cells were added at a 1:1 ratio to the original number of B cells. Cells were collected 24 hours later and analyzed for T-cell apoptosis by Annexin V-based, three-color flow cytometry. Data shown are mean ± standard deviation of triplicate samples. Ab, antibody; AnnV, Annexin V; APC, antigen-presenting cell; DR4, human class II major histocompatibility complex DRB1*0401; PI, propidium iodide.