Peripheral autoantigen binding B cells are poorly competitive with naive B cells for survival and undergo rapid cell death. However, in monoclonal Ig-transgenic mice lacking competitor B cells, autoantigen binding B cells can survive for extended periods. The basis for competitive elimination of autoantigen binding B cells has been unknown. Here we demonstrate that autoantigen binding B cells have increased dependence on BAFF for survival. In monoclonal Ig-transgenic mice, each autoantigen binding B cell receives elevated amounts of BAFF, exhibiting increased levels of NFkappaB p52 and of the prosurvival kinase Pim2. When placed in a diverse B cell compartment, BAFF receptor engagement and signaling are reduced and the autoantigen binding cells are unable to protect themselves from Bim and possibly other death-promoting factors induced by chronic BCR signaling. These findings indicate that under conditions where BAFF levels are elevated, autoantigen-engaged cells will be rescued from rapid competitive elimination, predisposing to the development of autoimmune disease.

We report that B cell–activating factor of the tumor necrosis factor (TNF) family (BAFF) is expressed in the normal human brain at ∼10% of that in lymphatic tissues (tonsils and adenoids) and is produced by astrocytes. BAFF was regularly detected by enzyme-linked immunosorbent assay in brain tissue lysates and in normal spinal fluid, and in astrocytes by double fluorescence microscopy. Cultured human astrocytes secreted functionally active BAFF after stimulation with interferon-γ and TNF-α via a furin-like protease-dependent pathway. BAFF secretion per cell was manifold higher in activated astrocytes than in monocytes and macrophages. We studied brain lesions with B cell components, and found that in multiple sclerosis plaques, BAFF expression was strongly up-regulated to levels observed in lymphatic tissues. BAFF was localized in astrocytes close to BAFF-R–expressing immune cells. BAFF receptors were strongly expressed in situ in primary central nervous system (CNS) lymphomas. This paper identifies astrocytes as a nonimmune source of BAFF. CNS-produced BAFF may support B cell survival in inflammatory diseases and primary B cell lymphoma.

PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.

Blockade of the CD154-CD40 co-stimulatory pathway with anti-CD154 mAbs has shown impressive efficacy in models of autoimmunity and allotransplantation. Clinical benefit was also demonstrated in systemic lupus erythematosus (SLE) and idiopathic thrombocytopenia patients with the humanized anti-CD154 mAb, 5C8 (hu5C8). However, thromboembolic complications that occurred during the course of the hu5C8 clinical trials have proven to be a major setback to the field and safe alternative therapeutics targeting the CD154-CD40 pathway are of great interest. Recently, effector mechanisms have been shown to play a part in anti-CD154 mAb-induced transplant acceptance in murine models, while this issue remains unresolved for humoral-mediated models. Herein, aglycosyl anti-CD154 mAbs with reduced binding to FcgammaR and complement were used as a novel means to test the role of effector mechanisms in non-human primate and murine models not amenable to gene knockout technology. While aglycosyl hu5C8 mAb was relatively ineffective in rhesus renal and islet allotransplantation, it inhibited primary and secondary humoral responses to a protein immunogen in cynomolgus monkeys. Moreover, an aglycosyl, chimeric MR1 mAb (muMR1) prolonged survival and inhibited pathogenic auto-antibody production in a murine model of SLE. Thus, the mechanisms required for efficacy of anti-CD154 mAbs depend on the nature of the immune challenge.