B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model for human CD20 (hCD20) expression in which treatment of hCD20+ mice with anti-hCD20 mAbs mimics B cell depletion observed in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cell-activating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune diseases.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoformspecific effects on the deposition or clearance of amyloid ␤ (A␤) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on A␤ production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased A␤ production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in A␤ production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptorrelated protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which abolish apoE4 intramolecular domain interaction. Thus, apoE4 appears to modulate APP processing and A␤ production through both the LRP pathway and domain interaction. These findings provide insights into why apoE4 is associated with increased risk for Alzheimer's disease and may represent a potential target for drug development.Alzheimer's disease ͉ neurodegeneration

Alzheimer's disease is a progressive neurodegenerative disease characterized by senile plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. Activated microglia are found to be intimately associated with senile plaques and may play a central role in mediating chronic inflammatory conditions in Alzheimer's disease. Activation of cultured murine microglial BV2 cells by freshly sonicated A␤42 results in the secretion of neurotoxic factors that kill primary cultured neurons. To understand molecular pathways underlying A␤-induced microglial activation, we analyzed the expression levels of transcripts isolated from A␤42-activated BV2 cells using high density filter arrays. The analysis of these arrays identified 554 genes that are transcriptionally up-regulated by A␤42 in a statistically significant manner. Quantitative reverse transcription-PCR was used to confirm the regulation of a subset of genes, including cysteine proteases cathepsin B and cathepsin L, tissue inhibitor of matrix metalloproteinase 2, cytochrome c oxidase, and allograft inflammatory factor 1. Small interfering RNA-mediated silencing of the cathepsin B gene in A␤-activated BV2 cells diminished the microglial activation-mediated neurotoxicity. Moreover, CA-074, a specific cathepsin B inhibitor, also abolished the neurotoxic effects caused by A␤42-activated BV2 cells. Our results suggest an essential role for secreted cathepsin B in neuronal death mediated by A␤-activated inflammatory response.

[1][2][3][4] Since approval of a depleting anti-CD20 monoclonal antibody (mAb) (rituximab [Genentech, South San Francisco, CA; Biogen-IDEC, Cambridge, MA; Roche, Basel, Switzerland)] in 1997 for the treatment of non-Hodgkin lymphoma (NHL), almost 1 million patients have been treated with rituximab as a first or second line therapy, either alone or in combination with chemotherapy. In addition, rituximab maintenance therapy significantly prolongs tumor remission and patient survival in patients with indolent B-cell NHL or chronic lymphocytic leukemia (CLL). 5,6 More recently, rituximab has demonstrated clinical benefit in a variety of autoimmune diseases including rheumatoid arthritis, pemphigus vulgaris, immune thrombocytopenia and autoimmune hemolytic anemia. 4,7,8 As a result, understanding the contribution of B lymphocytes to human autoimmune diseases received revived interest, and several mechanisms have been postulated to participate in disease pathogenesis, including autoantibody production, B-cell antigen presentation, cytokine generation, and lymphorganogenesis. 2,3,9,10 Inhibition of different combinations of these mechanisms is probably responsible for clinical benefit.The success of anti-CD20 B-cell immunotherapy spearheaded a large number of preclinical and clinical efforts to understand in vivo mechanisms of drug activity. Direct elimination of malignant B cells through antigen-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and apoptosis have been demonstrated as the main mechanisms of action in a variety of systems including mouse xenotumors and normal mouse and nonhuman primate (NHP) B-cell subsets. 11-15 Two major determinants affecting normal mouse B-cell depletion have been identified. 13,16 First, the kinetics of B-cell recirculation determines the speed and magnitude of anti-CD20 mAb-mediated B-cell depletion. Cells with higher recirculatory kinetics from blood, lymph nodes, and spleen follicular areas are depleted faster and more completely than cells with lower recirculatory kinetics (eg, peritoneal cavity [PEC], marginal zone [MZ], germinal centers [GC]). Second, the local microenvironment influences the extent of B-cell depletion. Marginal zone, Peyer patches (PP), germinal center, memory, and peritoneal cavity B cells exhibit greater resistance to depletion in mice and nonhuman primates. Reduced recruitment of effector mechanisms in the peritoneal cavity as well as intrinsic B1 B cells properties appear to cause the slower kinetics and B-cell reduction after anti-CD20 mAb treatment. 16 Differences across mouse strains and epitopes recognized by anti-CD20 antibodies used for depletion might explain the differential effects seen on mouse splenic marginal zone B cells. 13,16 Small but consistent numbers of residual B cells can be detected in most lymphoid organs in mice and primates treated with anti-CD20 mAbs. One possibility of achieving a more complete B-cell reduction would be to block B-cell survival signals in addition to Submitted April 30, 2007...