Germinal centre and marginal zone B cells expand quickly in a second Plasmodium chabaudi malaria infection producing mature plasma cells

Antibodies and B cells are critical in the protective immune response to the blood stage of the malaria parasite, Plasmodium chabaudi. However, little is known about the development of memory B cells and their differentiation into plasma cells during infection or after re-infection. Here we have shown that B cells with phenotypic characteristics of memory cells (CD19+IgD− CD38+, IgG1+) are generated in a primaryPlasmodium chabaudi chabaudi infection of mice. In addition, we observed that germinal centre cells (CD19+, GL7+, MHCIIhi) and Marginal Zone B cells (CD19+CD23−IgD−) show faster expansion on re-infection than in the primary, though other subsets do not. Interestingly, though both IgM− and IgM+ memory cells are produced, IgM+ memory cells do not expand on second infection. The second infection quickly produced mature bone marrow plasma cells (intracellular Ighi, CD138hi, CD9+, B220−), compared to primary infection; which generates a very large population of immature splenic plasma cells (B220+). This analysis suggests that a memory B cell population is generated after a single infection of malaria, which on re-infection responds quickly producing germinal centres and generating long-lived plasma cells making the second encounter with parasite more efficient

Complement Activation on B Lymphocytes Opsonized with Rituximab or Ofatumumab Produces Substantial Changes in Membrane Structure Preceding Cell Lysis.

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death

Fractionated subcutaneous rituximab is well-tolerated and preserves CD20 expression on tumor cells in patients with chronic lymphocytic leukemia

A pilot study previously demonstrated that thrice-weekly, fractionated-dose intravenous rituximab (RTX) limits CD20 loss from chronic lymphocytic leukemia (CLL) B cells, thereby enhancing immunotherapeutic targeting. Here, we investigated the feasibility of giving 20 mg rituximab subcutaneously thrice weekly for up to 12 weeks in 4 previously treated CLL patients. Subcutaneous rituximab was well-tolerated with minimal injection site reactions; a variable degree of efficacy was observed, likely influenced by the size of the patients’ B cell/CD20 burden. Subcutaneous RTX largely preserved CD20 expression on leukemic cells but the most effective therapeutic dosing regimen needs to be established (ClinicalTrials.gov Identifier: NCT00366418)