Murine model of CD40-activation of B cells.
Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (>10(9) cells per patient) from healthy donors as well as cancer patients. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro. Furthermore they efficiently take up, process and present antigens to T cells. CD40-B cells were shown to not only prime naíve, but also expand memory T cells. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa). Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (>90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach.
Liebig
Tanja M
TM
Department I of Internal Medicine, University Hospital of Cologne.
Fiedler
Anne
A
Klein-Gonzalez
Nela
N
Shimabukuro-Vornhagen
Alexander
A
von Bergwelt-Baildon
Michael
M
D000818:Animals; D017951:Antigen Presentation; D001402:B-Lymphocytes / Q000276:immunology*; D019013:CD40 Antigens / Q000276:immunology*; D023201:CD40 Ligand / Q000235:genetics / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D014162:Transfection
D019013:CD40 Antigens; D023201:CD40 Ligand