Generation of human CD40-activated B cells.
CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a). CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (>10(9) cells per patient) from healthy donors as well as cancer patients (Fig.1c,d). In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3). To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function. CD40-B cells efficiently take up, process and present antigens to T cells. They do not only prime naïve, but also expand memory T cells. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors.
Laboratory for Tumor and Transplantation Immunology and Stem Cell Transplantation Program, University Hospital of Cologne, Department I of Internal Medicine.
Tanja M
TM
Liebig
Anne
A
Fiedler
Shahram
S
Zoghi
Alexander
A
Shimabukuro-Vornhagen
Michael S
MS
von Bergwelt-Baildon
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D019013:CD40 Antigens / Q000276:immunology*; D023201:CD40 Ligand / Q000276:immunology / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D016572:Cyclosporine / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D015847:Interleukin-4 / Q000276:immunology / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology
D019013:CD40 Antigens; D011994:Recombinant Proteins; D023201:CD40 Ligand; D015847:Interleukin-4; D016572:Cyclosporine