PMID- 19838159
OWN - NLM
STAT- MEDLINE
DCOM- 20091228
LR  - 20211020
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 32
DP  - 2009 Oct 16
TI  - Generation of human CD40-activated B cells.
LID - 1373 [pii]
LID - 10.3791/1373 [doi]
AB  - 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 naive, 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.
FAU - Liebig, Tanja M
AU  - Liebig TM
AD  - Laboratory for Tumor and Transplantation Immunology and Stem Cell Transplantation 
      Program, University Hospital of Cologne, Department I of Internal Medicine.
FAU - Fiedler, Anne
AU  - Fiedler A
FAU - Zoghi, Shahram
AU  - Zoghi S
FAU - Shimabukuro-Vornhagen, Alexander
AU  - Shimabukuro-Vornhagen A
FAU - von Bergwelt-Baildon, Michael S
AU  - von Bergwelt-Baildon MS
LA  - eng
PT  - Journal Article
PT  - Video-Audio Media
DEP - 20091016
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (CD40 Antigens)
RN  - 0 (Recombinant Proteins)
RN  - 147205-72-9 (CD40 Ligand)
RN  - 207137-56-2 (Interleukin-4)
RN  - 83HN0GTJ6D (Cyclosporine)
SB  - IM
MH  - Animals
MH  - B-Lymphocytes/*cytology/*immunology
MH  - CD40 Antigens/*immunology
MH  - CD40 Ligand/immunology/pharmacology
MH  - Cell Culture Techniques/methods
MH  - Cyclosporine/immunology/pharmacology
MH  - Humans
MH  - Interleukin-4/immunology/pharmacology
MH  - Mice
MH  - NIH 3T3 Cells
MH  - Recombinant Proteins/immunology/pharmacology
PMC - PMC3164064
EDAT- 2009/10/20 06:00
MHDA- 2009/12/29 06:00
PMCR- 2011/10/16
CRDT- 2009/10/20 06:00
PHST- 2009/10/20 06:00 [entrez]
PHST- 2009/10/20 06:00 [pubmed]
PHST- 2009/12/29 06:00 [medline]
PHST- 2011/10/16 00:00 [pmc-release]
AID - 1373 [pii]
AID - 10.3791/1373 [doi]
PST - epublish
SO  - J Vis Exp. 2009 Oct 16;(32):1373. doi: 10.3791/1373.