I want to simulate a germinal center reaction with human cells in vitro. Ideally, I want to use human naive B cells, and simulate Antigen-stimulation and further GC reaction in vitro in order to look at AG-specific clonal B cell expansion, SHM and CSR.
From what I read CD40L supports BC growth in vitro nicely, but how can I induce AG-specific clonal expansion from a relatively small sample of PBMC (say 30 ml of whole blood, or 10 million PBMC)? I want to generate clonal expansion, and I think CPG works for polyclonal expansion, correct? Can you recommend a model antigen to add to PBMC to mimic GC reaction, for example Influenza or CMV particle? And will these stimulate naive B cells? Are the reagents commercially available? Also is there a commercially available CD40L-expressing human fibroblast feeder cell line?
Many, many thanks
Dear Loisa. Perhaps you can find some solution for your problem in these papers.
I can not to share here the full text because they are not Open acess but I can send you them by e-mail.
Fang X1, Tong Y1, Tian H1, Ning H1, Gao X2, Yao W3 Rapid de novo generation of antigen specific human B cells with expression of Blimp-1 and AID by in vitro immunization.Exp Cell Res. 2017 Mar 1;352(1):53-62. doi: 10.1016/j.yexcr.2017.01.020.
Ingvarsson S, Lagerkvist AC, Mårtensson C, Granberg U, Ifversen P, Borrebaeck CA, Carlsson R. Antigen-specific activation of B cells in vitro after recruitment of T cell help with superantigen. Immunotechnology. 1995 May;1(1):29-39.
Best regards,
Hi Louisa,
May be I can not answer some parts of your question but for second half of your question please see Figure 6 of my paper (link given). We have stimulated CD8 T cells with Flu and CMV peptide. We haven't looked in B cells. But the materials and concentrations you can easily follow from the paper.
https://www.nature.com/articles/s41467-018-04832-2
For the second question you might would like to check LL8 cell line which express CD40L after 3-4h treatment of 10ug/ml Mitomycin.
Best.
Alexander Batista Duharte, thanks for the papers, that might be just what I was looking for!
Vishal Khairnar, thanks for the link to your paper!
A couple of years later... I am facing the same doubts! It would be great if Louisa or Alexander could send me those papers!! ;)