Hello. I have a question.
I want to generate 2C CD8 memory T cells by culture with hIL15. I need to generate very high number of these cells for cell transfer. I think my protocol is not good.
I tried several protocols but they were not successful. I stimulated 2C cells by 2C peptide for 16h and then cells were washed 3 times to remove peptide. then 10^5 cells in 24 wells plate (1ml medium/2ng hIL-15) were cultured for several days. at the beginning, cells proliferate well but after day 4 most of cells dies. I tried the same protocol with OTI cells and they survive better.
Please if you have experience guide me.
Thank you
Masoud
Dear Masoud, 1) Stimulation by p2Ca may be successful if this peptide is complexed with H2-Ld molecule. 2C TCR is originally allospecific and stimulation of T cells will be successful if you stimulate them by allogeneic molecule (Ld) plus allogeneic peptide (p2Ca). 2) What is genetical background of your 2C TCR mice? If they are H-2b mice, you will need to use stimulators from H-2Ld expressing mice. If they are H-2d, 2C TCR cells may be deleted in thymus. 3) Do you use 2C TCR T cells from RAG KO mice? If the answer is "no", the most probably, you see propagation of unrelated T cells expressing TCRs with endogenously rearranged alpha- and beta-chains.
Thank dear Dmitry
I used to activate 2C mice with peptide. I just cultured total splenocytes of 2C mice with peptide and after 3 days there are almost 90% activated 2C cells in culture. my problem is the generation of memory cells by IL15. I tried different concentration of IL15 but my 2C cells die after 5days. same protocol of IL15 culture can make almost high number of OTI cells.my 2C cells have B6 background H-2b and can be activated in vivo by transferring them in to B6 mice and and then injection of balb/C splenocytes. but this in vivo activation can not make enough 2C cells for my experiment so I need in vito activation for making memory cells.
Dear Masoud, try to activate 2C T cells in vitro by irradiated or MitC treated splenocytes from BALB/c mice.
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