Dear all, I've done this experiment 6 times: isolate cd11b with MACS beads, culture in RPMI medium, and after 3 days add CD8 isolated cells (Macs beads), IL-2 and anti-CD3. My results are always the same: huge induction of proliferation of CD8 (CFSE) compared with CD8+IL2+anti CD3 culture, during the same time. The results are the same, either I use cells from the same donor or from different donors. Can anyone help explain why this is happening? I can't find literature about it.
Thanks a lot,
Best regards,
The CD11b monocytes, which are probably in the process of differentiating to macrophages in the flask after 3 days, provide secondary signals required for optimal activation and proliferation of CD8 T cells.
CD3 stimulation alone is far from optimal and tends to induce apoptosis of T cells. Adding APCs such as these monocyte-derived macrophages will provide B7.1/B7.2-CD28 interactions at the very least, that synergize with CD3 stimulation.
Dear Ana Claudia, aCD3 can bind to CD11b monocytes via Fc receptors. This results to formation of artificial APC, capable to form immunological synapses with T cells and non-specifically activate almost all CD3+ T lymphocytes. CD8 T cells proliferate much more quickly than CD4 T cells. Obviously, as result you see their prevalence in cultures. Especially, this prevalence is more robust at the presence of IL-2 providing very good conditions for proliferation of CD8 T cells (in the past, clones of CD8 CTLs were successfully grown in vitro at the presence of high concentrations of IL-2 only, even without additional stimulation via MHC/peptide/TCR). The presence of IL-2 allows to obviate the dependence of T cells on costimulatory signals of APC. Michel is right too, saying about CD80/CD86 interactions with CD28. The most important outcomes of costimulation via CD80/CD28 are IL-2 production and stimulation of viability of T cells via production of antiapoptotic molecules in T cells.
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