Can I look at human naive T cell polarization using DCs and T cells in a allo-setting? Hence, induce T cell activation and proliferation with DCs displaying non-self MHC complexes. Given that the DCs are activated, will this induce proper T cell proliferation and cytokine production?
Dear Annette, if you use allogeneic dendritic cells you will stimulate only the alloreactive lymphocyte, that is around 10% of all lymphocytes. These 10% lymphocytes will be properly stimulated and proliferating if your DCs were mature.
Hi Annette,
What do you mean by "T cell polarization" exactly? Are you trying to look at Th1/ Th2 differentiation or just effector T cell generation? I assume you are also looking at CD4+ T cells.
If you have the option of selecting DC donors with known HLA types, your best bet of finding alloreactive recipients would be to choose individuals who are either DR4 or DR17. These should stimulate the highest number of naive T cell donors as long as they themselves are not DR4 or DR17 themselves. If you do not have the luxury of knowing the HLA type of your T cell donors, it may help to know that DR4 is relatively rare in people of African decent and DR17 is somewhat less common in those of Asian decents.
So to summarize: Ideal DC donors - DR4 and/or DR17, naive T cell donors - anything other than DR4 or DR17.
Good luck!
Dear Annete,
What is your aim?
If you like to investigate the differentiation process of naïve human T cells, the source of T cells is essential. In our hands, cordblood-derived CD4+CD25- T cells are the best (J Exp Med. 2000 Nov 6;192(9):1213-22) for these studies.
The use of allogeneic DC is the standard for analysis of human T cell polarization studies in vitro. T cell alloreactivity is in principal not different from any other antigen-specific stimulation. However, polarization needs several steps: activation – proliferation – differentiation. Therefore, analysis of polarized T cells (distinct cytokine profiles) needs repetitive stimulation with DC from the same donor.
Hi Annette,
I have just started my project with the culture of monocytes-derived DCs and used it to stimulated B cells. To your question, I think you can measure Th1/Th2 shifting by detecting intracellular IL-4 and IFN-gama in CD4+T gate after coculture with DC.
Here, I have a question too. does anyone know how to selectively drive naive CD4T differentiate into Th2 deviation in vitro?
Many thanks.
Thanks for the answers, I will look at the cytokine production from the DC-stimulated naive T cells both in FACS and with ELISA - hopefully that will be a start. I also want to detect the proliferation with CFSE staining, but due to the varying protocols, I guess it will be complicated to combine cytokine profile and CFSE proliferation in the same sample.
Shiqiang, the addition of IL-4 to CD3-CD28 stimulated T cells would be the easy answer, but I know it does not always work and might need some optimization. Look for additional cytokines or co-stimulatory molecules. IL-33 might drive the Th2 phenotype as well. Good luck!
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