CD4+ T helper (Th) cell subset generation in vivo requires T cell receptor activation and surface CD28 co-stimulation in the presence of one or more cytokines. Similarly, Th cells can be generated in vitro by activating naïve CD4+CD25-T cells with plate bound-anti-CD3 monoclonal antibody (mAb) (pbCD3) and soluble-anti-CD28 mAb (sCD28) in the presence of polarizing recombinant (r) cytokines and anticytokine mAbs. In comparison to in vitro CD4+CD25-T cells, memory CD4+CD25-CD45RO+ T cells have been shown to convert to Th9 cells more efficiently. Here, protocol for in vitro generation of human Th9 cells by activating CD4+CD25−CD45RO+ memory T cells with pbCD3 and sCD28 in the presence of polarizing recombinant interleukin-4 (rIL-4) and transforming growth factor (rTGF-β) is described.
Putheti, P. (2017). Polarizing cytokines for human Th9 cell differentiation. In Methods in Molecular Biology (Vol. 1585, pp. 73–82). Humana Press Inc. https://doi.org/10.1007/978-1-4939-6877-0_6