I'm desperately trying to generate skewed populations of human CD4+ T cells. I start with ~95% pure naive CD4+ T cells and activate with anti-CD3/28 magnetic beads in iMDM and in the presence of IL2 and polarizing cytokines/neut antibodies towards Th1 (IL12, aIL4), Th2 (IL4, aIFNg, aIL12) and Th17 (IL1b, IL6, IL23, TGFb). At this point, I only seem able to get strong Th1's (I've checked d3, 7, 14, 21 with varying stim/restim time courses). My Th2's have no IFNg but also no IL4. The Th17s have no IL4, some IFNg (less than Th1s) but no Th17. I get very strong activation (nearly 100% CD69+ CD25+ 24h post stim), and robust proliferation (3rd generation by d3 post stim and past 5th or 6th generation by d7 post stim according to CFSE). For my polarizing conditions, I'm basing them on Current Protocols in Immunology and adjusting concentrations of cytokines/Abs according to the lit.
To troubleshoot the Th2s I've tried IL4 at increasing concentrations, from 2 different suppliers (eBioscience and R&D). I also tested my IL4 antibody and it works on PMA/iono-stimmed PBMCs. I have no idea where to start troubleshooting the Th17s. I'm at my wit's end with this. Any advice would be greatly appreciated. Thanks.
Edited by Greg S, 12 July 2011 - 04:43 PM.