3 replies to this topic

[Greg S]

Hi all,

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.

[Greg S]

All neutralizing Abs=10ug/mL, 20ng/mL IL2

Th1: 20ng/mL IL12, anti-IL4
Th2: 50ng/mL IL4 (also tried 20ng/mL), anti-IFNg, anti-IL12
Th17: .5ng/mL TGFb, 10ng/mL IL1b, IL23, IL6

CellSpecific.com, on 13 July 2011 - 06:30 PM, said:

Can you give information about the concentration of skewing reagents? The more details the better.

[Greg S]

Oddly enough, I tried plate-bound, but it wasn't activating my naive CD4s (but they are definitely sticking to the bottom of the plate which suggests there is Ab coated). I have samples running now using PBMCs and both soluble and plate-bound Abs in the same skewing cocktails.

The viability of the cells is great post-skewing (used both Trypan and a Live/Dead flow cytometry stain).

CellSpecific.com, on 15 July 2011 - 04:44 PM, said:

Have you tried skewing with plate bound anti-CD3 and soluble anti-CD28? Bead-based activation is sure to stimulate but I wonder if this might be too strong/much. Have you checked viability of cells post skewing? If I'm right, the cells could be dead/dying from AICD (activation induced cell death).

Hope this helps.

Protocols and Methods at CellAssays.com

[tk2509]

Hi guys!! I'm in need of a protocoll to skew naive T cells to Th2 cells in cell cultures. Do you have any reliable one?