Hello there,
I am trying many in vivo experiments and most of them includes CD4 T cell adoptive transfer between mice. But not many results show significant difference and now I am worried if the T cells I transferred might be defective.
My concern is that anti-CD4 fluorescent monoclonal antibodies I am using for flow cytometry sorting CD4+ T cells might block CD4 signaling and inactivate transferred T cells in recipient mice. In my lab, we have GK1.5 and RM4-5. Application notes on BioLegend website say they both can block CD4 T cell activation, or even deplete CD4 T cells.
I hope anyone with experience in CD4 T cell transfer can answer if my concern is just or irrelevant.
Thank you.
I would transfer untouched cd4 cells, depleted from all other immune cells
How are you isolating the CD4+ cells prior to adoptive transfer
Sure! Mabs to CD4 block T cell signaling. Agree to Maud! You need to use negative selection to obtain purified CD4 T cells. Appropriate way would be immunomagnetic separation to deplete subpopulation of interest from all other cells.
Probably, using negative selected CD4+ T-cells purified by MACS is enough for your needs.
I would also suggest staining with CFSE; that would allow you monitor for proliferation in vivo. And definitely use a negative selection separating method
I agree that MACS sorting CD4 T cells via negative selection for adoptive transfer is the best way to go. If you are unfamiliar with this technique here is the website for the negative selection kit we use in our lab: http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-reagents/t-cells/cd4-t-cell-isolation-kit-mouse.aspx
I would indeed go for negative selection, such as for example described for a kit by StemCell Technologies, Vancouver (see link).
Best of luck!
http://www.stemcell.com/en/Products/All-Products/EasySep-Mouse-CD4CD62L-T-Cell-Isolation-Kit.aspx
Thank you for your answers! One more question, anti-CD4 mAb clone RM4-4 does not seem to have any reference for CD4 signal blocking. Have anyone tried positive selection with RM4-4 for in vivo assays such as adoptive transfer? I know negative selection is much better choice, but in some experiments CD4 T cell purity is important, such as IBD induction where Treg or B cell contamination can prevent IBD.
if you need very high purity CD4+ Foxp3- , using MACS you can make an enrichment in CD4+ first by negative selection and then separate Tregs by positive selection based on CD25 expression (use anti-CD25-PE, + anti-PE magnetic beads).Miltenyi has a kit for this.
Also you can try FACS for purification of CD4+CD25neg Tregs.
But, if you have access to a Foxp3-EGFP reporter mice that would improve a lot your purification, just sorting Tconv cells as CD4+Foxp3-EGFPneg.
Then if you have problems with Tregs after transfer, you can inject anti-CD25 (PC61.5) to avoid expansion of induced Tregs.
good luck!
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