T Regulatory Cells

Isolation of Viable Treg Cells

T regulatory cells were originally identified as a CD4+CD25+ T cell population with the capacity to suppress an immune response. The identification of Foxp3 as the “master-regulator” of Tregs was a critical step in defining Tregs as a distinct T cell lineage. However, it has become clear that Foxp3 expression may also be induced in T cells that lack Treg function. Moreover, the localization of Foxp3 to the nucleus prevents its use as a marker for isolation of viable Tregs. The identification of additional antigenic markers on the surface of Tregs has enabled identification and FACS sorting of viable Tregs to greater purity, resulting in a more highly-enriched and suppressive Treg population. In addition to CD4 and CD25, it is now known that both mouse and human Tregs express GITR / AITR, CTLA-4, but express only low levels of CD127 (IL-7Ra). Moreover, Tregs can exist in different states which can be identified based on their expression of surface markers. Tregs which develop in the thymus from CD4+ thymocytes are known as “natural” Tregs, however Tregs can also be induced in the periphery from naïve CD4+ T cells in response to low-dose engagement of the TCR, TGF beta and IL-2. These “induced” Tregs secrete the immunosuppressive cytokine IL-10. The phenotype of Tregs changes again as they become activated, and markers including GARP in mouse and human, CD45RA in human, and CD103 in mouse have been shown to be useful for the identification of activated Tregs.

See all reagents for T Regulatory Cell analysis

Sorting Viable T Regulatory Cells

eBioscience offers the most extensive portfolio of antibodies, in a wide selection of formats, against targets critical for the accurate identification and sorting viable Treg populations. Indicated below are combinations of markers which can be used for the isolation of various sets of human and mouse Tregs. eBioscience provides reagents for both cell surface and intracellular phenotyping of mouse, human, rat and non-human primate Tregs.

Improved T Regulatory Cell Purity with Additional Markers

In human Tregs, the addition of markers including CD45RO, CD39 and CD73 improve the purity of Tregs obtained from identification using CD4/CD25/CD127 alone. Furthermore, CD45RA has proven to be a useful marker for the identification of activated Tregs.

Similarly, in the mouse model, the addition of CD39, CD101, FR4 and CD73 to any basic CD4/CD25/CD127 enrichment cocktail for sorting Tregs can be useful to enhance the purity of Tregs (as defined by Foxp3 expression). In the data below, Foxp3 identified by intracellular staining, has been used to identify Tregs. By using the series of key markers indicated it is possible to selectively enrich viable Treg cells without using cell permeabilizing techniques. The identification of live Tregs enables downstream analysis and assessment of Treg suppressive function.

References

Cell-surface IL-7 receptor expression facilitates the purification of FOXP3(+) regulatory T cells. Banham AH. Trends Immunol. 2006 Dec;27(12):541-4. Epub 2006 Oct 12. Review.

CD101 surface expression discriminates potency among murine FoxP3+ regulatory T cells. Fernandez I, Zeiser R, Karsunky H, Kambham N, Beilhack A, Soderstrom K, Negrin RS, Engleman E. J Immunol. 2007 Sep 1;179(5):2808-14. Erratum in: J Immunol. 2007 Oct 15;179(8):5605.

Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, Giometto R, Höpner S, Centonze D, Bernardi G, Dell'Acqua ML, Rossini PM, Battistini L, Rötzschke O, Falk K. Blood. 2007 Aug 15;110(4):1225-32.

GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells. Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, Shevach EM. Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13445-50.