Regulators of the immune system

Regulatory T cells (Tregs) are critical to the maintenance of immune cell homeostasis as evidenced by the catastrophic consequences of genetic or physical ablation of the Treg population. Specifically, Treg cells maintain order in the immune system by enforcing a dominant negative regulation on other immune cells. Broadly classified into natural or adaptive (induced) Tregs; natural Tregs are CD4+CD25+ T-cells which develop, and emigrate from the thymus to perform their key role in immune homeostasis. Adaptive Tregs are non-regulatory CD4+ T-cells which acquire CD25 (IL-2R alpha) expression outside of the thymus, and are typically induced by inflammation and disease processes, such as autoimmunity and cancer.

Precise understanding of the immunosuppressive mechanism of T regulatory cells remains elusive, although there is increasing evidence that Tregs manifest their function through a myriad of mechanisms that include the secretion of immunosuppressive soluble factors such as IL-9, IL-10 and TGF beta, cell contact mediated regulation via the high affinity TCR and other costimulatory molecules such as CTLA-4, GITR, and cytolytic activity. Understanding the mechanisms by which Treg cells exert their influence is an area of intense research with broad implications for the development of therapeutic strategies for many disease processes including cancer, diabetes, and Immune mediated diseases.

Under the influence of TGF beta, adaptive Treg cells mature in peripheral sites, including mucosa-associated lymphoid tissue (MALT), from CD4+ Treg precursors, where they acquire the expression of markers typical of Tregs, including CD25, CTLA4 and GITR / AITR. Upon up-regulation of the transcription factor Foxp3, Treg cells begin their suppressive effect. This includes the secretion of cytokines including IL-10 and TGF beta which may induce cell-cycle arrest or apoptosis in effector T cells, and blocking co-stimulation and maturation of dendritic cells.

T regulatory cell properties

Property Natural Treg (nTreg) Induced Treg (iTreg) - Tr1 Induced Treg (iTreg) - Th3
Development Thymus Periphery (MALT) Periphery (MALT)
Phenotype CD4+CD25+CD127low CD4+CD25- CD4+CD25+ from CD25- precursors
Other Associated Markers CTLA-4+GITR+Foxp3+ CD45RBlowFoxp3- CD25low-variableCD45RBlowFoxp3+
Suppression Contact-, Granzyme-B dependent, makes TGF beta IL-10 mediated TGF beta mediated
Target Cells APC and Effector T Cells Effector T Cells Unknown
CD28 Involvement

Thymic development and maintenance in periphery

Unnecessary for development or function Unnecessary for development or function
in vivo Role Suppression of autoreactive T cells Mucosal immunity, inflammatory response Mucosal immunity, inflammatory response
in vitro Expansion TCR/CD28 stimulation and IL-2 CD3, IL-10, Retinoic Acid CD3, TGF beta

References

  1. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, Shimizu J, Sakaguchi S. Int Immunol. 1998 Dec;10(12):1969–80.
  2. Regulatory T cells in autoimmmunity. Shevach EM. Annu Rev Immunol. 2000;18:423–49. Review.
  3. Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor? Curotto de Lafaille MA, Lafaille JJ. Immunity. 2009 May;30(5):626–35. Review.

In vitro expansion of Treg cells

To completely understand the mechanism of Treg-mediated immune suppression, protocols have been developed for the in vitro expansion of Tregs isolated from both mouse and human. Tregs have been found to express high levels of growth inhibitory genes including SOCS1 and 2, CTLA-4, and PD1. Therefore, strong signals through TCR / CD28 and IL-2R are thought to be required to overcome the cell cycle arrest of Tregs. Even with established protocols, the challenge to prepare high quality functionally suppressive Tregs with a minimum contamination of effector and non-suppressive T cells remains. To expand Tregs, it has been shown that high concentrations of key cytokines including IL-2 and TGF beta as well as TCR signaling are the essential components required to generate large numbers of either human or murine Tregs. In this approach, IL-2 and TCR signaling antibodies induce T cell proliferation and the addition of TGF beta directs cells towards the T regulatory phenotype by preventing apoptosis and driving conversion of naive CD4 T cells into Tregs with preferentially high expression levels of Foxp3. Experiments have shown that the intensity and sustainability of induced Foxp3 expression is directly proportional to the amount of TGF beta added, which in turn determines the efficacy of Treg suppression.

References

  1. GITR engagement preferentially enhances proliferation of functionally competent CD4+CD25+FoxP3+ regulatory T cells. Liao G, Nayak S, Regueiro JR, Berger SB, Detre C, Romero X, de Waal Malefyt R, Chatila TA, Herzog RW, Terhorst C. Int Immunol. 2010 Feb 5. 
  2. Development of CD4+CD25+FoxP3+ regulatory T cells from cord blood hematopoietic progenitor cells. Hutton JF, Gargett T, Sadlon TJ, Bresatz S, Brown CY, Zola H, Shannon MF, D'Andrea RJ, Barry SC. J Leukoc Biol. 2009 Mar;85(3):445-51. 
  3. Isolation, expansion, and characterization of human natural and adaptive regulatory T cells. Gregori S, Bacchetta R, Passerini L, Levings MK, Roncarolo MG. Methods Mol Biol. 2007;380:83-105. Review.

T regulatory cell suppression

Following the isolation of Tregs, and subsequent expansion, it is critical to assess the efficiency of T regulatory cell production. This can be done by analyzing the cytokine expression profile of human and mouse in vitro-derived Treg populations. Cells which have maintained a Treg phenotype in culture will express the Treg effector molecules IL-10 and TGF beta. However, other T cells will express cytokines uncharacteristic of the Treg lineage including IFN gamma and TNF alpha. Addressing Treg suppression can readily be achieved using a variety of approaches to profile Treg /Teffector cytokine secretion.

References

  1. GITR engagement preferentially enhances proliferation of functionally competent CD4+CD25+FoxP3+ regulatory T cells. Liao G, Nayak S, Regueiro JR, Berger SB, Detre C, Romero X, de Waal Malefyt R, Chatila TA, Herzog RW, Terhorst C. Int Immunol. 2010 Feb 5. 
  2. Development of CD4+CD25+FoxP3+ regulatory T cells from cord blood hematopoietic progenitor cells. Hutton JF, Gargett T, Sadlon TJ, Bresatz S, Brown CY, Zola H, Shannon MF, D'Andrea RJ, Barry SC. J Leukoc Biol. 2009 Mar;85(3):445-51. 
  3. Isolation, expansion, and characterization of human natural and adaptive regulatory T cells. Gregori S, Bacchetta R, Passerini L, Levings MK, Roncarolo MG. Methods Mol Biol. 2007;380:83-105. Review.

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.

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.

Human viable T regulatory cell sorting guide

Marker Natural Treg cells Induced Treg cells Activated Treg cells
CD4 + + +
CD25 (IL-2Ra) + + high
CD127 (IL-7Ra) low low low
CD45RA + +
CD45RO + + +
CD39 + + +
CD73 + + +
AITR + + +
CD101 low low high

Mouse viable T regulatory cell sorting guide

Marker Natural Treg cells Induced Treg cells Activated Treg cells
CD4 + + +
CD25 (IL-2Ra) + + high
CD127 (IL-7Ra) low low low
GARP +
CD45RB low low low
CD103 +
FR4 (Folate Receptor 4) + + +
CD39 + + +
CD73 + + +
CD101 low low high
GITR + + +

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

  1. 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. 
  2. 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. 
  3. 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. 
  4. 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.