New tools to identify regulatory T cells

Authors

  • Luis Graca

    Corresponding author
    1. Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Portugal
    2. Instituto Gulbenkian de Ciência, Oeiras, Portugal
    • Cellular Immunology Unit, Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Avenida Professor Egas Moniz, 1649-028 Lisbon, Portugal, Fax: +351-21-7985142
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Abstract

The lack of tools for direct identification of regulatory T cells (Treg cells) at the single-cell level has been one of the major hurdles for the study of Treg cells and their involvement in human disease. The identification of the transcription factor Foxp3 as a molecular correlate for Treg function offered the opportunity to directly identify Treg cells, but until recently adequate reagents were not available. The tools promising the solution for this problem have emerged through the development of transgenic mice in which Foxp3 expression drives the production of fluorescent proteins, as well as the development of mAb that are able to identify Foxp3+ cells by histology or flow cytometry. With these new tools, the mAb in particular, it will become possible for the first time to directly probe the participation of Foxp3+ Treg cells in human pathology in a quantitative fashion.

See accompanying article: http://dx.doi.org/10.1002/eji.200526189

Abbreviation:
Treg cell:

Regulatory T cell

Introduction

The impact of the study of regulatory T cells (Treg cells) has increased exponentially since the identification of phenotypic markers allowing the isolation of a T cell population with potent suppressive effect – CD4+CD25+ Treg cells 1. It has been reported that CD4+CD25+ Treg cells, representing approximately 10% of peripheral CD4+ T cells in experimental animals and in humans, are important for the prevention of different types of immunopathology. Several studies have identified a role for such Treg cells in the prevention of autoimmune diseases and inflammatory bowel disease 2. But it has also been shown that Treg cells can be elicited therapeutically to suppress allograft rejection, being capable of maintaining transplant survival in the absence of pharmacological immunosuppression 3. Treg cells isolated from bone-marrow donors may also be used therapeutically to prevent graft-versus-host disease 4. Furthermore, it has been suggested that Treg cells may have a deleterious effect in suppressing anti-tumor immune responses 5.

The identification of Treg cells

The important role attributed to CD4+CD25+ Treg cells in such diverse immunopathologies prompted the study of their involvement in many different human diseases, as well as in equivalent animal models. However, a major hurdle for such studies has been the difficulty in identifying the "true" Treg cells among the CD4+CD25+ T cell population. It has always been a concern that despite the strong suppressive effect of the CD4+CD25+ cells – tested as a population – they are heterogeneous, containing both Treg cells and also non-regulatory CD4+CD25+ cells, such as recently activated T cells. The proportion of true Treg cells within the CD4+CD25+ T cell population has been impossible to establish with the reagents available. Occasional excitement has followed the identification of phenotypic markers claimed to allow a better discrimination of Treg cells, such as CTLA-4, GITR, CD62L, CD103 or LAG-3 (on activated Treg cells). However, since these same molecules can also be present on non-regulatory T cells, their usefulness for pinpointing the true Treg cells is limited. More recently, neuropilin-1, a semaphorin III receptor, was claimed to be a useful surface marker to discriminate between CD4+CD25+ Treg cells and both naive and recently activated CD4+CD25 T cells 6.

The transcription factor Foxp3 is generally accepted as the best available marker for CD4+CD25+ Treg cells 79. It was shown to be mainly expressed by the CD4+CD25+ Treg cells and, significantly, the ectopic expression of Foxp3 by non-regulatory CD4+CD25 T cells induces a change of phenotype and function as these cells become CD4+CD25+ Treg cells 7. Furthermore, humans and mice defective for Foxp3 gene develop a disease associated with autoimmunity and allergic disregulation 10. However, the usefulness of Foxp3 as a marker for Treg cells has been hampered by its intracellular expression and lack of anti-Foxp3 antibodies suitable for histological and flow-cytometry studies. Without the capacity to identify Foxp3+ cells at the single-cell level, it has been difficult to study the contribution of the true Treg cells within the entire CD4+CD25+ T cell pool in human pathologies like autoimmunity or cancer. In a similar way, the study of Treg cells also present in the CD4+CD25 population has been hampered by the difficulty in identifying these Treg cells from a large population of non-regulatory cells 11, 12.

New tools to identify Treg cells

Recent reports have demonstrated the ability to identify Treg cells at the single-cell level. Two groups have developed mouse models in which expression of Foxp3 is associated with the expression of red or green fluorescent proteins 13, 14. In this issue, Roncador et al. provide a powerful new tool for the study of Treg contribution in human pathology 15. They describe a panel of anti-Foxp3 mAb that can be used to directly identify Foxp3+ Treg cells in histological sections or flow cytometry. Some of the mAb clones, generated to human Foxp3, can also be used for murine studies.

These new tools have allowed, for the first time, the identification of true Treg cells within the CD4+CD25+ population, as it was previously shown that only the CD4+CD25+Foxp3+, and not the CD4+CD25+Foxp3, T cells can behave in a regulatory fashion 13. Whereas in murine lymph nodes Foxp3+ cells represent ∼70% of the CD4+CD25+ T cell population 13, in human peripheral blood the proportion is ∼55% 15. The major difference between these studies concerns the proportion of Foxp3+ T cells within the CD4+CD25 population: a minority in human peripheral blood, but a significant population in murine lymph node, spleen, and especially lung. Interestingly, the suppressive function of different populations of Foxp3+ cells is equivalent regardless of CD25 co-expression 13.

Foxp3 and different Treg populations

Treg cells can either develop as a distinct thymic lineage or be generated in the periphery (Fig. 1). In mice where transplantation tolerance is induced with therapeutic mAb it was shown that CD4+CD25 and CD4+CD25+ regulatory T cells, when studied as global populations, can have equivalent suppressive function 16. It will be important to clarify the relationship between naturally occurring Treg cells and induced Treg cells – both CD25 and CD25+; in particular, whether Foxp3 expression is also required for the acquisition of suppressive function by therapeutically induced CD4+CD25 Treg cells and those infiltrating tolerated allografts 17.

Figure 1.

Possible lineage relationship between CD4+ T cells with different phenotypes (conventional T cells are shown in red, Treg cells in green). It is accepted that naturally occurring CD4+CD25+Foxp3+ Treg cells are produced in the thymus, whereas the thymic CD4+CD25 T cells seem to be devoid of regulatory activity. Upon antigenic stimulation in the periphery, CD4+CD25Foxp3 T cells can acquire an "aggressive" function (becoming CD4+CD25+Foxp3) but they can equally become CD4+CD25+Foxp3+ Treg cells 18. CD4+CD25Foxp3+ Treg cells can be found in the periphery and they can acquire CD25 expression 19. These cells may represent a stage of differentiation of CD4+CD25+Foxp3+ Treg cells or a different lineage of Treg cells. It is equally unclear whether CD4+CD25Foxp3 Treg cells can be induced therapeutically.

Conclusions

These recently developed tools for the direct identification of Treg cells represent an important breakthrough, yet more are still eagerly anticipated to address different issues. For instance, the development of a mouse model with a bicistronic reporter expressing a surface marker protein under the control of Foxp3 may allow not only the direct identification of Treg cells, but also in vivo studies where Treg cells are specifically ablated with depleting antibodies targeting the marker protein.

In the case of the report by Roncador et al., it is anticipated that the anti-Foxp3 mAb, by allowing direct identification of true Treg cells, will drive exciting new findings concerning Treg biology, in particular concerning their role in human pathology.

Footnotes

  1. 1

    WILEY-VCH

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