Transient regulatory T-cells: A state attained by all activated human T-cells
Introduction
CD4+CD25+/high regulatory T (Treg) cells are an important arm of the immune system that downregulate potentially harmful effector immune responses [1]. They have been shown to play a role in autoimmune disorders, infections, tumors, asthma, allergy and transplantation, and hence their modulation in these diseases is thought to be of great potential benefit [2]. Further elucidation of this subset has been hampered by the lack of a specific surface marker to isolate and study these cells. Markers identified as being expressed on Tregs are also expressed by activated T-cells. Hence, the discovery of the transcription factor FOXP3 as a marker that is expressed in Tregs but not on activated T-cells held major significance. Predominantly through murine studies, it was shown that FOXP3 is necessary and sufficient for the development and function of Tregs[3], [4], [5], [6], [7]. Transduction of FOXP3 in human CD4+CD25− T-cells appeared to confer regulatory properties to those cells [7]. FOXP3 regulates T-cell activation by interacting with NF-AT or NF-κB and consequently repressing IL-2 secretion [8], [9].
CD4+CD25+/high Tregs can be subdivided into natural Tregs and induced Tregs. Natural CD4+CD25+FOXP3+ Tregs are thought to arise in the thymus and suppress harmful immune responses in the periphery [1]. While FOXP3 expression is thought to be a unique feature of natural Tregs in mice [3], [4], [5], several human studies and some murine studies have suggested that CD4+CD25− T-cells may give rise to induced CD4+CD25+FOXP3+ regulatory T-cells following stimulation [10], [11], [12], [13]. However, due to the lack of antibodies against intranuclear FOXP3 at that time, it had been repeatedly suggested that such cells merely represent an expansion of contaminating populations of natural Tregs[14], [15], [16], [17]. This continues to be a controversy in the field, with some studies reporting no upregulation of FOXP3 expression [7] and others proposing that induced FOXP3+ T-cells may not be regulatory in function [18]. However, these studies did not directly evaluate suppressor function. Also, it is currently unclear if the induced and natural Tregs represent separate lineages or if they belong to the same lineage but just differ in their location and timing of their origin. In either case, it is generally believed that FOXP3-expressing T-cells, induced or natural, are a stable population of T-cells with immune regulatory functions. Thus, the presence of FOXP3 and the ability to suppress effector T-cell responses in-vitro have been used as the hallmarks for the detection and quantification of this population [15]. This approach has been widely used recently in human disease settings where presence or absence of FOXP3+ T-cells at the disease site or in the blood (with or without the presence of concomitant suppressive activity) is interpreted as evidence for involvement of Tregs in the disease pathogenesis/modulation [19], [20], [21], [22], [23].
In this study, we evaluated the immune biology of adaptively induced FOXP3+ T-cells by using polychromatic flow cytometry and recently developed robust anti-FOXP3 antibodies including one that recognizes a specific spliced isoform of human FOXP3 [24]. Using CFSE staining of highly purified T-cell populations, we tracked their dynamics and function following activation with different stimuli. Using this approach, we conclusively show that virtually all activated CD4+ and CD8+ T-cells transiently upregulate FOXP3 and show transient suppressive activity. We further show that this transient regulatory state and certain effector functions are differentially regulated, suggesting that this state might be a general immune mechanism of fine-tuning an ongoing immune response.
Section snippets
Cell preparation and bead sorting
PBMC were isolated from fresh buffy coats from healthy blood donors using Ficoll Hypaque density gradient. “Untouched” CD4+ T-cells and CD8+ T-cells were negatively selected using negative selection kits and AutoMacs (DEPLETE program) from Miltenyi Biotech. Total CD3+ T-cells were negatively selected using MagCellect negative selection kits from R and D systems. All of these were negatively selected to greater than 85% purity. CD25+ T-cells were depleted from the purified CD4+ and CD8+ T-cells
All CD4+CD25− T-cells upregulate FOXP3 transiently following activation
First, we negatively selected “untouched” human CD3+CD25− (or CD4+CD25−) T-cells and stained them with CFSE. These cells were stimulated either in a mixed lymphocyte reaction using allogeneic, T-cell-depleted antigen-presenting cells (APC) or with anti-CD3 and autologous APC to stimulate the formation of induced FOXP3+ T-cells, as previously described [10], [11]. Following activation, these cells were monitored longitudinally for CFSE dilution and FOXP3/CD25 expression (to determine their
Discussion
It is known that CD25+FOXP3+ T-cells with regulatory properties can be induced following activation [10], [11], [12], [13]. For example, using PCR-based analysis, it has been demonstrated that CD4+CD25− T-cells can generate FOXP3+ regulatory T-cells [11]. However, it was widely proposed that these cells arose from a contaminating population of natural Tregs[14], [15], [16]. Moreover, the proportion of activated T-cells that become FOXP3+ and the stability and significance of such expression are
Acknowledgments
This work was supported by grants (to N.J.K.) from the NIH and National MS Society (NMSS). N.J.K. is a Harry Weaver Neuroscience Scholar of the NMSS. We thank Drs. Ellen Vitetta, Mihail Firan and Mr. Ethan Baughman for review and discussions and Ms. Bonnie Darnell for assistance with flow sorting.
References (52)
- et al.
Regulatory T cell lineage specification by the forkhead transcription factor foxp3
Immunity
(2005) - et al.
Scurfin (FOXP3) acts as a repressor of transcription and regulates T cell activation
J. Biol. Chem.
(2001) - et al.
Mucosal but not peripheral FOXP3+ regulatory T cells are highly increased in untreated HIV infection and normalize after suppressive HAART
Blood
(2006) - et al.
High prevalence of autoreactive neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay
Blood
(2004) - et al.
Persistence of naive CD45RA+ regulatory T cells in adult life
Blood
(2006) - et al.
Regulatory T cell compartmentalization and trafficking
Blood
(2006) - et al.
Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans
Hum. Immunol.
(2005) - et al.
CD28 superagonists put a break on autoimmunity by preferentially activating CD4+CD25+ regulatory T cells
Autoimmun. Rev.
(2006) - et al.
Transient accumulation of human mature thymocytes and regulatory T cells with CD28 superagonist in “human immune system” Rag2(−/−)gammac(−/−) mice
Blood
(2006) - et al.
Functional avidity directs T-cell fate in autoreactive CD4+ T cells
Blood
(2005)
Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self
Nat. Immunol.
CD4+ regulatory cells as a potential immunotherapy
Philos. Trans. R. Soc. London, Ser. B Biol. Sci.
Foxp3 programs the development and function of CD4+CD25+ regulatory T cells
Nat. Immunol.
Control of regulatory T cell development by the transcription factor Foxp3
Science
An essential role for Scurfin in CD4+CD25+ T regulatory cells
Nat. Immunol.
Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells
Int. Immunol.
Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells
Proc. Natl. Acad. Sci. U. S. A.
De novo generation of antigen-specific CD4+CD25+ regulatory T cells from human CD4+CD25− cells
Proc. Natl. Acad. Sci. U. S. A.
Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25− T cells
J. Clin. Invest.
Conversion of peripheral CD4+CD25− naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3
J. Exp. Med.
Inducing and expanding regulatory T cell populations by foreign antigen
Nat. Immunol.
CD4+ Tregs and immune control
J. Clin. Invest.
A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3
Nat. Immunol.
Regulatory/suppressor T cells in health and disease
Arthritis Rheum.
CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells
J. Exp. Med.
Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development
Proc. Natl. Acad. Sci. U. S. A.
Cited by (305)
Spatial immunophenotyping of the tumour microenvironment in non–small cell lung cancer
2023, European Journal of CancerDifference between CD25<sup>+</sup>Tregs and Helios<sup>+</sup>Tregs in a murine model of allergic rhinitis
2021, Brazilian Journal of OtorhinolaryngologyCitation Excerpt :This work found fewer CD25+Tregs in AR mice, consistent with previous studies.15–17 However, studies have found that this strategy does not represent the true inhibitory function of Treg cells, even in specific T-cell induction scenarios, because CD25 and Foxp3 (two important Treg markers) are also present in activated non-Treg cells.18,19 In 2006, Sugimoto et al. first suggested that Helios, a member of the Ikaros family of zinc-finger proteins, was a marker of natural Treg.20
Exploring the controversial role of PI3K signalling in CD4<sup>+</sup> regulatory T (T-Reg) cells
2020, Advances in Biological RegulationIncreased infiltration of CD4<sup>+</sup>IL-17A<sup>+</sup>FOXP3<sup>+</sup> T cells in Helicobacter pylori-induced gastritis
2024, European Journal of Immunology