Transient regulatory T-cells: A state attained by all activated human T-cells

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Abstract

CD4+CD25+FOXP3+ regulatory T-cells (Tregs) form an important arm of the immune system responsible for suppressing untoward immune responses. Tregs can be thymically derived or peripherally induced, even from CD4+CD25FOXP3 T-cells. FOXP3 expression and in vitro suppressive activity are considered unique hallmarks of this dedicated and stable lineage of regulatory cells. Here we show that virtually all human CD4+CD25FOXP3 T-cells and CD8+CD25FOXP3 T-cells attain a transient FOXP3+CD25+ state during activation. In this state of activation, these cells possess the classic phenotype of Tregs, in that they express similar markers and inhibit in vitro proliferation of autologous CD4+CD25 T-cells. This state is characterized by suppressed IFN-γ production and robust TNF-α and IL-10 production. Interestingly, the great majority of the activated cells eventually downregulate FOXP3 expression, with a concomitant drop in suppressive ability. Our results show that, in humans, FOXP3 expression and Treg functionality are not exclusive features of a stable or unique lineage of T-cells but may also be a transient state attained by almost all T-cells. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts and suggest that attempts to induce “Tregs” may paradoxically result in induction of effector T-cells, unless stability is confirmed.

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.

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