The maintenance of self-tolerance involves central and peripheral mechanisms, but the process of thymic negative selection is imperfect. Self-reacting clones that escape central tolerance are kept in check by a variety of peripheral tolerance mechanisms, in which the dominant tolerance exerted by CD4⁺CD25⁺ regulatory T (T_reg) cells plays an important role. The development and function of T_reg cells crucially depends on the forkhead/winged helix transcription factor FOXP3. This dependence is compellingly illustrated by the fact that loss of function of FOXP3 leads to the development of the fatal autoimmune lymphoproliferative disorder IPEX (immunodysregulation, polyendocrinopathy, and enteropathy, X-linked) syndrome 1 2 . Similarly, loss of function of FoxP3 in mice, either natural (scurfy) or recombinant, results in an analogous immune pathology due to a lack of T_reg cells 3 4 5 . The dominant role of FOXP3 is further corroborated by the fact that the gain of function induced by ectopic expression in conventional CD4⁺CD25^- helper T (T_h) cells leads to the acquisition of suppressor function and the induction of a partial regulatory phenotype in mice and humans 3 6 7 8 .

Despite advances in our understanding of T_reg-cell lineage commitment and function, gained mainly by the study of FoxP3 knock-out/knock-in mice 4 5 9 10 11 , the dominant role of FOXP3 in the human system and its suitability as a bona fide marker of human T_reg cells have been questioned 8 12 13 14 .

The reasoning that more than FOXP3 is necessary for fully explaining the regulatory phenotype in humans is supported by several observations. First, T-cell receptor (TCR) stimulation of CD4⁺CD25^-FOXP3^- T cells leads to the induction of FOXP3 without interfering with the expression of effector cytokines such as interleukin-2 and interferon-γ 15 16 17 . Second, the expression of FOXP3 by conventional CD4⁺CD25^- T_h cells and even by T_h lines and clones does not necessarily indicate the acquisition of suppressor function 8 12 17 18 19 . Third, and more specifically, it has recently been found that demethylation of a conserved FOXP3 intronic region may be a better marker for suppressor function than the differential expression of FOXP3 at the mRNA or protein level in T_reg and T_h cell clones 20 . Fourth, ectopic expression of FOXP3 in human T_h cells does not lead to the establishment of a stable regulatory phenotype 8 12 21 . Finally, although the enhancement of FOXP3 expression by transforming growth factor-β1 (TGF-β1) in activated human CD4⁺CD25^- T_h cells generates so-called induced T_reg (iT_reg) cells, this phenotype is rapidly lost 19 21 22 . Altogether, these observations suggest that more than FOXP3 is necessary for fully explaining the regulatory phenotype.

A plausible explanation for the qualitative and quantitative differences in the expression of FOXP3 in human T_h and T_reg cells, with their mutually exclusive effector and regulatory functions, is the presence of a T_reg-specific higher-order regulatory network 13 14 23 . The possibility that T_reg-specific control mechanisms can maintain sustained high levels of FOXP3 and can control the regulatory function has been addressed only recently with the identification of the glycoprotein-A repetitions predominant (GARP or LRRC32) receptor 21 24 25 26 27 28 .

GARP was identified based on the analysis of gene expression profiling of T_reg and T_h cells following TCR stimulation, since TCR stimulation does lead to the induction of their mutually exclusive functions 21 24 . GARP is specifically induced in CD4⁺CD25^hiFOXP3^hi T_reg cells and thus is a T_reg-specific activation marker 21 24 25 26 27 . Because the expression of GARP is up-regulated in FOXP3-transduced T_h cells, GARP obviously depends on FOXP3, and this finding suggests a potential contribution to the regulatory phenotype 21 29 .

The function of GARP has been elucidated by ectopic over-expression in human alloantigen-specific T_h cells and down-regulation of GARP in human antigen-specific T_reg cells 21 29 . Retroviral over-expression of GARP in T_h cells, after some rounds of TCR stimulation, leads to an efficient and stable reprogramming/transdifferentiation of the established effector toward the regulatory program. This finding is associated with constitutive expression of FOXP3, the β-galactoside binding protein lectin, galactoside-binding, soluble, 3 (LGALS3) 8 30 , and the cysteine-endoprotease legumain (LGMN) 8 and with an extended T_reg-signature with suppression of effector cytokine production and acquisition of regulatory functions similar to those of T_reg cells 21 29 . Thus, a FOXP3-regulating gene has been identified in the human system, and this finding suggests that the GARP signaling pathway may have direct therapeutic applications 28 31 similar to those that have been described for CD83 in the murine system 32 .

In contrast, lentiviral down-regulation of GARP in human alloantigen-specific T_reg cells by specific small interfering RNA (siRNA) substantially impairs suppressor function and FOXP3 expression that is associated with impaired induction of CD83 and CD27, both known to regulate FOXP3 21 32 33 . More striking is the fact that similar changes are induced by the down-regulation of FOXP3 in T_reg cells, a finding that provides compelling evidence for a GARP-FOXP3 positive feedback loop in T_reg cells. Because this feedback loop seems to be interrelated with other FOXP3-regulating systems such as CD83 and CD27, the existence of a higher-order regulatory network as discussed recently by Hori 13 , can be speculated (Figure 1a).

Thus, GARP is a specific marker of activated T_reg cells and can explain the qualitative and quantitative differences in FOXP3 expression and function in T_reg cells and T_h cells. Lack of GARP expression further differentiates and explains the transient nature of TGF-β-induced iT_reg cells 19 , suggesting that natural and iT_reg cells may represent alternative differentiation stages of suppressor cells 21 26 . In line with these observations, a specifically hypomethylated region in intron 1 of GARP and two differentially methylated regions with enhancer functions in T_reg cells have recently been characterized. These findings have identified an epigenetic basis for the lineage-specific difference in GARP expression 34 .

Members of the TGF-β family are pleiotropic cytokines with crucial functions in differentiation, morphogenesis, and immune homeostasis. Their function in immune homeostasis is evidenced by the fact that dysregulation of TGF-β functions is associated with autoimmunity 35 . Like many other cell types, human T_reg and T_h cells can secrete latent TGF-β 20 36 . However, latency-associated peptide (LAP) prevents the activation of latent TGF-β toward the active mature TGF-β 37 . The selective induction of LAP and the activation of active TGF-β upon TCR stimulation has been reported only for human T_reg cells and clones 20 37 38 . This finding further explains the TGF-β specific transcriptional signature and detection of expression of phosphorylated SMAD2 in T_reg cells of human and murine origin 20 23 37 39 . Moreover, specific up-regulation of LAP on activated human T_reg cells has been shown to allow improvements in the purity of T_reg cell isolation procedures by separating activated LAP⁺FOXP3⁺ T_reg cells from contaminating effector LAP^-FOXP3^+/lo T_h cells 38 . Thus, besides being a marker of activated T_reg cells in complex with LAP, TGF-β1 is an important modulator of FOXP3 expression 19 21 40 , and a potential mediator of infectious tolerance by its action in converting FoxP3^- murine CD4⁺ T cells into functional FoxP3⁺ iT_reg cells 41 .

The issue of cell-surface binding of LAP/latent TGF-β on human T_reg cells has been addressed only recently with the identification of GARP as a receptor of this complex 26 27 . Therefore, two important regulatory circuits of T_reg cells come together: the GARP-FOXP3 feedback loop and the autocrine TGF-β loop (Figure 1b). With that, the potential synergy of many of the T_reg signature components that are essential for the regulatory properties that have been ascribed to TCR activation, interleukin-2, TGF-β, and FOXP3 itself 20 23 could be explained by this particular spatiotemporal interplay of interrelated signaling systems on T_reg cells.

The identification of GARP as a receptor for LAP/latent TGF-β opens speculation about a potential common denominator. The reason for such speculation is that neuropilin 1 (Nrp1), which has been characterized as a marker of CD4⁺CD25⁺ T_reg cells in mice that enhances T_reg cell/dendritic cell contact during antigen recognition 42 43 , has also been characterized as a receptor for LAP/latent TGF-β 39 . Moreover, murine sorted Nrp1^- T cells capture soluble Nrp1 (applied as a constant fragment [Fc]-fusion protein), and the captured Nrp1 increases their ability to bind LAP/TGF-β1. Such coated T cells acquire strong regulatory activity 39 . Thus, Nrp1 is a TGF-β1 receptor that, unlike GARP 27 , also activates the latent form of TGF-β1.

The structural differences between GARP, a Toll-like receptor homologue with leucine-rich repeats, and the multi-domain protein Nrp1, a receptor for class-3 semaphorin-family proteins and vascular endothelial growth factor (Figure 1c) 44 , necessitate further biochemical and molecular analyses to identify specific binding interactions and potentially associated molecules. The issue of GARP expression in murine T_reg cells at the protein level remains to be elucidated.

Because GARP is constitutively expressed on platelets 26 45 , and because a potential function of GARP on thrombus formation has recently been reported 46 , important questions about GARP signaling and parallel function in platelets compared to T_reg cells could be considered. Concerning the dependence of GARP expression on FOXP3 as described above, it has been reported that FOXP3 is expressed by human and murine megakaryocytes 47 . Therefore, the thrombocytopenia and platelet abnormalities experienced by some patients with IPEX syndrome can be explained by loss of function of FOXP3 47 ; however, this explanation would suggest a concomitant impairment of GARP expression and function on IPEX platelets, an impairment that has not yet been shown.

A controversial issue is the potential suppressor function of GARP, suggested recently 24 , because platelets as a natural source of GARP-expressing cells do not function as suppressor cells 21 . Therefore, differences in the function of GARP in platelets and T_reg cells can be suggested. This question is important for the potential design of GARP-selective drugs that can specifically target only T_reg cells and not platelet functions. As of now, neither the ligand nor the signaling system or potential co-receptor(s) of GARP has been identified, and identifying them is the most important challenge for the future.

Reviewer 1: Dr. Jim Di Santo, Cytokines and Lymphoid Development Lab, Institut Pasteur, Paris, France

Reviewer's comment: I have read your comment for Biology Direct entitled "FOXP3 and GARP (LRRC32): the master and its minion". I find this comment to be interesting and suitable for publication in Biology Direct. I do not have any specific comments for web publication.

Reviewer 2: Dr. Benedita Rocha, Institut National de la Santé et de la Recherche Médicale (INSERM) U591, Insitut Necker, France

Reviewer's comment: Regulatory cells have a fundamental role in many disease processes, and extensive experimental and clinical data indicate that tools preventing or increasing regulatory function will have a fundamental therapeutic role. While FOXP3 expression generally correlates with regulation in the mouse, this is not so in human T cells. This comment reviews a fundamental aspect, the factors besides FOXP3 expression that ensure the induction of a stable regulatory function on human cells, their correlation with FOXP3 regulation and their possible mechanisms of action. I found the topic actual and important, the comment clear and comprehensive and strongly support publication.

Reviewer 3: Dr. Werner Solbach, Insitute for Medical Microbiology and Hygiene, University Lübeck, Germany

Reviewer's comment: The ms. deals with the connectivity of FOXP3 and "glycoprotein - A repititions predominant receptor (GARP or LRRC32) in the context of the functioning of regulatory T cells (Tregs). This topic is of great relevance in the context of clarifying the equivocal role of FOXP3 and its partners for explanation of regulatory T cell circuits. It is clear that not only FOXP3 or GARP alone, but also latently activated TGF-β is crucial for the suppressive activity of Treg cells. The authors now very nicely bring together the GARP - FOXP3 regulatory circuit with the autocrine TGF-β loop which helps to explain many Treg features. In their perspective, they also try to open thoughtful avenues how to bring together (human) GARP with its possible functional homologue in mice, neuropilin 1. In summary, they present a valuable conceptual framework for understanding the regulatory program in the T cell reactivity system and beyond. The ms. is well written and easy to understand. I recommend publication in Biology Direct.