CD4+FoxP3+ regulatory T-cells in human systemic lupus ... - CORE

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enteropathy, X-linked (IPEX) disease with autoimmune manifestations and .... T-cells and disease activity, including SLE Disease Activity Index (SLEDAI) score.

Journal of the Formosan Medical Association (2012) 111, 465e470

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CD4DFoxP3D regulatory T-cells in human systemic lupus erythematosus Jau-Ling Suen a,b, Bor-Luen Chiang c,* a

Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC Department of Microbiology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC c Department of Pediatrics, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC b

Received 15 November 2011; received in revised form 9 May 2012; accepted 21 May 2012

KEYWORDS FoxP3; regulatory T-cell; systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of immune tolerance to self antigens and by the persistent production of pathogenic autoantibodies. Recent studies have suggested a dysregulation of regulatory T-cells (Tregs), particularly CD4þCD25highFoxP3þ (forkhead box P3) Tregs, as one of the major factors conferring the risk for expression of human autoimmune diseases, including SLE. However, detailed studies of CD4þFoxP3þ T-cells in patients with SLE remain limited. We attempt here to integrate the current experimental evidence to delineate the role of CD4þCD25high and other subsets of CD4þFoxP3þ T-cells in human SLE. Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.

Introduction Immune tolerance to autoantigens is a tightly regulated process. Deletion of self-reactive T-cells in the thymus is an important mechanism for self-tolerance. However, some autoreactive cells still can escape negative selection into the periphery. Peripheral tolerance is maintained a number of ways, including CD4þCD25þ regulatory T-cells (Tregs) that actively * Corresponding author. Department of Pediatrics, National Taiwan University Hospital, 1 Chang-Teh Street, Taipei, Taiwan, ROC. E-mail address: [email protected] (B.-L. Chiang).

suppress autoimmunity and control immune homeostasis. Tregs represent 5e10% of CD4þ T-cells in both humans and mice.1,2 They are characterized by the constitutive expression of CD25 [interleukin-2 (IL-2) receptor alpha chain], cytotoxic T-lymphocyte antigen 4 (CTLA-4), glucocorticoidinduced tumor necrosis factor receptor-related protein (GITR), and transcriptional repressor forkhead box P3 (FoxP3), and a low level or nonexpression of CD127 (IL-7 receptor).3 CD25 used to be seen as the only reliable marker for Tregs; however, activated CD4þ T-cells also express CD25. In contrast, FoxP3 has been shown to be specifically expressed by Tregs and able to program the development and function of Tregs.4,5 Further, CD127 expression inversely correlates with FoxP3 irrespective of CD25 expression in both

0929-6646/$ - see front matter Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.

466 humans and mice.3,6 Thus, a combination of CD127, CD3, and CD4 expression can be used to isolate viable FoxP3þ T-cells (CD3þCD4þCD127e) for functional studies of Tregs. In addition, to further characterize human CD4þFoxP3þ T-cells, they can be separated into three subsets by the expression of CD45RA, FoxP3, and CD25.7 These subsets are (1) CD45RAþFoxP3low(CD25low) resting Tregs, (2) CD45RAeFoxP3high(CD25high) activated Tregs, and (3) CD45RAeFoxP3low(CD25low) non-Tregs. Resting Tregs, which come from the thymus, can be differentiated into activated Tregs in vitro and in vivo. Both resting Tregs and activated Tregs have suppressive activity in vitro. In contrast, CD45RAeFoxP3low T-cells can secrete IL-2 and interferongamma (IFN-g) and exhibit little suppressive activity. Thus, the identification of FoxP3 and CD45RA expression as a marker for Tregs is important in order to further analyze their role in disease states. Thus, FoxP3 combined with CD127 and CD45RA is currently the best marker for the identification of different subsets of human FoxP3þ Tregs.

The role of FoxP3 in Tregs Based on the studies of scurfy mutant mice, which have autoimmune lymphoproliferative disease, the Foxp3 gene has been identified, and it has been shown that FoxP3 is responsible for the scurfy phenotype.8 Several lines of evidence demonstrate that FoxP3 is necessary and sufficient for the development and function of Tregs in mice. First, using FoxP3 reporter mice that were knockin with an allele of the Foxp3-green fluorescent protein gene,9 it was shown that the predominant type of FoxP3þ cells were abTCRþCD4þ T-cells irrespective of CD25 expression. In addition, both CD25þFoxP3þ and CD25eFoxP3þ T-cells can act as suppressors in vitro.4 Second, ectopic expression of FoxP3 in conventional CD4þCD25e T-cells converted those cells to a regulatory phenotype. These converted FoxP3þ T-cells can function as Tregs both in vitro and in vivo.4 Third, when mice lack functional FoxP3 proteins, via either the scurfy mutation or a targeted mutation, they do not display Treg activity and develop severe systemic autoimmune diseases.5,9,10 In humans, the FOXP3 mutation also leads to immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) disease with autoimmune manifestations and defective Treg function.11,12 It has been demonstrated in both mice and humans that FoxP3-expressing CD4þCD25þ T-cells can act as suppressors. However, further characterization of FoxP3 expression shows two major differences between humans and mice. First, human CD4þCD25þ T-cells, but not murine cells, can express two isoforms of FoxP3.13 One isoform represents the homolog to murine FoxP3, whereas the other isoform is encoded from an mRNA lacking exon 2.14 The sequences encoded by exon 2 fall within the repressor domain of the FoxP3 protein.14 To date, the role of the 6exon 2 isoform in the function and development of Tregs, and whether or not the same T-cells express both isoforms simultaneously, remains unclear. Second, the regulation of FoxP3 expression is different between humans and mice. Activationinduced expression of FoxP3 has been reported for in vitro-stimulated human CD4þ T-cells14,15 but not mouse cells.4 A recent study has demonstrated that human

J.-L. Suen, B.-L. Chiang CD4þFoxP3þ T-cells contain cytokine-secreting, nonsuppressive effector T-cells that are CD45RAeFoxP3low.7 These nonsuppressive CD45RAeFoxP3lowCD4þ T-cells may correspond to in vitro activation-induced FoxP3-expressing cells.14,15 In addition, not only the CD45RAeCD25high subset,2 but also the CD45RAþCD25low subset in human FoxP3þCD4þ T-cells displays suppressive activity in vitro and in vivo.7 Thus, human FoxP3þ T-cells are heterogeneous in function and consist of not only suppressive Tregs, but also nonsuppressive T-cells.

Natural Tregs and adaptive Tregs There are at least two populations of CD4þ Tregs in humans and mice, defined by their origin. The first population of CD4þ Tregs is natural Tregs, which are generated during normal T-cell maturation in the thymus. These typically express CD25, as well as CTLA-4 and GITR.16 This subset is self-reactive and is involved in protection from autoimmune responses. Recently, two subsets of FoxP3þ natural Tregs according to differential inducible costimulator (ICOS) expression have been found in the human thymus and periphery.17 ICOSþFoxP3þ Tregs secrete IL-10 to suppress dendritic cell (DC) function, and transforming growth factor beta (TGF-b) to suppress T-cell function, whereas ICOSeFoxP3þ Tregs mainly secrete TGF-b to mediate the suppressive function. The second population is adaptive Tregs, which originate from the thymus but are developed throughout the course of the immune response in vivo. This population includes TGF-b-expressing type 3 T-helper cells, IL-10-producing type 1 regulatory T-cells, and peripherally converted FoxP3þ Tregs.18 Research has shown that naı¨ve CD4þ T-cells can be converted, de novo, into CD25þFoxP3þ and CD25eFoxP3þ suppressor T-cells on subimmunogenic stimulation.19 This de novo Treg conversion may be mediated either by DCs with tolerogenic properties20 or by intestinal DCs from lamina propria.21

CD4DFoxP3D T-cells in systemic lupus erythematosus Systemic lupus erythematosus (SLE) is characterized by dysregulated immunity with hyperactive T-cells and Bcells. Lupus-prone mice with CD4þCD25þ Treg depletion from thymectomy have an enhanced expansion of autoreactive T-cells and accelerated autoantibody production.22 In contrast, treatment with CD4þCD25þ Tregs from syngeneic normal mice can effectively abrogate the progress of autoimmune disease,23 as well as supplement with in vitro-expanded Tregs24 and TGF-b-generated Tregs.25 Thus, the dysregulated immunity in lupus may be correlated with the altered homeostasis or defective function of Tregs. A few studies have been performed to quantify the frequency of CD4þCD25þ/high T-cells in patients with SLE. These studies have shown that the frequency of CD4þCD25þ/high T-cells is decreased in patients with SLE.26,27 However, no correlation has been established between the level of CD4þCD25þ/high T-cells and disease

Regulatory T-cells in systemic lupus erythematosus activity. Lee et al have shown an inverse correlation between percentages of CD4þCD25þ T-cells and disease activity, including SLE Disease Activity Index (SLEDAI) score and serum anti-double-stranded DNA levels in pediatric patients.28 Interestingly, this finding showed higher FoxP3 mRNA levels in CD4þ T-cells in active SLE compared with normal controls and those with inactive SLE. A recent study by Miyara et al29 also observed that CD4þCD25high T-cell depletion in lupus patients was associated with the clinical severity of the flare. These Tregs with normal suppressive function did not redistribute to lymph nodes or tissues and were sensitive to Fas-induced apoptosis.29 This study suggests that the inappropriate induction of Treg apoptosis is, at least in part, associated with the depletion of CD4þCD25high T-cells in lupus patients and thus relevant to SLE pathogenesis. However, Valencia et al showed that CD4þCD25high T-cells from patients with active SLE expressed reduced levels of FoxP3 and displayed a poor inhibitory activity on the proliferative response of responding T-cells.30 Alvarado-Sanchez et al showed that about one-third of lupus patients exhibited a diminished suppressive function of CD4þCD25high T-cells, but had a normal frequency of Tregs in their peripheral blood.31 These conflicting results may be due to an imprecise phenotypic definition of Tregs. Due to the unique expression pattern of human CD25 on activated CD4þ T-cells and Tregs, it is difficult to draw the line between CD25high and CD25low cells on fluorescence-activated cell sorting plots (Fig. 1). However, by analyzing CD25 and FoxP3 expression on CD4þ T-cells in conjunction, it becomes easier to distinguish different subsets of T-cells.32 Therefore, if only CD25 serves as a Treg marker, sorted CD25high Tregs could display poor suppressive ability in vitro if they were contaminated by CD25low effector T-cells. In contrast, strict gating for high CD25 expression on CD4þ T-cells may result in falsely low Treg numbers/frequency in human peripheral blood mononuclear cells. Five studies have examined FoxP3-expressing CD4þ Tcells in patients with SLE (Table 1). All the studies showed that SLE patients had higher CD4þCD25eFoxP3þ and/or CD4þCD25þFoxP3þ T-cell frequencies than normal controls.7,32e35 According to these studies, and based on the findings of heterogeneous subsets in the function of human FoxP3þ cells,7 SLE patients may have a defect in the homeostatic control of different subsets of FoxP3þ cells. These human FoxP3þ subsets are described separately as follows.


CD4DCD25lowFoxP3D T-cells As shown in Table 1,32e35 several studies have shown that patients with active lupus also have an increased percentage of CD4þCD25low/þFoxP3þ T-cells (the R2 region in Fig. 1) in their peripheral blood. This population may contain at least two subsets e CD45RAþ resting Tregs and CD45RAe cytokine-secreting non-Tregs (Fig. 2).7 There are two possible mechanisms to explain their high percentage in patients with active lupus. The first one is that CD45RAþ resting lupus Tregs (CD45RAþCD25lowFoxP3þ) may accumulate due to the global depletion of activated Tregs (CD25highFoxP3þ), because activated Tregs can suppress the proliferation of resting Tregs by negative feedback (Fig. 2).7 The second mechanism is that lupus CD45RAe cytokinesecreting non-Tregs (CD45RAeCD25lowFoxP3þ) may consist of a large portion of autoreactive CD4þ T-cells specific for nuclear antigens, such as nucleosomes36 as these autoreactive T-cells continue expansion in vivo, especially in patients with active SLE. Glucocorticoid treatment in patients with inactive disease may be able to control this expansion, so that the frequency of CD4þCD25lowFoxP3þ T-cells in patients with inactive disease is lower than that with active SLE.32 However, the functional delineation and

CD4DCD25highFoxP3D Tregs Patients with active SLE have a significantly decreased percentage and number of CD4þCD25highFoxP3þ Tregs (corresponding to activated Tregs7; the R1 region in Fig. 1) with normal suppressive activity in their peripheral blood. This decrease is also correlated with disease activity.29,32,34 The global depletion of CD4þCD25highFoxP3þ Tregs may be associated with their hypersensitivity to Fas-induced apoptosis (Fig. 2).29 However, the mechanisms responsible for the exacerbated susceptibility to apoptosis of human lupus CD4þCD25highFoxP3þ Tregs still need to be clarified.

Figure 1 Characterization of different subsets of FoxP3þ Tcells in a patient with systemic lupus erythematosus (SLE) and a healthy individual. Peripheral blood mononuclear cells were stained with fluorochrome-labeled anti-CD3, anti-CD4, antiCD25, and anti-FoxP3 monoclonal antibodies and analyzed using a flow cytometer (LSRII, BD Biosciences). Dot plots show the expression of FoxP3 (x-axis) and CD25 (y-axis) on gated CD3þCD4þ T-cells from one healthy individual (upper panel) and one patient with SLE (lower panel). Quadrants were established using appropriate isotype controls. R1 Z CD25highFoxP3þ; R2 Z CD25lowFoxP3þ; R3 Z CD25eFoxP3þ; R4 Z CD25þFoxP3e.


J.-L. Suen, B.-L. Chiang Analysis of CD4þFoxP3þ T-cell subpopulations in human systemic lupus erythematosus.

Table 1 Reference Lin et al


Zhang et al35 Yan et al34 Miyara et al7

Suen et al32

Percentage of CD4þ T-cells þ

FoxP3 [ CD25þFoxP3þ [ CD25eFoxP3þ [ CD25þFoxP3þ / CD25eFoxP3þ [ CD25þFoxP3þ(CD127low) [ CD25eFoxP3þ e NA CD45RAeFoxP3high(CD25high)Y CD45RAþFoxP3low(CD25þ)[ CD45RAeFoxP3low(CD25þ)[ CD25highFoxP3þ(CD127low) Y CD25lowFoxP3þ(CD127low) [ CD25eFoxP3þ(CD127low) [ CD25þFoxP3e(CD127þ) (Teff)[

Ratio of FoxP3þ/FoxP3e

Disease correlation

Treg function





Anti-dsDNA IgG





CD4þCD25þ e normal suppressive activity CD4þCD25high e normal suppressive activity NA

CD25highFoxP3þ/Teff Y CD25lowFoxP3þ/Teff Y CD25eFoxP3þ/Teff /

Anti-dsDNA IgG C3/C4 level

CD4þCD25high e normal suppressive activity

dsDNA Z double-stranded DNA; NA Z not analyzed; Ig Z immunoglobulin; SLEDAI Z Systemic Lupus Erythematosus Disease Activity Index; Teff Z effector T-cell (represented by CD4þCD25þFoxP3e T-cells); Treg Z regulatory T-cell.

differentiation dynamics of CD4þCD25lowFoxP3þ T-cells in lupus still need to be explored.

CD4DCD25eFoxP3D T-cells Another important subset of FoxP3þ T-cells that has been found to show a significant increase in frequency in SLE patients is CD4þFoxP3þ T-cells without CD25 expression (the R3 region in Fig. 1).32 The CD4þCD25e T-cells, irrespective of their CD45RA expression, express little FoxP3 in normal

individuals.7 Thus, this subset seems relatively unique to SLE. However, the origin and function of this subset are at present largely unknown. There are several issues worth discussing here. The first one is the function and origin of this unique subset in lupus. Their FoxP3 protein may be transiently induced by activation, and this subset may display non-Treg activity. Second, systemic inflammation can induce the differentiation of naı¨ve CD4þ T-cells into CD25þFoxP3þ and CD25eFoxP3þ Tregs, or so-called adaptive Tregs.18,19,32,37 This subset may represent the adaptive Tregs in patients with SLE. Finally,

Figure 2 Altered homeostasis of CD4þFoxP3þ T-cell subsets in patients with active systemic lupus erythematosus. Patients with active lupus have a significantly decreased frequency of activated regulatory T-cells (Tregs; CD25highCD45RAeFoxP3high), increased CD25lowFoxP3low, CD25eFoxP3low, and CD25lowFoxP3e T-cell subsets. The depletion of activated Tregs may be associated with their hypersensitivity to Fas-induced apoptosis. Activated Tregs can suppress the proliferation of resting Tregs via negative feedback. CD25lowFoxP3þ T-cells may contain resting Tregs and FoxP3þ non-Tregs; however, the CD45RA expression level of this subset has not yet been clarified. Another unique subset, CD25eFoxP3low T-cells, also has been observed in patients; however, their origin and function are largely unknown. This unique subset may represent adaptive Tregs that are differentiated from naı¨ve T-cells or FoxP3-expressing nonTregs. In addition, a significantly increased number of activated T-cells (CD25lowCD45RAeFoxP3e) also exist in the peripheral blood of patients. This subset may contain a large number of autoreactive T-cells that are activated by nucleosome-presenting dendritic cells (DCs) in the interferon-alpha-rich environment. Ag Z antigen; APC Z antigen-presenting cell; DC Z dendritic cell; IFN Z interferon.

Regulatory T-cells in systemic lupus erythematosus the increased frequency of CD25eFoxP3þ T-cells in patients may compensate for the loss of CD25highFoxP3þ Tregs in active SLE. However, this compensation may not be enough to regulate the autoimmune response, as SLE patients have altered relative ratios of CD25highFoxP3þ Tregs and CD25lowFoxP3þ T-cells versus effector T-cells (Fig. 2).32 In contrast to those with SLE, Rheumatoid arthritis (RA) patients have similar frequencies of CD4þFoxP3þ T-cells irrespective of their CD25 expression compared with normal controls.32,33 In addition, Franz et al showed that patients with cutaneous lupus erythematosus had normal levels of CD4þFoxP3þ T-cells in their peripheral blood, but decreased levels in local skin lesions.38 This suggests that the global dysregulation of decreased CD25highFoxP3þ Tregs and increased CD25low/eFoxP3þ CD4þ T-cells seems to be relatively unique to SLE. The mechanism clearly needs to be investigated further. Several pieces of evidence suggest that lupus DCs may be responsible for this dysregulation.39,40 It has been shown that IFN-a-producing antigen-presenting cells, such as plasmacytoid DCs, play a vital role in the pathogenesis of SLE.40 These IFN-a-producing antigen-presenting cells may block Treg cell-mediated suppression in SLE patients.34 In addition, the overproduction of IL-6 by DCs in lupus-prone mice may mediate the impaired Treg function.41 Another major issue is the antigen specificity of Tregs in SLE patients. Hahn et al demonstrated that functional human Tregs can be induced by exposure to anti-DNA immunoglobulin-based peptides.42 In addition, the FoxP3 expression level in lupus CD4þCD25high T-cells also clearly increased when peripheral blood mononuclear cells from patients stimulated with these self-peptides in vitro. Two possibilities may explain the increase in number or FoxP3 level of CD4þCD25high T-cells with in vitro culture. First, self peptides may directly promote the expansion of lupus CD4þCD25high T-cells in vitro. Second, CD4þCD25high T-cells may be derived from FoxP3-expressing CD25low or CD25e T-cells after in vitro stimulation, which leads to the change in phenotype from CD25low/e to CD25high without losing FoxP3 expression. Thus, these studies suggest that dysregulated Treg homeostasis may play an important role in the pathogenesis of SLE.

Potential implications of Treg immunotherapy in SLE patients In humans, small-scale trials have demonstrated that in vitroexpanded Tregs under good-manufacturing practice conditions can provide a beneficial effect in the management of bone marrow transplantation.43e45 This suggests that Treg therapy is another therapeutic strategy to control systemic autoimmunity. Two approaches can be considered regarding Treg therapy in patients with SLE. First, we could isolate patients’ resting Tregs (CD25lowCD45RAþCD127eFoxP3þ) and expand them in vitro to perform Treg therapy. Second, it has been found that administration of a histone deacetylase inhibitor in vivo significantly increases FoxP3 gene expression and the suppressive function of Tregs through epigenetic modification of FoxP3.46 Thus, it is worth examining whether histone deacetylase inhibitor treatment can increase the FoxP3 expression level in FoxP3-expressing lupus subsets

469 (as shown in Fig. 2) and whether the increased FoxP3 expression enables those cells to display the suppressive activity in vivo. If so, the autoimmune responses in patients may be downregulated by these high percentages of lupus FoxP3-expressing subsets.32

Summary SLE patients have altered homeostasis in their CD4þFoxP3þ T-cell subsets, that is, a decreased frequency of CD4þCD25highFoxP3þ Tregs and an increased number of CD4þFoxP3þ T-cells with CD25low or CD25e expression. One should remember that FoxP3 is also transiently expression in human nonregulatory T-cells.12,13 In contrast, murine FoxP3 expression is sufficient for the suppressive function of Tregs.4 Thus, functional characterization is required for each subset of lupus FoxP3þ T-cells in humans. Furthermore, analyzing the function and origin of each subset will help us to better understand the immune responses in cases of lupus and to design antigen-specific immunotherapy for SLE.

Acknowledgments This study was supported by grants from the National Science Council (NSC 95-2320-B-037-009) and the Kaohsiung Medical University Research Foundation (KMU-M098007). We apologize for excluding a significant number of relevant references due to space limitations. The authors declare that they have no competing interests.

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