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. Author manuscript; available in PMC: 2017 Dec 27.
Published in final edited form as: Ann Rheum Dis. 2016 Dec 7;76(5):898–905. doi: 10.1136/annrheumdis-2016-210070

An expanded population of pathogenic regulatory T-cells in giant cell arteritis is abrogated by IL-6 blockade therapy

Chie Miyabe 1, Yoshishige Miyabe 1, Klemen Strle 1, Nancy D Kim 1, John H Stone 1, Andrew D Luster 1,*, Sebastian Unizony 1,*
PMCID: PMC5744591  NIHMSID: NIHMS921263  PMID: 27927642

Abstract

Objectives

Randomized-controlled trials have recently proven the efficacy of the interleukin-6 receptor antagonist tocilizumab (TCZ) in GCA. However, the mechanism of action of IL-6 blockade in this disease is unknown. Moreover, the role of regulatory T-cells (Treg) in the pathogenesis of GCA remains underexplored. Given the plasticity of Tregs and the importance of IL-6 in their biology, we hypothesized that TCZ might modulate the Treg response in GCA. We therefore characterized the Treg compartment of GCA patients treated with TCZ

Methods

We classified 41 GCA patients into 3 groups: active disease (aGCA, n=11); disease remission on corticosteroids (rGCA-CS, n=19); and disease remission on TCZ (rGCA-TCZ, n=11). Healthy controls were included for comparison. We determined the frequency, phenotype and function of peripheral blood Tregs.

Results

aGCA patients demonstrated a hypoproliferating Treg compartment enriched in IL-17-secreting Tregs (IL-17+Tregs). Tregs in aGCA patients disproportionally expressed a hypofunctional isoform of Foxp3 that lacks exon 2 (Foxp3Δ2). Foxp3Δ2-expressing Tregs co-expressed CD161, a marker commonly associated with the Th17 linage, significantly more often than full-length Foxp3-expressing Tregs. Compared to those of healthy controls, GCA-derived Tregs demonstrated impaired suppressor capacity. Treatment with TCZ, in contrast to CS therapy, corrected the Treg abnormalities observed in aGCA. In addition, TCZ treatment increased the numbers of activated Tregs (CD45RA−Foxp3high) and the Treg expression of markers of trafficking (CCR4) and terminal differentiation (CTLA-4).

Conclusions

TCZ may exert its therapeutic effects in GCA by increasing the proliferation and activation of Tregs, and by reverting the pathogenic Treg phenotype seen during active disease.

Keywords: giant cell arteritis, regulatory T cells, tocilizumab, interleukin-6, interleukin-17, Foxp3

INTRODUCTION

Giant cell arteritis (GCA) is the most frequent primary vasculitis in Western countries [1]. The main histopathologic feature of the disease comprises a granulomatous inflammatory process rich in CD4+T-cells and macrophages that involves large- and medium-sized arteries [1]. Most patients develop relapsing courses despite prolonged treatments with corticosteroid (CS), which invariably lead to drug-related toxicity [2]. Agents that maintain disease remission and spare the use of CS are therefore the greatest unmet need for this patient population [3–6].

An imbalance among CD4+T helper (Th)1, Th17, and regulatory T (Treg) cells is thought to contribute to the pathogenesis of GCA [7–9]. Patients with new-onset disease demonstrate Th1 and Th17 cell infiltrates in their arteries and an expansion of these cell subsets in peripheral blood [7–9]. Conversely, decreased numbers of Tregs in the peripheral circulation are found in GCA patients, regardless of the state of disease activity [8, 9]. Although the Th17 axis is sensitive to CS treatment [7–10], some reports suggest that the abnormalities described in both the Th1 and Treg subsets are resistant to CS therapy [7, 8], thereby accounting for the high relapse rate in GCA following CS tapering.

The interleukin (IL)-6 pathway is a novel target in GCA. GCA patients demonstrate increased IL-6 RNA expression within inflamed arteries [11, 12] and elevated IL-6 protein levels in the peripheral blood during active disease [13]. Recently, two randomized controlled trials showed that tocilizumab (TCZ), a monoclonal antibody against the IL-6 receptor (IL-6R), is effective in maintaining disease remission in absence of CS [14, 15]. However, the mechanism of action of IL-6 signaling blockade in GCA remains unknown.

Considerable phenotypic and functional plasticity exists within the Treg and the Th17 cell subsets [16]. Th17 cells and Tregs develop from a common naïve CD4+T-cell precursor under the influence of transforming growth factor-β (TGF-β) [17]. In the presence of pro-inflammatory mediators (e.g., IL-6 or IL-21), TGF-β-stimulated CD4+T-cells differentiate into Th17 cells, whereas in the absence of an inflammatory microenvironment these TGF-β-stimulated precursors are induced to become Tregs [18]. Furthermore, under specific circumstances, fully-differentiated Tregs may lose their suppressive function and become IL-17-producing cells [16] (e.g., “pathogenic Tregs” [19, 20] and exFoxp3 Th17 cells [21]).

One mechanism regulating the divergent fates between Tregs and Th17 cells involves the molecular antagonism of RORγt (RORC in humans) by Foxp3 through the domain encoded by the exon 2 of the FOXP3 gene [22]. Tregs that express a spliced variant of Foxp3 lacking exon 2 (Foxp3Δ2) are less suppressive [23], and more likely to become IL-17 producing Tregs. Increased numbers of Foxp3Δ2+Tregs have been reported in ANCA-associated vasculitis [24]. It is not known, however, whether this abnormality is also present in GCA patients. In addition, cells that express both Foxp3 and IL-17 have been detected in inflamed GCA arteries [10], but whether this cell population is present in peripheral circulation, and most importantly, whether disease treatment reverts the pathogenic phenotype of those Tregs has been insufficiently studied.

We aimed to characterize the regulatory CD4+T-cell compartment in peripheral blood of GCA patients and to investigate the effects of IL-6R blockade therapy with TCZ on the frequency, phenotype and function of those cells.

MATERIALS AND METHODS

Study population

We evaluated 41 GCA patients in a cross-sectional study. GCA patients were classified into one of three categories based on disease activity and treatment: active disease (aGCA, n=11); disease remission on CS monotherapy (rGCA-CS, n=19); and disease remission on TCZ therapy (rGCA-TCZ, n=11). Among the subjects with aGCA, 3 had new-onset disease and 8 were sampled during a disease relapse. We also evaluated samples from 10 healthy controls (HC). Upon diagnosis, all patients had been treated with CS according to the standard of care for GCA [1]. Patients in the TCZ group (rGCA-TCZ group) received their IL6R blockade therapy because of relapsing disease or prohibitive CS-related toxicity during previous treatment courses. Once on TCZ, patients underwent a prednisone taper of variable rate, but generally faster than the standard of care in order to ameliorate or prevent CS-related toxicity. Other clinical information is provided in the Supplementary Text. The study was approved by our institutional IRB. All participants provided informed consent.

Cell isolation, culture and flow cytometry

CD4+T-cells were purified (>90% purity) from whole blood using RosetteSep CD4+enrichment antibody cocktail (StemCell Technologies) according to manufacturer’s instructions. Cells were labeled with Pacific Blue-conjugated anti-CD4, fluorescein isothiocyanate (FITC)-conjugated anti-CD45RA, phycoerythrin (PE)-Cy7-conjugated anti-CCR4, PE-conjugated anti-CTLA4, allophycocyanin (APC)-Cy7-conjugated anti-IL-17A, PE-Cy7-conjugated anti-CD25, PE-conjugated anti-Ki67, APC-conjugated anti-CD161 (BioLegend); Alexa Fluor 488-conjugated anti-Foxp3 and Alexa Fluor 700-conjugated anti-Foxp3. Foxp3Δ2 was detected using clone PCH101 (eBiocience) that recognizes the N-terminus portion of the protein and clone 150D (BioLegend) that recognizes the exon 2 [24, 25]. Data were acquired on a LSRFortessa cell analyzer (BD) and analyzed with FlowJo software.

Regulatory T-cell suppression assays

CD4+CD25+Tregs were isolated from a pool of CD4+T-cells utilizing CD25 MicroBeads (Miltenyi Biotec). CD4+CD25− conventional T-cells were incubated for 10 min at 37°C in 10 μM CFSE (Invitrogen), washed with phosphate buffered saline containing 2% FCS, and re-suspended in complete RPMI. CFSE-labeled CD4+CD25−cells (1×105) were co-incubated with varying concentrations of autologous CD4+CD25+Tregs to create conventional T-cell to Treg ratios of 8:1, 4:1, 2:1 and 1:1. Cultures were stimulated for 4 days with Treg Suppression Inspector (Miltenyi Biotec), or left unstimulated. Proliferation of conventional CD4+T-cells was measured by assessing CFSE dilution by flow cytometry.

Statistical analysis

Categorical variables were compared between groups using Fisher’s exact test. Continuous variables were compared between groups using paired and unpaired Student’s t-test, Mann-Whitney’s test, ANOVA, or Kruskal-Wallis test as appropriate. In order to account for confounders on the number of specific CD4+T-cell subsets (e.g., CS dose) we used linear regression. Statistical significance cut-off was 0.05. P-values were two-sided. Stata 13 (StataCorp LP) was used for all analyses.

RESULTS

Baseline characteristics of GCA patients and healthy controls

The baseline characteristics of GCA patients and HC are shown in Table 1. There were no significant differences among patient groups (aGCA, rGCA-TCZ and rGCA-CS) with regard to demographic features, disease type, or disease duration. The mean daily dose of prednisone at the time of blood sampling was 15.7 mg in rGCA-CS patients, 0.2 mg in rGCA-TCZ patients and 8.0 mg in aGCA patients (P=0.02). Patients in the rGCA-TCZ group had received TCZ for a median period of 18 months. HC were younger than the GCA patients (mean age 59 versus 72 years; P<0.01), but there were no other important differences.

Table 1.

Characteristics of the GCA patients and the healthy individuals at baseline.

rGCA-CS (n= 19) rGCA-TCZ (n = 11) aGCA (n =11) P-value Controls 1 (n = 10) P-value
Age, years: mean (SD) 73 (10) 69 (8) 72 (10) 0.41 59 (10) <0.01
Sex, females: number (%) 12 (63) 9 (82) 9 (82) 0.48 4 (40) 0.07
Race, white: number (%) 17 (89) 10 (91) 11 (100) 0.78 11 (100) 1.00
Relapsing disease: number (%) 12 (63) 11 (100) 8 (73) 0.06 - -
Biopsy-proven disease: number (%) 11 (58) 5 (45) 7 (64) 0.78 - -
Image compatible with large vessel vasculitis (%) 2 2 (11) 4 (36) 4 (36) 0.16 - -
Disease duration, months: median (IQR) 25.5 (9.2; 54.1) 35.7 (32.7; 70.4) 34.9 (3.7; 60.3) 0.73 - -
Duration of CS treatment, months: median (IQR) 18.4 (9.2; 54.1) 28.4 (9.9; 67.9) 34.5 (1.0; 58.0) 0.90 - -
Duration of TCZ treatment, months: median (IQR) - 18 (14.2; 28.5) - - - -
Prior MTX use: number (%) 6 (32) 4 (36) 3 (27) 1.00 - -
CS dose at time of sampling, mg/day: mean (SD) 15.7 (18.3) 0.2 (0.4) 8.0 (6.8) 0.02 3 - -
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GCA = giant cell arteritis; CS = corticosteroids (prednisone); TCZ = tocilizumab; MTX = methotrexate; rGCA-CS = GCA in remission on CS; rGCA-TCZ = GCA in remission on TCZ without or without CS; aGCA = active GCA; SD = standard deviation; IQR = interquartile range; Analysis: ANOVA, Kruskal Wallis, Student’s t-test, and Fisher’s exact test;

1

controls versus all GCA patients;

2

indicates magnetic resonance angiography, computer tomography angiography, or positron emission tomography;

3

rGCA-CS versus rGCA-TCZ

TCZ increases the frequency of activated Tregs

We first measured the population of Tregs defined as CD4+T-cells expressing Foxp3 and found no significant differences among groups (Supplementary Figure 1A–B). We then classified Tregs into 3 functionally distinct subpopulations based on the level of expression of Foxp3 and CD45RA [26]: 1) CD45RA−Foxp3high (activated Treg, aTreg), 2) CD45RA+Foxp3low (resting Treg, rTreg) and, 3) CD45RA−Foxp3low (non-suppressive Foxp3low cells) cells (Figure 1A). We observed that the mean percent of aTregs was significantly greater in rGCA-TCZ patients (1.3% [SD 0.9]) compared to rGCA-CS patients (0.6% [SD 0.4]) (P<0.01) (Figure 1B). There were no significant differences among groups in terms of rTregs and non-suppressive Foxp3low cells (Supplementary Figure 2). The phenotype of cellular activation observed in Tregs derived from rGCA-TCZ patients was then confirmed by measuring on all CD4+Foxp3+ cells the expression of CCR4 and CTLA-4, markers of the most terminally differentiated activated effector Tregs [26–30] (Figure 1B). The differences between rGCA-TCZ patients and rGCA-CS patients in terms of the numbers of aTregs, CD4+Foxp3+CCR4+ cells and CD4+Foxp3+CTLA-4+ cells remained statistically significant after analyses adjusted for age and CS dose. As expected, aTregs demonstrated higher expression of CD25, CCR4 and CTLA-4 when compared to rTregs and non-suppressive Foxp3low cells (Figure 1C). These findings demonstrate that in GCA patients, remission maintenance with IL-6 blockade therapy is associated with increased Treg activation.

Figure 1. TCZ therapy increases the numbers of activated Tregs.

Figure 1

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CD4+ T-cells were purified from peripheral blood of GCA patients and healthy controls (HC) by negative selection. A, Representative flow cytometry plots of CD4+ T-cells classified according to the expression of CD45RA and Foxp3 in 1) resting Tregs (rTregs, subset I), 2) activated Tregs (aTregs, subset II), and 3) non-suppressive Foxp3low cells (subset III). B, Frequencies of aTregs, CD4+Foxp3+CCR4+ cells and CD4+Foxp3+CTLA-4+ cells in GCA patients (rGCA-TCZ, n = 9; rGCA-CS, n = 18; aGCA, n = 11) and HC (n = 10). C, Representative histograms showing the expression of CD25, CCR4 and CTLA-4 in rTregs (I), aTregs (II) and non-suppressive Foxp3low cells (III). Analysis: Student’s t-test. Error bars represent means and SD. CS = corticosteroids; TCZ = tocilizumab; rGCA-CS = GCA in remission on CS; rGCA-TCZ = GCA in remission on TCZ without or without CS; aGCA = active GCA.

TCZ restores the impaired proliferative capacity of Tregs

Tregs are among the most actively replicating cells within the CD4+T-cell compartment, and impaired Treg proliferation has been implicated in the pathogenesis of autoimmunity [31]. Thus, we investigated the proliferative capacity of Tregs in GCA patients and HC by measuring the expression of Ki67, a marker of cellular replication. We observed that the mean percent of Ki67+Tregs was equivalent in rGCA-TCZ patients (27.7% [SD 9.7]) and HC (30.0% [SD 8.6]) (p=0.71). In contrast, rGCA-TCZ patients demonstrated significantly higher numbers of Ki67+Tregs when compared to both, rGCA-CS patients (15.4% [SD 6.4]; p=0.02) and aGCA patients (16.8% [SD 3.5]; p=0.04) (Figure 2A–B). These differences persisted in age- and CS dose-adjusted analysis. The expression of Ki67 in CD4+Foxp3−T-cells, however, did not differ significantly among groups (Supplementary Figure 3). These results suggest that Treg proliferation is impaired in GCA and that TCZ, in contrast to CS, selectively restores the Treg replicative potential without influencing the proliferation of non-regulatory CD4+T-cells.

Figure 2. TCZ therapy restores impaired Treg proliferation.

Figure 2

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A, Representative flow cytometry plots of Ki67+ cells within CD4+Foxp3+ T-cells in patients with GCA and HC. B, Frequencies of Ki67+ cells within CD4+Foxp3+ T-cells in GCA patients (rGCA-TCZ, n = 5; rGCA-CS, n = 7; aGCA, n = 5) and HC (n = 5). Analysis: Student’s t-test. Error bars represent means and SD. See figure 1 for other definitions

TCZ decreases the number of Foxp3Δ2+ Tregs

During Treg ontogeny, the exon 2 of Foxp3 directly inhibits key transcription factors that drive the Th17 cell differentiation program [22, 32]. Recently, less suppressive Tregs that disproportionately express Foxp3Δ2 have been reported in human autoimmune disease [24]. We therefore analyzed the expression of full-length Foxp3 and Foxp3Δ2 in proliferating Tregs of GCA patients and HC (Figure 3A). We observed that the mean percent of Ki67+Foxp3Δ2+Tregs was not significantly different between rGCA-TCZ patients (26.3% [SD 11.4]) and HC (25.3% [SD 6.7]) (p=0.88) (Figure 3B). In contrast, rGCA-TCZ patients demonstrated significantly lower numbers of Ki67+Foxp3Δ2+ Tregs when compared to both, rGCA-CS patients (54.9% [SD 21.4]; p=0.03) and aGCA patients (63.2% [SD 16.2]; p=0.01) (Figure 3B). These differences persisted in age- and CS dose-adjusted analysis. These results demonstrate that the increased Foxp3Δ2 Treg expression seen in patients with GCA is not corrected by CS, but is abrogated upon IL-6 signaling inhibition with TCZ.

Figure 3. Effects of TCZ on Foxp3Δ2 expression in proliferating Tregs and phenotype of Foxp3Δ2+ Tregs.

Figure 3

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A, Representative flow cytometry plots of Foxp3Δ2 expression within CD4+Foxp3+Ki67+ T-cells in GCA patients and HC. B, Frequencies of Foxp3Δ2+ cells within CD4+Foxp3+Ki67+ T-cells in GCA patients (rGCA-TCZ, n = 5; rGCA-CS, n = 7; aGCA, n = 5) and HC (n = 5). C, Representative flow cytometry plots of the expression of CD25 and CD161 in full-length Foxp3- and Foxp3Δ2-expressing CD4+Foxp3+Ki67+ T-cells in a patient with aGCA. D, Surface expression of CD25 (left panel) and CD161 (right panel) in full-length Foxp3- and Foxp3Δ2-expressing CD4+Foxp3+Ki67+ T-cells of GCA patients and HC (n = 22). Analysis: unpaired Student’s t-test in B; paired Student’s t-test in C. Error bars represent means and SD. Foxp3-FL = full-length Foxp3 isoform. See figure 1 for other definitions

Foxp3Δ2+ Tregs co-express CD161

It has been demonstrated that IL-6-stimulated Tregs may become IL-17 producing cells [16, 20, 21]. In addition, IL-17-producing Tregs may also express other Th17-related markers such as CD161 [20]. For this reason, we further characterized the phenotype of proliferating Tregs that either expressed full-length Foxp3 or Foxp3Δ2 by measuring the co-expression of CD25 and CD161 (Figure 3C). We found that Tregs that expressed full-length Foxp3 co-expressed high amounts of CD25 (i.e., CD25high) significantly more often than did Tregs expressing Foxp3Δ2 (mean 74.88% [SD 13.26] versus 39.63% [SD 18.39]; p<0.01). In contrast, Foxp3Δ2-expressing Tregs co-expressed CD161 significantly more often than did full-length Foxp3-expressing Tregs (mean 7.54% [SD 7.00] versus 0.66% [1.17]; p<0.01) (Figure 3D). The correlation between full-length Foxp3 and CD25 and between Foxp3Δ2 and CD161 was equivalent across all groups (aGCA, rGCA-CS, rGCA-TCZ and HC) (Supplementary Figure 4). In summary, Foxp3Δ2-expressing Tregs demonstrated decreased co-expression of CD25 and increased co-expression of CD161, a phenotype that suggests the potential for IL-17 production.

TCZ reduces the population of IL-17-producing Tregs

Because our data showed that proliferating Tregs derived from patients with active disease preferentially expressed Foxp3Δ2, and these cells were also characterized by CD161 co-staining, we examined the Treg production of IL-17 (Figure 4A). We found that the mean percent (SD) of IL-17+Tregs in aGCA, rGCA-CS, rGCA-TCZ and HC was 4.40% (1.29), 2.68% (1.36), 1.29% (1.69), and 1.94% (1.14), respectively (Figure 4B). Whereas no significant differences in the numbers of IL-17+Tregs existed between HC and rGCA-TCZ patients (p=0.39), rGCA-TCZ patients demonstrated significantly lower numbers of IL-17+Tregs than aGCA patients (p<0.01) and a trend towards fewer of these cells compared to rGCA-CS patients (p=0.06). The differences among GCA groups persisted in age- and CS dose-adjusted analysis. In concordance with prior reports [20, 26], the main source of IL-17 within the Treg population of patients with active disease resided in the CD45RA−Foxp3low non-suppressive cell subset (Figure 4C–D). These results demonstrate that the IL-17-producing Treg population is expanded in peripheral blood during periods of GCA activity, and that TCZ abrogates this abnormality more efficiently than do CS.

Figure 4. TCZ corrects the expansion of IL-17-producing Tregs.

Figure 4

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A, Representative flow cytometry plots of IL-17+ cells within CD4+Foxp3+ T-cells in GCA patients and HC. B, Frequencies of CD4+Foxp3+IL-17+ T-cells in GCA patients (rGCA-TCZ, n = 7; rGCA-CS, n = 16; aGCA, n = 10) and HC (n = 10). C, Representative flow cytometry plots of IL-17 expression within rTregs (subset I), aTregs (subset II), and non-suppressive Foxp3low cells (subset III) in a patient with aGCA. D, Frequencies of IL-17 expressing non-suppressive Foxp3low cells (subset III) within CD4+Foxp3+IL-17+ T-cells (subsets I+II+III) in GCA patients (rGCA-TCZ, n = 7; rGCA-CS, n = 16; aGCA, n = 10) and HC (n = 10). Analysis: Student’s t-test. Error bars represent means and SD. See figure 1 for other definitions

Treg function is impaired in GCA

Previous research has shown that IL-6 may decrease Treg function [33]. In patients with new-onset GCA, however, Tregs have been reported to be competent regardless of disease activity or CS treatment [8]. To investigate whether GCA patients in our cohort had normal or impaired Treg function and to examine whether IL-6 blockade influenced this function, we performed suppression assays co-culturing CD4+CD25−conventional T-cells and CD4+CD25+Tregs. The results showed no significant differences in Treg function among the GCA groups (Figure 5A–B). However, Tregs derived from GCA patients taken together demonstrated significantly impaired suppressive ability when compared to Tregs derived from HC (Figure 5A–B). To assess for the potential confounding effect of pro-inflammatory CD25+ effector cells that could have been included in the population of CD4+CD25+ cells utilized for functional assays, we analyzed the number of non-Tregs (CD4+CD25+CD45RA− cells) and non-suppressing Foxp3low cells (CD4+CD25++CD45RA−cells) in comparison to the number of aTregs (CD4+CD25+++CD45RA−cells) and rTregs (CD4+CD25++CD45RA+ cells) within the CD4+CD25+ pool [26] and found no significant differences among groups (Supplementary Figure 5).

Figure 5. Treg function in GCA patients and healthy controls.

Figure 5

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105 CFSE-labeled CD4+CD25− conventional T-cells stimulated with anti-CD3/CD28 antibodies were incubated for 4 days with varying concentrations of autologous CD4+CD25+ Tregs (HC:HC; GCA:GCA) to create ratios of 8:1, 4:1, 2:1 and 1:1. Proliferation of conventional T-cells was measured by determination of CFSE dilution by flow cytometry. A, Conventional T-cell proliferation plots from representative GCA patients and HC (conventional T-cell to Treg ratio 1:1). B, Suppression assays in GCA patients (rGCA-TCZ, n = 4; rGCA-CS, n = 9; aGCA, n = 4) and HC (n = 5). Analysis: Student’s t-test. Error bars represent means and SD. Asterisks denote statistically significant differences compared to HC. Teff = conventional T-cells. See figure 1 for other definitions

DISCUSSION

We sought to characterize the peripheral Treg compartment in GCA and to evaluate whether IL-6R blockade was associated with modulation of the Treg response. Our results showed that patients with active disease have a defective and likely pathogenic Treg population that demonstrates decreased proliferation, over-expression of Foxp3Δ2, and increased IL-17 production. In addition, our study revealed a mechanism by which IL-6 signaling inhibition may exert its therapeutic effects in GCA [14]. Unlike therapy with low to moderate doses of CS, treatment with TCZ not only restored the Treg proliferative capacity, but also reverted the pathogenic Treg phenotype (Foxp3Δ2 and IL-17 expression) and increased the expression of markers of Treg activation, trafficking and terminal differentiation (Foxp3high, CD25high, CCR4, and CTLA-4).

Foxp3 largely controls the phenotype and function of Tregs [34]. Three variants of Foxp3 have been described, a full-length and two spliced forms (Foxp3Δ2 and Foxp3 lacking exon 2 and 7 [Foxp3Δ2Δ7]) [35, 36]. Although the regulation and function of Foxp3Δ2 and Foxp3Δ2Δ7 are poorly understood [35, 36], exon 2 is known to encode a repression domain that blocks the activity of the transcription factors RORγt (RORC in humans) and RORα, which are involved in the differentiation of CD4+ cells towards the Th17 phenotype [22, 32, 37]. Foxp3Δ2 is regarded as a hypofunctional isoform of Foxp3 [23, 32] and increased expression of this spliced variant has been reported as a mechanism of immune dysregulation in ANCA-associated vasculitis [24]. Herein, we demonstrate for the first time that Tregs in GCA patients preferentially express Foxp3Δ2 over full-length Foxp3. Moreover, we show that Foxp3Δ2 Tregs are often CD161highCD25low, which suggests potential for IL-17 production [20]. We therefore predict that Foxp3Δ2 Tregs in GCA not only lose their suppressive function, but also themselves become pathogenic as a source of IL-17.

The functional plasticity of Tregs is highly dependent on the surrounding microenvironment [17, 18, 20], and the stability of Tregs is thought to play a role in the pathogenesis of inflammatory disorders [20, 21, 38, 39]. IL-17-producing Tregs have been detected in inflamed tissues of patients with autoimmune conditions such as rheumatoid arthritis, in which IL-6 may induce Tregs to become IL-17+ cells [20]. In some cases, IL-17-producing Tregs seem to “relax” their suppressive function [20], but in others, they retain full regulatory capacity [20, 40, 41]. Of note, IL-17+Foxp3+ cells have also been found infiltrating arteries of GCA patients [10]. However, their functional characterization, contribution to disease pathogenesis and response to treatment have not been fully elucidated. Here we show that IL-17-producing Tregs are also present in peripheral blood of GCA patients during periods of disease activity, and that their expansion normalizes following IL-6R blockade therapy. In accordance to prior reports, we found that IL-17-producing Tregs in GCA also express other markers commonly associated with the Th17 lineage (e.g., CD161) [20] and reside within the CD45RA−Foxp3low non-suppressive cell subset [20, 26]. Because we observed that TCZ led to pronounced reduction of both, Foxp3Δ2 and IL-17 expression, we speculate that by a yet undefined mechanism, IL-6 promotes the transcription of Foxp3Δ2 in Tregs with subsequent polarization of these cells towards the Th17 phenotype. The function of IL17-producing Tregs and Foxp3Δ2 Tregs in GCA remains to be determined.

The reasons why Samson et al. [8] found reduced frequencies of functional Tregs in GCA patients and we did not are not apparent. Possible explanations include differences in the methods used to isolate Tregs as well as differences in the characteristics of the populations analyzed. Our cohort was composed of GCA patients with long disease duration and prolonged CS exposure (mean 27 months). In contrast, the cohort studied by Samson et al. was comprised of newly-diagnosed patients, whose CS treatment was relatively short (mean 3.4 months). It is possible that early phases of the disease are characterized by decreased numbers of competent Tregs, which tend to normalize in number over time, but become functionally deficient under the influence of chronic inflammatory stimuli or prolonged CS exposures.

Tregs from GCA patients undergoing TCZ therapy did not demonstrate enhanced ability to suppress the proliferation of conventional T-cells despite the increased expression of effector molecules (e.g., CTLA-4). Although this apparent discrepancy could represent a type II error, other possibilities also exist. First, an augmented regulatory response can be achieved not only by increasing Treg function, but also by increasing the trafficking of Tregs to the sites of inflammation. CCR4 is involved in Treg migration [42] and has been shown to direct Tregs into cardiovascular allografts, the allergic lung and certain tumors [43–45]. It could be hypothesized that a highly proliferative Treg compartment that expresses CCR4 may form the basis for increased Treg cell migration into inflamed arteries. In addition, other important in-vivo mechanisms by which Tregs exert their function (e.g., through CTLA-4 competition with CD28 for binding to CD80/86 [30, 46]) could not be assessed in the functional assay utilized.

Two randomized controlled trials have recently demonstrated that TCZ is effective in maintaining disease remission and sparing CS in GCA [14, 15]. Our findings complement the results of these trials and provide a pathophysiologic rationale for the use of IL-6 blockade therapy in this disease. In addition, given that several of the Treg abnormalities observed in patients with active disease were not fully reversed upon treatment with CS monotherapy, our results may also provide insight into the reason why the great majority of CS-treated patients relapse upon CS dose reduction.

In summary, we found that GCA is associated with marked abnormalities in the peripheral Treg compartment. In addition, we demonstrated that unlike CS treatment, TCZ-therapy not only abrogated the pathogenic Treg phenotype seen during periods of disease activity, but also increased Treg activation, proliferation and terminal differentiation. Limitations of our study include its cross sectional nature and the relatively small sample size; therefore, larger studies that include longitudinal follow up of new onset GCA patients treated with TCZ will be required to continue to improve our understanding of the beneficial effects of blocking IL-6 in this disorder.

Supplementary Material

SI 1

Supplementary Figure 1. Treg frequencies in GCA patients and healthy controls. A, Treg (CD4+Foxp3+ T-cells) gating strategy. B, Frequencies of total Tregs in GCA patients (rGCA-TCZ, n = 9; rGCA-CS, n = 18; aGCA, n = 11) and HC (n = 10). Analysis: ANOVA. Error bars represent means and SD. FSC = forward scatter; SSC = side scatter; H = height; A = area; GCA = giant cell arteritis; CS = corticosteroids (prednisone); TCZ = tocilizumab; rGCA-CS = GCA in remission on CS; rGCA-TCZ = GCA in remission on TCZ without or without CS; aGCA = active GCA; HC = healthy controls.

Supplementary Figure 2. Frequencies of resting Tregs and non-suppressive Foxp3low cells in GCA patients and healthy controls. A, Frequencies of CD45RA+Foxp3low resting Tregs (rTregs or subset I) in GCA patients (rGCA-TCZ, n = 9; rGCA-CS, n = 17; aGCA, n = 9) and HC (n = 10). B, Frequencies of CD45RA−Foxp3low non-suppressive cells (non-suppressive Foxp3low cells or subset III) in GCA patients (rGCA-TCZ, n = 9; rGCA-CS, n = 17; aGCA, n = 7) and HC (n = 10). Analysis: ANOVA. Error bars represent means and SD.

Supplementary Figure 3. Ki67 expression in CD4+Foxp3− cells in GCA patients and healthy controls Frequencies of Ki67+ cells within CD4+Foxp3− T-cells (non Tregs) in GCA patients (rGCA-TCZ, n = 5; rGCA-CS, n = 13; aGCA, n = 10) and HC (n = 6). Analysis: Student’s t-test. Error bars represent means and SD.

Supplementary Figure 4. Co-expression of CD25 and CD161 within full-length Foxp3− and Foxp3Δ2-expressing CD4+Foxp3+Ki67+ T-cells. Expression of CD161 and CD25 in full-length (FL) Foxp3- and Foxp3Δ2-expressing CD4+Foxp3+Ki67+ T-cells in GCA patients (rGCA-TCZ, n = 5; rGCA-CS, n = 7; aGCA, n = 5) and HC (n = 5). Analysis: Paired Student’s t-test. Error bars represent means and SD. Shapes and colors of the symbols identify individual patients and HC.

Supplementary Figure 5. Distribution of activated Tregs, resting Tregs, non-suppressive CD25+ cells, and non-Tregs within the CD4+CD25+ cells. Graph displaying the ratio between the percent of non-Tregs (subset IV, CD4+CD25+CD45RA− cells) plus non-suppressing CD25+ cells (subset III, CD4+CD25++CD45RA−cells) compared to the percent of activated Tregs (subset II, CD4+CD25+++CD45RA−cells) plus resting Tregs (subset I; CD4+CD25++CD45RA+ cells) in GCA patients (rGCA-TCZ, n = 8; rGCA-CS, n = 6; aGCA, n = 5) and HC (n = 5). Analysis: ANOVA. Error bars represent means and SD.

SI 2

Acknowledgments

Funding info:

Arthritis Foundation CRTA grant #5924

Footnotes

Competing interests:

There are no competing interests

Contributorship Statement:
  1. Substantial contributions to the conception or design of the work, or the acquisition, analysis or interpretation of data: Miyabe C, Miyabe Y, Strle K, Kim N, Stone JH, Luster AD, and Unizony S
  2. Drafting the work or revising it critically for important intellectual content: Miyabe C, Miyabe Y, Strle K, Kim N, Stone JH, Luster AD, and Unizony S
  3. Final approval of the version published: Miyabe C, Miyabe Y, Strle K, Kim N, Stone JH, Luster AD, and Unizony S
  4. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: Luster AD and Unizony S

Data Sharing Statement:

All data related with this study are presented in the revised manuscript and and supplementary material

Ethical approval information:

IRB number 2012P002348

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SI 1

Supplementary Figure 1. Treg frequencies in GCA patients and healthy controls. A, Treg (CD4+Foxp3+ T-cells) gating strategy. B, Frequencies of total Tregs in GCA patients (rGCA-TCZ, n = 9; rGCA-CS, n = 18; aGCA, n = 11) and HC (n = 10). Analysis: ANOVA. Error bars represent means and SD. FSC = forward scatter; SSC = side scatter; H = height; A = area; GCA = giant cell arteritis; CS = corticosteroids (prednisone); TCZ = tocilizumab; rGCA-CS = GCA in remission on CS; rGCA-TCZ = GCA in remission on TCZ without or without CS; aGCA = active GCA; HC = healthy controls.

Supplementary Figure 2. Frequencies of resting Tregs and non-suppressive Foxp3low cells in GCA patients and healthy controls. A, Frequencies of CD45RA+Foxp3low resting Tregs (rTregs or subset I) in GCA patients (rGCA-TCZ, n = 9; rGCA-CS, n = 17; aGCA, n = 9) and HC (n = 10). B, Frequencies of CD45RA−Foxp3low non-suppressive cells (non-suppressive Foxp3low cells or subset III) in GCA patients (rGCA-TCZ, n = 9; rGCA-CS, n = 17; aGCA, n = 7) and HC (n = 10). Analysis: ANOVA. Error bars represent means and SD.

Supplementary Figure 3. Ki67 expression in CD4+Foxp3− cells in GCA patients and healthy controls Frequencies of Ki67+ cells within CD4+Foxp3− T-cells (non Tregs) in GCA patients (rGCA-TCZ, n = 5; rGCA-CS, n = 13; aGCA, n = 10) and HC (n = 6). Analysis: Student’s t-test. Error bars represent means and SD.

Supplementary Figure 4. Co-expression of CD25 and CD161 within full-length Foxp3− and Foxp3Δ2-expressing CD4+Foxp3+Ki67+ T-cells. Expression of CD161 and CD25 in full-length (FL) Foxp3- and Foxp3Δ2-expressing CD4+Foxp3+Ki67+ T-cells in GCA patients (rGCA-TCZ, n = 5; rGCA-CS, n = 7; aGCA, n = 5) and HC (n = 5). Analysis: Paired Student’s t-test. Error bars represent means and SD. Shapes and colors of the symbols identify individual patients and HC.

Supplementary Figure 5. Distribution of activated Tregs, resting Tregs, non-suppressive CD25+ cells, and non-Tregs within the CD4+CD25+ cells. Graph displaying the ratio between the percent of non-Tregs (subset IV, CD4+CD25+CD45RA− cells) plus non-suppressing CD25+ cells (subset III, CD4+CD25++CD45RA−cells) compared to the percent of activated Tregs (subset II, CD4+CD25+++CD45RA−cells) plus resting Tregs (subset I; CD4+CD25++CD45RA+ cells) in GCA patients (rGCA-TCZ, n = 8; rGCA-CS, n = 6; aGCA, n = 5) and HC (n = 5). Analysis: ANOVA. Error bars represent means and SD.

NIHMS921263-supplement-SI_1.pdf (30KB, pdf)
SI 2
NIHMS921263-supplement-SI_2.pdf (365.6KB, pdf)

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