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Keywords:

  • Arthritis;
  • Autoimmune disease;
  • FOXP3;
  • Ki67;
  • Methylation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Regulatory T cells (Tregs) are important for maintaining immune homeostasis, but many studies suggest that Tregs are functionally impaired in autoimmune and chronic inflammatory disorders. In addition, effector T cells may vary in sensitivity toward Treg suppression. Herein, we have studied the interplay between T effectors and Tregs in the rheumatic joint. Synovial Tregs demonstrated a high degree of FOXP3 demethylation and displayed only marginal IL-17 and virtually no IFN-γ production following in vitro stimulation, altogether indicating suppressive capacity. Still, the frequency of FOXP3 expression could not predict the degree of suppression. Instead, the inflammatory milieu in the joint, i.e. proliferative capacity of effector T cells and in situ levels of pro-inflammatory cytokines influenced Treg function. Indeed, blocking IL-6 or TNF increased the suppression by Tregs in co-cultures. Additionally, approximately 30% of the synovial FOXP3+ T cells were Ki67+ and hence actively dividing, but proliferation did not overlap with cytokine production, suggesting that these cells represent functional Tregs having met their cognate antigen and expanded in an attempt to alleviate joint inflammation. Overall, our data argue against a general functional deficit in joint-derived Tregs and instead emphasize the importance of the inflammatory milieu to set the threshold for immune regulation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Naturally occurring CD4+CD25+ suppressor or regulatory T cells (Tregs) play an active role in establishing and maintaining immunological self-tolerance and controlling various immune responses to non-self-antigens. The forkhead transcription factor 3 (Foxp3; FOXP3 in humans) is essential for Treg development and congenital mutations in FOXP3 result in severe autoimmunity, multi-organ immune pathology and allergy in humans 1. The development of FOXP3 monoclonal antibodies and knock-in or transgenic mouse strains where fluorescent proteins are co-expressed with Foxp3 has greatly enhanced our ability to define and characterize Tregs both in mice and in humans 2.

However, there are limitations in defining Tregs based on FOXP3 since naïve human CD4+CD25 cells can be induced to express FOXP3 through polyclonal activation via the T-cell receptor in the presence of TGF-β, but such induced FOXP3 expression is transient, declining to baseline amounts with prolonged culture 3. Therefore, functional assays are critical to assess the suppressive function of Tregs. In addition, the degree of DNA methylation in the FOXP3 locus can also be used to distinguish between Tregs and activated T cells temporarily expressing FOXP3 4.

The autoimmune disease under investigation here is inflammatory arthritis, which is characterized by activated immune cell infiltration into the joint, which can manifest into chronic inflammatory diseases such as rheumatoid arthritis (RA) 5. RA is considered a Th1- and/or Th17-associated disease and T cells comprise a large proportion of the inflammatory cells invading synovial tissue and accumulating in synovial fluid (SF) in a relapsing manner. Today, many different therapeutic strategies for treating arthritis are available in clinical practice, but none are remedial or curative. Phenotypic and functional characterization of immune cells, both inflammatory and Tregs, at the site of inflammation may contribute to a better understanding of the effect of new biological agents in arthritis patients.

We have recently shown that FOXP3 expression in SF T cells is not confined to CD25bright T cells as seen in blood, but included CD25intermediate (CD25int) and even some CD25 T cells 6. Indeed, when isolating Tregs, the cut-off for CD25 intensity could be lowered without losing suppressive function, indicating the presence of functional Tregs in T cells with a lower intensity of CD25 expression 6, 7. However, it has been demonstrated that the addition of IL-7 and TNF, cytokines commonly elevated in joint fluid, to Treg co-cultures can reduce the suppressive function of Tregs 8, 9, suggesting that Treg function might be compromised locally. Indeed, neutralization of TNF activity (receptor antagonist or neutralizing antibody) has proven to be very successful in breaking the feedback loop of chronic inflammation 10 with possible effects on the function of Tregs in vivo 11, 12. In addition, several studies (in various autoimmune settings both in mice and in humans) have demonstrated that the effector T cells may not be sensitive to immune regulation by Tregs 13–15, but so far this aspect has not been addressed in inflammatory arthritis.

In the current study, we have utilized longitudinal patient samples, i.e. studied several relapses, to dissect both the frequency of FOXP3+ cells in CD4+ T cells with different levels of CD25 expression as well as the level of demethylation of the FOXP3 locus. We could demonstrate a consistent increase in the frequency of FOXP3+ CD4+ T cells in the SF compared with the peripheral blood (PB) over several relapses and, further, a high demethylation pattern in CD25bright T cells. Additionally, while a large proportion of the FOXP3+ T cells in the joint were actively dividing when analyzed ex vivo, only a few of these cells could be pushed into IL-17 and IFN-γ production following in vitro stimulation. Suppressive function by Tregs could not be correlated with frequency of FOXP3 expression but instead correlated inversely with the proliferative capacity of the effector T cells. Thus, we found that the inflammatory milieu could override suppression by CD25bright Tregs even in patients with a high frequency of FOXP3. Indeed, addition of blocking antibodies to TNF or IL-6R enhanced suppressive activity by Tregs in co-culture, suggesting that patients with established chronic arthritis (who rarely achieve disease remission with current treatment strategies) could benefit from a combination of anti-inflammatory and Treg therapy.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Tregs from SF express higher levels of FOXP3 compared with Tregs from PB

Intranuclear FOXP3 staining was carried out in paired and longitudinal PB and SF samples isolated from nine patients with arthritis at time points of active knee inflammation (Fig. 1A). The frequency of FOXP3 in the CD4+ T-cell population was consistently higher in synovial fluid mononuclear cells (SFMCs) compared with peripheral blood mononuclear cells (PBMCs) (Fig. 1B) but also tended to vary more in the joint, while the CD8+ subset contained virtually no FOXP3+ cells (Fig. 1B). In addition to the frequency of FOXP3+ cells being higher in CD4+ T cells from SF as compared with blood (p<0.01), the median fluorescence intensity (MFI) of FOXP3 was also brighter (p<0.01) (Fig. 1C).

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Figure 1. Comparison of the frequency of FOXP3+ CD4+ T cells between PB and SF over several relapses in patients with rheumatic disease. (A) Dot plot of FOXP3 and isotype control staining in PBMCs and SFMCs from one representative patient. Numbers indicate frequencies of FOXP3+ cells among gated CD4+ T cells and the median fluorescence intensity (MFI) in parentheses. Data are the representative of eight patients. (B) PBMCs (n=8) and SFMCs (n=9) were collected from patients over 3–5 relapses and the frequency of FOXP3 among CD4+ (top) and CD8+ (bottom) T cells was analyzed. Each line represents the frequency of FOXP3 in one patient. (C) Direct comparison of FOXP3 frequency (left) and intensity, i.e. MFI (right) in gated CD4+ T cells of paired PBMC and SFMC samples from eight patients. Each line represents the median of all the time points for each patient. Wilcoxon signed-rank test **p<0.01.

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Further analysis of FOXP3 expression was carried out by subdividing the CD4+ T cells into CD25high (only PB) or CD25bright (only SF), CD25int and CD25 and again both frequencies and MFI were analyzed (see Fig. 2A for gating strategy). As expected, the highest frequency of FOXP3+ cells was observed in T cells expressing the highest levels of CD25 in both PBMCs and SFMCs, but there was no statistical difference between the two compartments (Fig. 2B). Still, a significantly higher MFI of FOXP3 was observed in SFMCs compared with paired PBMCs (p<0.01). The CD25int and CD25 T cells displayed lower FOXP3 MFI than CD25bright, but consistently the MFI was higher in SF as compared with blood (Fig. 2B). We also observed substantially higher frequencies of FOXP3 in the CD25int and CD25 compartments in SF as compared with PB (p<0.01). In summary, our results show that FOXP3 is expressed both more frequently and at a higher level in CD4+ T cells from SF compared with CD4+ T cells from PB.

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Figure 2. Comparison of the frequency and intensity of FOXP3 and demethylation in the FOXP3 gene locus with varying expression of CD25 in PBMCs and SFMCs. (A) Representative plot showing the gating strategy for analysis of CD4+CD25high (PB only) CD25bright (SF only), CD25int or CD25 CD4+ FOXP3+ T cells in PB and SF. (B) Both the frequency of FOXP3 among the T cells with varying intensity of CD25 (left) and MFI (right) were evaluated in PBMCs and SFMCs collected from patients over several time points. Each line represents the median of all the time points for each patient. Wilcoxon signed-rank test *p<0.05, **p<0.01. (C) CD25 and CD25high T cells/CD4+CD25bright T cells were sorted from blood (five patients) and SF (six patients). The degree of demethylation in the FOXP3 gene locus is depicted for the different subsets. (D) Longitudinal analysis of demethylation in the FOXP3 gene locus was evaluated in sorted SF CD4+CD25bright, CD25int and CD25 T cells from a single patient at five separate relapses.

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DNA methylation as a marker for stable expression of FOXP3 in CD4+ CD25+ T cells

Demethylated regulatory elements in the FOXP3 locus support stable expression of FOXP3 in Tregs isolated from PB of healthy individuals 16 and we have previously described DNA methylation analysis of the FOXP3 promoter region as a tool for distinguishing Tregs from activated T cells with transient FOXP3 expression 4. We performed methylation analysis of both CD25 and CD25hi/bright cells isolated from both PB and SF. Our results demonstrate 71–96% demethylation at the evolutionarily conserved −77 position in CD4+CD25bright T cells (putative Tregs) from six different patients, with a median value of 89%, while the corresponding median for the CD25high T cells from PB was only 48% and with substantial variation between the patients (Fig. 2C). As a comparison the levels detected in CD25 T cells were more homogenous but also here a higher demethylation level was found in the joint-derived compared with PB samples (median 17 versus 53%). In addition, we performed a temporal study of CD4+CD25bright, CD25int and CD25 T cells from one of the patients at five relapses over a period of 4 y to investigate the variation at relapses in disease activity. As shown in Fig. 2D, the data were consistent over time with CD4+CD25bright T cells (n=5) displaying extensive demethylation (median level of demethylation at 92%, range 88–95) in comparison with CD25int and CD25 T cells (79 versus 52% demethylation respectively). Based upon the high and constant level of demethylation in sorted CD25bright cells, we conclude that these cells display a stable FOXP3 phenotype, which is a hallmark of bonafide Tregs.

Joint-derived FOXP3+ T cells are often dividing but rarely produce pro-inflammatory cytokines

Given the higher frequency and intensity of FOXP3 in joint-derived CD25int and CD25 T cells compared with blood, we wondered whether these cells could represent recently activated T cells or maybe ex-Tregs that have converted into an inflammatory phenotype.

First, we assessed the degree of cells in division by staining for the proliferation marker Ki67. A higher frequency of proliferating CD4+ T cells was found in SF compared with PB as exemplified in Fig. 3A. FOXP3+ cells were proliferating to a higher degree than FOXP3 T cells in both PB (n=4, p<0.01) and SF (n=7, p<0.01) in Fig. 3B. We further subdivided the synovial FOXP3+ cells based on CD25 expression and could see that the CD25bright T cells express the highest frequency of Ki67 compared with CD25int or CD25 T cells (Fig. 3C), indicating that the dividing cells are not primarily activated T cells (which would be expected to be CD25int transiently expressing FOXP3).

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Figure 3. Frequencies of proliferating (Ki67+) CD4+ T-cell subsets in paired samples. (A) Representative intranuclear staining for Ki67 in PB and SF CD4+ T cells. The frequency of FOXP3+ and FOXP3 CD4+ T cells that were positive for Ki67 is indicated. (B) Summary of the frequency of Ki67+ T cells among the FOXP3+ and FOXP3 populations in PBMC (n=4) and SFMC samples (n=8). Wilcoxon signed-rank test **p<0.01. (C) The distribution of Ki67 expression in SF CD4+FOXP3+ T cells was analyzed based on CD25, CD25int and CD25bright expression in six patients. Wilcoxon signed-rank test *p<0.05. (D) Representative intranuclear staining for IL-17, IFN-γ, Ki67 and FOXP3 in SF CD4+ T cells. The two left panels depict the frequency of IL-17+ and IFN-γ+ cells respectively, among FOXP3 and FOXP3+ cells, while the two right-hand panel depict in gated FOXP3+ cells, the relationship between Ki67 and IL-17 and Ki67 and IFN-γ respectively. Wilcoxon signed-rank test *p<0.05. (E) Frequency of IL-17 or IFN-γ among FOXP3+ and FOXP3 SF cells from six patients (left) and the frequency of IL-17+ or IFN-γ+ cells among SF FOXP3+Ki67+ or FOXP3+Ki67 T cells (right). Wilcoxon signed-rank test *p<0.05.

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Second, as it has been demonstrated that FOXP3+ T cells could convert into cytokine-producing cells, we stimulated SF-derived cells in vitro and performed intracellular cytokine staining. Following a short TCR cross-linking, ample IL-17 and IFN-γ production by the FOXP3 T cells could be seen, but only very minor cytokine production by the FOXP3+ cells (Fig. 3D and E). Interestingly, when further assessing cytokine production with proliferation in FOXP3+ cells we saw virtually no overlap (Fig. 3D and E), further supporting the notion that the Ki67+FOXP3+ T cells are true Tregs.

Suppressive function of sorted CD25bright Tregs is influenced by the activation status of the CD25 T cells

As previously demonstrated by us and others, Treg suppression in synovial co-cultures is quite variable 6, 8, 17. In the present study, we searched for markers that could predict Treg function and the first candidate was FOXP3 expression. For this purpose we selected six patients with chronic arthritis; with five of them having sero-positive and one having sero-negative disease. When performing co-culture experiments (Fig. 4A), we found variable suppressive function of isolated Tregs between the patients, and this variation could not be predicted merely by FOXP3 frequency (Fig. 4B). For example, in patients 1 and 2, the CD25bright T cells, which had high frequencies of FOXP3+ cells, efficiently suppressed the proliferation of CD25 T cells. However, CD25bright T cells isolated from patients 3 and 8 suppressed CD25 T cells equally well (99 and 80% respectively), despite low frequencies of FOXP3 (41 and 42% respectively). Finally, CD25bright Tregs isolated from patients 9 and 10 did not suppress CD25 T cells, despite a high frequency of FOXP3 in the CD25bright compartment (82 and 60% respectively).

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Figure 4. The activation status of CD4+CD25 T cells influences the level of suppression by CD4+CD25bright T cells isolated from rheumatic joints. (A) Representative flow cytometry dot plot showing purity of sorted CD4+CD25 T cells and CD4+CD25bright Tregs with the frequency of FOXP3 in the two populations indicated. (B) Synovial CD4+CD25 T cells isolated from six patients were cultured alone and in the presence of CD4+CD25bright Tregs. Graph represents proliferation (CPM×103) of CD4+CD25 T cells alone (light gray bars), percentage of suppression by CD25bright T cells in co-culture (black bars) and FOXP3 frequencies in the CD25bright T cells (hatched bars). All values are expressed as mean+SD except for the frequency of FOXP3 in the CD25bright cells (right y-axis). (C) The % suppression plotted against the proliferation of CD25 T cells alone (left) and the % suppression plotted against frequency of FOXP3 in SF CD25bright T cells (right). The plots represent data from the same patients presented in (B). Correlation was evaluated by the Spearman correlation test. (D) Cell culture supernatants were collected from wells containing APCs and CD25 T cells and (E) co-culture wells with Tregs and analyzed for six different cytokines: IFN-γ, IL-17A, IL-6, TNF, IL-10 and IL-13 using the BioPlex cytokine detection array. The % secretion of cytokines in the co-cultures as compared with secretion by CD4+CD25 T cells alone is depicted. Wilcoxon signed-rank test *p<0.05.

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To further understand the variable suppression seen by CD25bright Tregs isolated from different patients and its relationship to FOXP3 expression we included in the analysis a third variable: the activation status of CD25 T cells in the individual patients. Indeed, proliferation of responder CD25 T cells in the absence of Tregs varied considerably between the patients. Sorted CD25 T cells isolated from patients 1 and 2 proliferated by a magnitude two-fold lower than those isolated from patients 9 and 10, which may explain the reduced suppressive function of Tregs seen in patients 9 and 10 compared with patients 1 and 2 (Fig. 4B). In line with this interpretation, suppressive function of Tregs was intact in cells from patients 3 and 8 in spite of a very low frequency of FOXP3+ cells in the CD25bright compartment due to lower activation of the CD25 T cells.

Indeed, when performing correlation analyses, it was clear that FOXP3 frequency in Tregs could not predict suppression while the proliferative capacity of the effector T cells could (r=0.89, p=0.03) (Fig. 4C).

Synovial Tregs suppress production of IFN-γ and TNF, but not IL-6 and IL-17

The functional capacity of joint-derived CD25bright T cells can be assessed both as a measure of suppression of proliferation (as discussed above) and of cytokine secretion. Although the majority of T cells in the inflamed joint are of effector/memory phenotype, only a minority of the FOXP3 T cells was in cell division when assessed directly ex vivo (median 15.5%, Fig. 3B). The pathology mediated by T cells at this inflammatory site can thus be predicted to be via their cytokine secretion pattern rather than T-cell expansion. We collected supernatants from our co-culture and control wells to screen for both T-cell-derived and APC-derived cytokines, namely IFN-γ, TNF, IL-6, IL-10, IL-13 and IL-17A. Overall, IFN-γ, TNF and IL-6 could readily be detected in the cultures, whereas IL-10, IL-13 and IL-17A varied substantially between patient samples (Fig. 4D).

The secretion of cytokines into the co-cultures was compared with secretion by CD25 T cells alone (Fig. 4D), and the ratio was used as a measure of suppression. Tregs from five out of six patients suppressed IFN-γ secretion by 50% or more, while strikingly only one patient (number 3) demonstrated suppression of IL-17A production (Fig. 4E).

The main IL-6 source in the cultures was from the APCs (median 1110 pg/mL) with only a minor contribution from the responder/effector CD25 T cells (49.5 pg/mL) and this cytokine was not suppressed by the Tregs, while TNF was seen from both APCs (138.5 pg/mL) and T cells (536.5 pg/mL) and was suppressed in three out of the six patients studied.

Finally, we also studied Th2 cytokines, since they could promote suppressive function of Tregs. However, we did not observe any reciprocal tendencies of increases in IL-10 and IL-13 with Treg function. Instead, Tregs further suppressed the secretion of both these cytokines in the majority of the patients studied (Fig. 4E).

In conclusion, IL-17A and IL-6 secretion by CD25 T cells was not suppressed in co-culture by Tregs as efficiently as IFN-γ, suggesting that these cytokines may contribute towards the chronic inflammation process in these patients.

Relation of FOXP3 expression to the de novo presence of pro-inflammatory cytokines in SF

SF from rheumatic joints is known for its high content of pro-inflammatory cytokines 10, a feature that will influence Treg function 8, 9, 18, 19. The differences in suppression among different patients prompted us to investigate the presence of the pro-inflammatory cytokines IL-1β, IL-6 and TNF in the joint fluid of the patients from whom cells were isolated and suppression assay or analysis of FOXP3 frequency in SF was performed. TNF and IL-6 were consistently found in SF of all patients, although at variable levels, while IL-1β was undetectable. Correlation analysis was carried out to study the relationship between FOXP3 expression and cytokine levels in the SF. Interestingly, there was an inverse correlation between the frequency of FOXP3 in the CD4+ T-cell compartment and the amount of IL-6 and TNF in the joint fluid (Fig. 5A).

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Figure 5. The influence of IL-6 and TNF on the FOXP3 frequency and the suppressive function of Tregs in SF. (A) The levels of IL-6 and TNF in SF were determined by ELISA in the same samples from which SFMCs were isolated. The concentrations are plotted against frequency of FOXP3 among CD4+ T cells, and Pearson correlation analysis shows significant inverse correlation between both cytokines and the frequency of FOXP3 in CD4+ T cells. (B) CD4+CD25 and CD4+CD25+ T cells (at different ratios) isolated from SF of three patients were cultured together with irradiated APCs and anti-CD3 antibody in the absence or presence of 100 μg/mL tocilizumab or adalimumab. The % suppression is shown for cultures without antibody (dotted line) and for cultures with tocilizumab or adalimumab (solid line). (C) The proliferative response of CD4+CD25 T cells alone is shown as the mean+SD (cpm×103) in the absence or presence of tocilizumab or adalimumab.

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IL-6 and TNF blockade increases Treg function in vitro

To assess the influence of IL-6 and TNF on Treg function, we added the commonly used drugs directed against IL-6 (tocilizumab, α-IL-6R) or TNF (adalimumab, α-TNF) to our co-cultures. We selected three patients for this study and two of them (patients 12 and 18) displayed improved suppression of proliferation by Tregs in the presence of both these agents (Fig. 5B). The third patient displayed good base line suppressive capacity (87% suppression at a 3:1 ratio), here no effect could be seen when adding tocilizumab and only a minor effect when blocking TNF (Fig. 5B).

Additionally, the effect of adalimumab or tocilizumab on the sorted SF CD25 T cells was analyzed. Here, TNF blockade, but not anti-IL-6R, reduced the CD25 proliferation in all three patients studied (patient 18, 54%; patient 12, 78% and patient 10, 50%) (Fig. 5C). Taken together, both IL-6R and TNF blockade could increase the suppressive capacity of Tregs; but probably by different mechanisms, where TNF blockade may act directly on the proliferation of responder T cells and hence facilitate suppression mediated by Tregs, whereas IL-6R blockade may act via APCs or directly on Tregs themselves.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

It is well established that changes in the frequency of FOXP3+ T cells in PB do not mirror the situation in the rheumatic joint 6, 20, thus it is important, albeit more difficult, to study FOXP3 expression at the site of inflammation. SF is accessible at time points of active inflammation in large joints, but cannot be retrieved during low or no disease activity. Such joint effusions are performed even in patients responding well to their treatment but maybe less frequently, and are thus representative of a broad arthritis population. Synovial CD25bright Tregs have the capacity to suppress both proliferation and cytokine secretion by activated effector T cells 17, 21 and are also significantly enriched at the site of inflammation. We saw only minor changes in FOXP3 frequencies when following our patients over time (up to 6 y) at consecutive relapses of active local disease. However, it has been questioned whether these FOXP3+ T cells are true Tregs and our data suggest that they are, based on four sets of data. First, the epigenetic analysis showed consistently high levels of demethylation in the FOXP3 locus, indicating that joint-derived Tregs are not distorted to any large degree by potentially activated non-regulatory T cells expressing FOXP3. Second, only a minor subset of the FOXP3+ T cells were cytokine producing (mainly IL-17) following in vitro activation. Third, our functional analysis revealed that the degree of suppression was inversely correlated with the proliferative capacity of the effector T cells and not with the level of FOXP3 in the Tregs. Finally, we could enhance the suppressive capacity of synovial Tregs by neutralization of either IL-6 or TNF. Overall, our data argue against a general functional deficit in joint-derived Tregs and instead emphasize the importance of the inflammatory milieu to set the threshold for immune regulation.

Increases in FOXP3 frequency at the site of inflammation have been reported in several inflammatory diseases and is presumably a general host response to chronic inflammation 22, 23. Such an increased frequency of FOXP3+ cells could either be due to the conversion of naïve T cells into FOXP3-expressing cells, or in situ proliferation of de novo FOXP3+ cells. To this end, Vukmanovic-Stejic et al. have demonstrated through deuterium labeling that memory CD4+FOXP3+CD25high cells in the circulation are more proliferative compared with memory CD4+FOXP3CD25 or even naive CD4+FOXP3CD25 populations 24. More recently, Miyara et al. demonstrated a higher frequency of proliferating FOXP3+ cells in the circulation of patients with chronic sarcoidosis than in healthy subjects 25. Hence, our observation of a high degree of Ki67+FOXP3+ T cells, even more in SF than in PB, appear similar to what has been reported in other non-autoimmune settings. Although Tregs are refractory in vitro to TCR stimulation and display anergic properties, in vivo FOXP3+ Tregs appear to proliferate, an observation that may account for their higher frequencies during ongoing inflammation.

Still, FOXP3 is not a foolproof marker of bonafide Tregs since activated CD25 T cells can transiently express FOXP3 3. To circumvent this issue alternative approaches have been developed, e.g. by studying the DNA methylation status of conserved regulatory elements in the FOXP3 locus 4. However, this approach is only valid for male subjects since the FOXP3 gene lies on the X chromosome and females always have one inactivated, and thus fully methylated, X chromosome. In this report, we investigated the evolutionarily conserved CpG site located −77 bp upstream of the FOXP3 transcription start site. Overall, we saw more demethylation in cells from SF as compared with PB, which probably is a reflection of the absence of naïve T cells in this inflammatory compartment. The patient samples in our study also demonstrated differences in the blood phenotype as compared with what has been shown in healthy subjects using the same method, which could maybe explained by systemic manifestations of the disease. Importantly, we show that the demethylation pattern was stable with only subtle variations in longitudinal samples (five relapses over 4 years), and we therefore believe that the reported phenotype is representative for patients with chronic inflammatory arthritis.

To what degree true Tregs are present in the CD25 and CD25int fraction in SF is difficult to clarify. The higher degree of FOXP3 may indicate this, however recently activated T cells temporary expressing FOXP3 would be expected to fall within this population. Indeed, when we stimulated SF cells in vitro and looked for cytokine production we could observe minor IL-17 production, and this came consistently from the CD25 and CD25int fraction. Still, the majority of the FOXP3+ but CD25low cells do not produce cytokines and could potentially still be Tregs, but the lack of surrogate markers makes this very difficult to dissect. IL-17 is a cytokine believed to actively contribute to joint inflammation and destruction in RA 26, 27, and treatment with a humanized monoclonal antibody targeting IL-17A has shown positive results 28. In this context, the ability of Tregs to suppress IL-17 production has been a matter of debate 29, 30. In our co-culture assay, the source of IL-17A was clearly T cell-derived; still in the presence of synovial Tregs we could not observe consistent IL-17A suppression. In fact, in the supernatants from some patients we could detect elevated IL-17A production even when IFN-γ was clearly suppressed. Varying suppression of IL-17 by Tregs is an unresolved issue and may become clearer by further dissecting Tregs into functional subgroups using new markers such as CD39 31.

In addition to IL-17A, the pro-inflammatory cytokines IL-6 and TNF might be key cytokines that antagonize Treg function in the rheumatic joint. IL-6 levels are often high in the joint and could associate to frequent relapses, either through the conversion of FOXP3+ T cells into Th17 18 or through its ability to make effector T cells refractory to suppression by Tregs 19. IL-6 has also been demonstrated to influence the methylation level of the FOXP3 gene 32. In our study, we report an inverse correlation between IL-6 and TNF levels in the SF and FOXP3 expression, and the rescue of suppressive function by Tregs upon blockade of these cytokines. This is interesting since both treatment regimes are commonly used to treat chronic arthritis today.

An outstanding question in the field is whether chronic autoimmune inflammation prevails due to Tregs being outnumbered by effector T cells, or by effector cells becoming refractory to Treg suppression, and our results favor the latter hypothesis. In line with this, it has been demonstrated in a mouse model of multiple sclerosis that autoantigen-specific Tregs migrated to the brain during autoimmune encephalomyelitis, but failed to exert suppressive effects due to the local inflammatory cytokine milieu 23. Similar defective regulation in patients with type 1 diabetes has also been suggested to be a result of decreased susceptibility of CD4+CD25 T-cell population to Treg-mediated suppression 14, 33.

In conclusion, our data do not support a general functional deficit in joint-derived Tregs; instead we could observe an increased frequency of functional FOXP3+ T cells in the affected joints. Still, their mere presence does not result in disease alleviation indicating that optimal Treg function cannot be achieved in a chronic inflammatory setting without first reducing the inflammatory pressure. However, there are reports of long-term remission following very early immunotherapy in RA and here de novo Tregs could play a critical role in re-establishing immune homeostasis 34. In contrast, established chronic arthritis (that rarely achieve disease remission with current treatment strategies) could benefit from a combination of anti-inflammatory and Treg therapy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Patient material

PB and SF samples were collected from the rheumatology clinic at time points of active inflammation (relapses) in large joints requiring joint effusion and mononuclear cells were prepared by Ficoll separation (Ficoll-Paque, Pharmacia, Sweden) and cryopreserved. A total of 21 patients with chronic inflammatory arthritis (the majority being either RA or juvenile idiopathic arthritis (JIA)) were included in this study (13 female) with a median age of 36 (range 23–69). For co-culture experiments, six patients were included (3 RF+RA, 2 RF+JIA and 1 RF−JIA), these patients were also included in the majority of the phenotypic analysis. Epigenetic analysis was performed on the six male patients (3 RF+RA, 2 spondarthropathy and 1 mono-arthritis), four out of these were also included in the phenotypic analysis. A total of eight patients were followed overtime (3 RF+RA, 2 RF+JIA, 1 RF−JIA and 2 spondarthropathy). Several of the patients changed treatment regimes over the study period, which lasted up to 6 y. The study was performed under informed consent and after ethical approval from the Karolinska University Hospital.

Flow cytometry

Single-cell suspensions of PB and SF were surface stained with FITC-conjugated anti-CD3, PerCP-conjugated anti-CD4 and phycoerythrin (PE) or allophycocyanin-conjugated anti-CD25 (Becton Dickinson Biosciences (BD), San José, CA, USA). Staining kits were used according to manufacturer's instructions for intranuclear staining with allophycocyanin or Pacific Blue-conjugated anti-FOXP3 (clone PCH101, eBioscience, San Diego, USA) and PE-conjugated anti-Ki67 (BD) or isotype-matched control antibody. Data acquired on a FACScalibur (BD) or on a Cyan (Beckman Coulter, Brea, CA, USA) instrument were analyzed with the FlowJo software (Treestar, Ashland, OR, USA).

Cell sorting

Cells were fluorescently labeled for sorting CD25bright, CD25int and CD25CD4+ T cells (for SF) or stained for sorting CD25high and CD25CD4+ T cells (for PB) using an MoFlo™ high-performance cell sorter (Beckman Coulter) as previously described 17. Sorted CD3 after irradiation (25 Gy) was used as antigen-presenting cells. Purity of the sorted cell populations was ∼99% for CD3, ∼98% for CD4+CD25, ∼94% for CD4+CD25int and ∼93% for CD4+CD25bright.

Proliferation and cytokine assay

Co-culture experiments (n=8) were set up with 2×104 irradiated autologous CD3 APCs, 5×103 CD25 CD4+ T cells and/or 1.5×104 CD25brightCD4+ cells (3:1 ratio of CD25bright:CD25 T cells) where indicated and stimulated with plate-bound anti-CD3 (0.5 μg/mL clone OKT-3) and cultured in RPMI 1640 supplemented with 5% heat-inactivated AB autologous serum, penicillin (100 U/mL), streptomycin (100 μg/mL), 2 mM L-glutamine, 10 mM HEPES. Some cells were cultured in the absence or presence of tocilizumab (100 μg/mL, Chugai Pharmaceuticals, Tokyo, Japan) or adalimumab (100 μg/mL, Abbott Laboratories, Abbott Park, IL, USA). All cells were incubated at 37°C for 6 days, the last 15–18 h in the presence of 3H thymidine. Cytokines in cell culture supernatants were detected using the Bio-Plex™ cytokine detection array (Bio-Rad, Hercules, CA, USA). The panel included the cytokines IL-6, IL-10, IL-13, IL-17A, IFN-γ and TNF and was run according to manufacturer's instructions.

ELISA

Cytokines (IL-1β, IL-6 and TNF) in SF of the same patients, where FOXP3 analysis was carried out, were assessed by sandwich ELISA. For IL-6 ELISA capture, anti-human IL-6 (clone MQ2-13A5) and detection biotin-conjugated anti-IL-6, clone MQ2-39C3 (Pharmingen (BD)) was used and for TNF and IL-1β ELISA kits were used according to manufacturer's instructions (Biosource, Invitrogen, USA). Briefly, SF samples were added in duplicates to 96-well plates pre-coated with antibodies against IL-6, TNF or IL-1β and incubated. The plates were then washed and incubated with biotinylated detection antibody, washed and finally streptavidin-horseradish peroxidase was added. After the addition of TMB (3,3′,5,5′-tetramethylbenzidine) (Bioscource), color development was measured at 490 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA) and the amount of cytokine in the samples was calculated using the SOFT max PRO software (Molecular Devices).

Analysis of DNA methylation levels of the FOXP3 promoter

Isolated CD4+CD25 and CD4+CD25bright T cells from SF (n=6) or CD4+CD25 and CD4+CD25high T cells from PB (n=5) from male arthritis patients were screened for the methylation status of the evolutionarily conserved CpG position –77 in the FOXP3 promoter region. Additionally, SF CD4+CD25bright CD25int and CD25 T cells from five longitudinal samples from one patient during a 4-year period were analyzed in the same way. Methylation analysis was conducted using bisulphate-sequencing 4. Genomic DNA was isolated using phenol:chloroform:isoamyl alcohol extraction (Sigma, St. Louis, MO, USA) following cell lysis (buffer, supplemented with Proteinase K (Qiagen, Valencia, CA, USA)). Extracted DNA was bisulphite converted using EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA) according to the manufacturer's instruction. The FOXP3 promoter region was PCR amplified using primers as previously described 4. PCR products were treated with exonuclease I and calf intestinal phosphatase (New England Biolabs, Ipswich, MA, USA) and used as template in the subsequent Methylation-Sensitive Single Nucleotide Primer Extension (Ms-SNuPE) reaction. The primer extension reaction was performed using SNaPshot Multiplex kit (Applied Biosystems, Foster City, CA, USA) and a probe directed toward the −77 CpG position. Primer extension products were loaded onto a 310 Genetic Analyzer (Applied Biosystems) and Gene Scan analysis was performed using the Gene Scan v3.7 application software (Applied Biosystems). The level of methylation was determined as the ratio of detected methylated DNA to total detected DNA in the fragment analysis.

Intracellular cytokine staining

Ficoll-separated SF cells from six patients diagnosed with arthritis were cultured in RPMI 1640 supplemented with 5% heat-inactivated AB autologous serum, penicillin (100 U/mL), streptomycin (100 μg/mL), 2 mM L-glutamine, 10 mM HEPES. The cells were cultured in the presence or absence of plate-bound anti-CD3 (1.0 μg/mL clone OKT-3) for 8 h. Brefeldin A (10 mg/mL) was added in the last 5 h of stimulation. Cells were harvested, washed and stained for surface markers using the following fluorochrome-conjugated antibodies: CD3-Cascade Yellow (Dako, Glostrup, Denmark), CD4-FITC, CD25-allophycocyanin, CD14-PerCp (all BD). Cells were washed twice, fixed and permeabilized using FOXP3 fixation/permeabilization solutions and buffers for FOXP3 staining (eBioscience). Briefly, FOXP3 fixation/permeabilization solution was added to the cells and incubated for 30 min on ice in the dark followed by two washes with the permeabilization buffer. Cells were then stained for IL-17-PE (clone P3, eBioscience), IFN-γ-PECy7 (clone B27, BD), Ki-67-Alexa Fluor 700 (clone B56, BD) and FOXP3-Pacific Blue (clone 206D, Biolegend, San Diego, CA, USA) or isotype-matched control antibody. Stained cells were acquired on a Gallios instrument (Beckman Coulter) and data were compensated and analyzed with the FlowJo software (Treestar).

Statistical analysis

Wilcoxon signed-rank test was used to evaluate statistics between paired patient samples. Correlation between data points was carried out using the Pearson or Spearman correlation analysis (Graphpad software, San Diego, CA, USA).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

The authors thank the staff and patients at the Rheumatology Clinic of Karolinska University Hospital, Eva Jemseby for organizing the sampling, storage, and administration of biomaterial, and Annika van Vollenhoven for excellent cell sorting. This study is supported by grants from the Margaretha af Ugglas Foundation, the Swedish Association against Rheumatism, the Swedish Medical Association, the King Gustaf V 80 year Foundation, the Swedish Research Council and the EU FP6 program AutoCure and EU FP7 program Masterswitch.

Conflict of interest:

The authors declare no financial or commercial conflict of interest.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
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