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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

A distinct subset of proinflammatory CD4+ T cells that produce interleukin-17 was recently identified. These cells are implicated in different autoimmune disease models, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, but their involvement in human autoimmune disease has not yet been clearly established. The purpose of this study was to assess the frequency and functional properties of Th17 cells in healthy donors and in patients with different autoimmune diseases.

Methods

Peripheral blood was obtained from 10 psoriatic arthritis (PsA), 10 ankylosing spondylitis (AS), 10 rheumatoid arthritis (RA), and 5 vitiligo patients, as well as from 25 healthy donors. Synovial tissue samples from a separate group of patients were also evaluated (obtained as paraffin-embedded sections). Peripheral blood cells were analyzed by multiparameter flow cytometry and immunohistochemistry. Cytokine production was examined by enzyme-linked immunosorbent assay and intracellular cytokine staining using specific monoclonal antibodies. Synovial tissue was examined for infiltrating T cells by immunohistochemical analysis.

Results

We found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (PsA and AS), but not in patients with RA or vitiligo. In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor–driven cytokine production.

Conclusion

These observations suggest a role of Th17 cells in the pathogenesis of certain human autoimmune disorders, in particular the seronegative spondylarthritides.

Recent studies have provided compelling evidence for a third CD4+ T cell effector subset besides the well-described Th1 and Th2 CD4+ T cells. The newly identified CD4+ T effector cells have been called Th17 cells (1, 2) based on their ability to secrete interleukin-17 (IL-17) (3). Through the ubiquitously expressed IL-17 receptor, IL-17 induces the secretion of proinflammatory cytokines, stimulates osteoclast formation and bone resorption (4), recruits neutrophils and monocytes, and triggers the production of granulocyte–macrophage colony-stimulating factor (5, 6). Th17 cells have been implicated in the host defense against various extracellular bacteria and fungi in mice and, more recently, in the antifungal immune response in humans (7–10). Th17 cells have been shown to be critical in the pathogenesis of various murine autoimmune diseases (11–13).

Rheumatoid arthritis (RA) and the seronegative spondylarthritides (SpA) are common chronic inflammatory systemic diseases of unknown origin, affecting mainly the joints. Their exact pathogeneses are still not fully understood. While some pathways of joint damage are common to both diseases, as demonstrated by the efficacy of tumor necrosis factor α (TNFα)–blocking agents (14, 15), RA and SpA are clearly different diseases that can be distinguished according to their clinical, radiologic, and serologic features (16).

IL-17 is believed to play a role in RA (17), and Th17 cells are involved in collagen-induced arthritis, a classic model of RA (18). To address the role of IL-17–producing T cells in humans, we examined the frequency and phenotype of Th17 cells in patients with chronic inflammatory arthritis and in patients with vitiligo. While we failed to demonstrate elevated levels of Th17 cells in the peripheral blood of patients with RA or vitiligo, as compared with healthy donors, we observed significantly increased levels in patients with ankylosing spondylitis (AS) and those with psoriatic arthritis (PsA). Furthermore, Th17 cells from patients with SpA were more differentiated toward effector cells and contained a distinct CCR6− subset. These findings suggest a direct involvement of Th17 cells in the pathogenesis of human SpA and may have novel therapeutic implications.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Cells.

Peripheral blood was obtained upon written informed consent from 10 PsA, 10 RA, 10 AS, and 5 vitiligo patients. Synovial fluid was also collected from 1 RA patient, and the cells were isolated as described for the blood samples. Paraffin-embedded synovial tissue sections from a separate group of RA patients were obtained from the Department of Pathology, University Hospital of Lausanne. As controls, peripheral blood was obtained from 25 healthy donors at the blood transfusion center (CHUV, Lausanne, Switzerland). Cord blood samples obtained from umbilical cord veins immediately after delivery of the placenta and thymus samples from children who had undergone corrective cardiac surgery were obtained from the University Hospital of Ghent (Ghent, Belgium). Mononuclear cells were purified and immediately frozen, as described previously (19).

Antibodies and flow cytometry.

Monoclonal antibodies were obtained from Becton Dickinson (Basel, Switzerland), except for anti-human IL-17 and anti-human forkhead box P3 (FoxP3) antibodies, which were from eBioscience (San Diego, CA), and anti-human CD39, which was from Abcam (Cambridge, UK). All monoclonal antibodies were used according to the manufacturers' recommendations.

CD4+ T cell clones.

CD4+CCR6+ T cells were isolated from peripheral blood by flow cytometry–based cell sorting and were then cloned by limiting dilution. Clones were screened for the release of IL-17 by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Abingdon, UK) and by intracellular cytokine staining (IL-17 and interferon-γ [IFNγ]) after 24 hours of stimulation with solid-phase anti-CD3 (OKT3, 1 μg/ml) and soluble anti-CD28 (CK248, IgM, 1 μg/ml) antibodies. For intracellular cytokine staining, 10 μg/ml of brefeldin A (Sigma-Aldrich, Buchs, Switzerland) was added after the first 2 hours of stimulation.

For immunohistochemical detection of intracellular IL-17 and IFNγ, clones were stimulated for 24 hours with 1 μg/ml of phorbol myristate acetate (Sigma-Aldrich) and 0.25 μg/ml of ionomycin (Sigma-Aldrich) on multichamber slides coated with poly-L-lysine (Sigma-Aldrich). Brefeldin A (10 μg/ml) was added after the first 2 hours of stimulation. After stimulation, cells were fixed with 4% paraformaldehyde, washed with phosphate buffered saline–2 mM EDTA (Gibco, Basel, Switzerland), and stained with goat anti-human IL-17 (R&D Systems) or mouse anti-human IFNγ (BioSource, Nivelles, Belgium) antibodies.

Ex vivo detection of cytokine-secreting cells.

In preliminary experiments, we ruled out the existence of directly detectable IL-17+ T cells in unstimulated lymphocytes from both the patients and the healthy donors. Subsequently, we stimulated T cells with solid-phase anti-CD3 (OKT3, 1 μg/ml) and soluble anti-CD28 (CK248, IgM, 1 μg/ml) antibodies. We identified a stimulation time of 24 hours as being optimal for the reliable detection of IL-17–producing T cells. We therefore hereinafter refer to these cells as ex vivo–detectable Th17 cells, as opposed to cells that become detectable only after several weekly rounds of stimulation.

Briefly, for detection of ex vivo cytokine–secreting T cells, peripheral blood lymphocytes (PBLs) were stimulated at 37°C for 24 hours in the presence or absence of coated anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies; 10 μg/ml of brefeldin A was added after the first 2 hours of stimulation. Cells were stained with cell surface antibodies, fixed, and then stained with anti–IL-17, anti–IL-2, anti-TNFα, and anti-IFNγ antibodies in the presence of 0.1% saponin (Sigma-Aldrich).

Immunohistochemical analysis of synovial tissue–infiltrating T cells.

Sections measuring 4 μm in thickness were prepared on glass slides from paraffin-embedded formaldehyde-fixed material. Sections were then deparaffinized in xylene and rehydrated through graded alcohols. Antigen retrieval was performed by heating under pressure in a microwave oven for 5 minutes in Tris–EDTA buffer (pH 9). The tissues were stained with mouse anti-human CD4 (Novocastra, Newcastle-upon-Tyne, UK) or goat anti-human IL-17 (R&D Systems) antibodies, followed by incubation for 30 minutes at room temperature with horseradish peroxidase–conjugated anti-mouse (Dako, Zug, Switzerland) or horseradish peroxidase–conjugated anti-goat (Histofine Bioscience, Tokyo, Japan), respectively, and diaminobenzidine (Dako). Finally, the tissues were counterstained with hematoxylin and dehydrated through graded alcohols and xylene.

CCR6+ and CCR6− cell sorting and in vitro stimulation.

Purified PBLs from the arthritis patients and the healthy donors were stained using a CCR6 monoclonal antibody (BD Biosciences, Basel, Switzerland). CCR6+ and CCR6− cells were sorted using a FACSVantage sorter (BD Biosciences). Sorted cells were stimulated for 24 hours and successively stained intracellularly as reported for the ex vivo experiments.

Proliferation assay.

Flow cytometry–based sorted CD25–CD127+CD4+ responder T cells or CD25+CD127–CD4+ Treg cells (4 × 104) were cultured for 5 days either alone or in coculture at a 1:1 ratio in triplicate with irradiated allogeneic PBLs. 3H-thymidine was added for the last 18 hours of culture. Cells were harvested, and the scintillation counts were measured.

Statistical analysis.

The significance of the results was determined using Student's t-test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Expansion of Th17 clones by T cell receptor–driven activation of human circulating CD4+ T cells.

Characterization of human T cells at the single-cell level is important for optimizing the methods for direct ex vivo monitoring of human circulating lymphocytes. In order to monitor human Th17 cells directly ex vivo with a validated labeling approach, we first sorted circulating CD4+ T cells from different donors and cloned them in the presence of low-dose IL-2. Clones were tested for their ability to secrete IL-17. First, in the IL-17 ELISA, a significant proportion of the clones were shown to produce detectable amounts of IL-17 upon stimulation for 24 hours with CD3-specific and CD28-specific antibodies (Figure 1A). In addition to IL-17, the majority of the clones tested also secreted TNFα and IFNγ when screened using a cytokine bead array, while the remaining clones were single producers of IL-17 (data not shown). IL-17–negative clones expressed a cytokine profile compatible with a Th1 phenotype (data not shown). Next, flow cytometric analysis of the clones was performed and showed similar results, confirming that the cytokines detected in the supernatant of stimulated clones could also be efficiently monitored by flow cytometry (Figure 1B). Finally, IL-17 and IFNγ were visualized in phorbol myristate acetate/ionomycin–stimulated Th17 and Th1 clones, respectively, using an immunohistochemical technique (Figure 1C). Together, these data suggest that IL-17 in human lymphocytes can be successfully detected using different approaches.

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Figure 1. Production of interleukin-17 (IL-17) by human CD4+ T cell clones. CD4+ T cell clones were assessed by enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry for their ability to secrete IL-17. A, Concentrations of IL-17 secreted by IL-17+ clones from 1 donor and by 5 representative IL-17− clones. The threshold was arbitrarily set at 100 pg/ml. Cells were left unstimulated (not stim) or were stimulated with anti-CD3/anti-CD28 (αCD3/αCD28) for 24 hours. B, Representative dot plots showing the production of IL-17 and interferon-γ (IFNγ) by unstimulated or anti-CD3/anti-CD28–stimulated clones. C, Representative photomicrographs showing immunohistochemical staining of a Th1 and a Th17 clone from the same donor. Clones were left unstimulated or were stimulated with phorbol myristate acetate (PMA)/ionomycin and then stained with anti-human IL-17 and IFNγ antibodies (original magnification × 600). Insets, In parallel, clones were analyzed by flow cytometry, and histograms for IL-17 and IFNγ are shown.

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Increased frequency of ex vivo–detectable IL-17–secreting CD4+ T cells in PBLs from PsA and AS patients.

We then assessed the frequency of PBLs that released IL-17 in samples from 60 individuals: 25 healthy donors, 5 vitiligo patients, and 30 patients with chronic inflammatory arthritis (10 with PsA, 10 with RA, and 10 with AS). (Clinical characteristics of the PsA, AS, and RA patients are available online at http://www.lau.licr.org/pages/RG-DCOI.htm.) Remarkably, low, but distinctly detectable, numbers of IL-17–secreting CD4+ T cells were detected following 24 hours of stimulation with CD3-specific and CD28-specific antibodies in all PBL samples studied (Figure 2A). Overall, the frequencies of ex vivo–detectable IL-17–secreting cells in the total CD4+ T cell population varied from 0.03% to 1.16%. No significant IL-17 secretion was observed in CD8+ T cells (detection limit of 0.01%). However, as expected, both CD4+ and CD8+ T cells produced significant amounts of other cytokines, such as IFNγ and IL-2 (Figure 2A).

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Figure 2. Ex vivo detection of interleukin-17 (IL-17)–secreting CD4+ T cells. A and B, Representative dot plots showing ex vivo–detected T cells producing IL-17, interferon-γ (IFNγ), and IL-2 in total human peripheral blood lymphocytes from healthy donors (HD), arthritis patients, and vitiligo patients (A) and from human thymus, cord blood, and peripheral blood (B). Values in the upper right compartment are percentages. C, Frequency of IL-17+, IFNγ+, and IL-2+ cells among total CD4+ T cells from healthy donors and patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS), rheumatoid arthritis (RA), and vitiligo (Vit). Bars show the mean. ∗ = P < 0.05; ∗∗ = P < 0.005; ∗∗∗ = P < 0.001 by Student's t-test. D, Representative photomicrographs showing immunohistochemical detection of CD4+ T cells and IL-17+ T cells in sections of synovial tissue from RA patients (original magnification × 200).

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We also investigated whether IL-17+ cells were present in thymus or cord blood samples from healthy donors. No IL-17+ cells could be directly detected by flow cytometric analysis (Figure 2B), whereas IFNγ-secreting and IFNγ- and IL-2–secreting CD4+ T cells were readily detectable ex vivo in thymus and cord blood, respectively (Figure 2B).

Both PsA patients and AS patients showed significantly increased frequencies of circulating Th17 cells (mean 0.65% and 0.49% of total CD4+ T cells, respectively) as compared with healthy donors (mean 0.15%; P < 0.001) and with RA patients (mean 0.23%; P = 0.009) (Figure 2C). No significant difference was observed for RA and vitiligo patients as compared with healthy donors. For patients with arthritis, no correlation between Th17 cell frequencies and disease activity, C-reactive protein level, or erythrocyte sedimentation rate was found, indicating that increased Th17 cell frequencies in SpA patients were disease specific rather than attributable to differential disease activity.

With regard to other cytokines, the frequency of IFNγ-secreting CD4+ T cells was also significantly higher in PsA patients and AS patients (5.9-fold and 4.3-fold increase, respectively, as compared with healthy donors) (Figure 2C). In addition, when compared with healthy donors, the patients with arthritis, irrespective of the arthritis type, displayed significantly increased levels of circulating IL-2–secreting cells (Figure 2C).

We also analyzed synovial fluid cells from 1 RA patient and observed that the frequency of IL-17–secreting cells in synovial fluid was comparable with that in PBLs (0.20% and 0.15% of total CD4+ T cells, respectively). We additionally performed immunohistochemical analyses of paraffin-embedded synovial tissue specimens and confirmed the presence of an infiltrate of CD4+ T cells loaded intracellularly with IL-17 (Figure 2D).

Finally, IL-17 could not be detected by ELISA in any of the serum samples from either the healthy donors or the patients (lower detection limit 15 pg/ml) (data not shown).

Increased frequencies of differentiated Th17 cells in PBLs from PsA and AS patients.

Multiparameter flow cytometric analysis with specific antibodies for cytokines and surface antigens (e.g., CD45RA, CCR7, CD28, CD27) allowed further ex vivo characterization of Th17 and Th1 cells from the arthritis patients and healthy donors. We found definite differences in circulating Th17 phenotypes between the patients with SpA and the healthy donors and RA patients (Figure 3).

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Figure 3. Phenotypic characterization of ex vivo–detected interleukin-17 (IL-17)–secreting and interferon-γ (IFNγ)–secreting CD4+ T cells. A, Representative dot plot showing CCR7 and CD45RA expression on ex vivo–detected IL-17+ CD4+ T cells (left), and frequency of naive (CCR7+/CD45RA+), central memory (CM; CCR7+/CD45RA–), and effector memory (EM; CCR7–/CD45RA–) cells in Th17 cells from healthy donors (HD) and patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA) (right). B, Representative dot plot showing CD27 expression on naive and memory Th17 cells (left) and frequency of highly differentiated effector memory CD27− cells among Th17 cells in healthy donors and patients with PsA, AS, and RA. C, Representative dot plot showing CCR7 and CD45RA expression by ex vivo–detected CD4+IFNγ+ T cells (left) and frequency of naive, central memory, and effector memory/effector cells among Th1 cells from healthy donors and patients with PsA, AS, and RA. D, Representative dot plot showing CD27 and CD28 expression on naive and effector memory Th1 cells (left) and frequency of highly differentiated effector memory/effector CD28–CD27− cells among Th1 cells from healthy donors and patients with PsA, AS, and RA. Bars show the mean. ∗ = P < 0.05; ∗∗ = P < 0.005 by Student's t-test.

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A small but significant population of CD45RA+ CCR7+ naive Th17 cells was observed in PBLs from the arthritis patients and healthy donors (Figure 3A). This fraction was reduced in PsA and AS patients (mean 2.7% and 2.2% of Th17 cells, respectively) but not in RA patients (mean 15.2% of Th17 cells), as compared with healthy donors (mean 9.6% of Th17 cells). Similarly, CD45RA–CCR7+ central memory Th17 cells were also significantly decreased in PsA and AS patients as compared with healthy donors and with RA patients. In parallel, a significant increase in the proportion of CD45RA–CCR7− effector memory cells was apparent in PsA and AS patients (mean 85.4% and 84.5% of Th17 cells, respectively), while no difference was detected in samples from RA patients (mean 55% of Th17 cells), as compared with healthy donors (mean 55.6% of Th17 cells) (Figure 3A).

Th17 cells were further characterized for expression of the costimulatory receptors CD27 and CD28, molecules that are down-regulated during CD4+ T cell differentiation. All cells expressed CD28 (data not shown). CD27 was homogeneously expressed by naive Th17 cells (Figure 3B), while different proportions of CD27+/CD27− cells were present in the memory Th17 cell compartment (Figure 3B). A population of highly differentiated effector memory CD27− Th17 cells was expanded in PBLs from both SpA patient groups (PsA and AS), while much lower and similar levels were observed in healthy donors and RA patients (Figure 3B). Moreover, memory Th17 cells expressed a 2-fold increase in intracellular IL-17 content as compared with their naive counterparts, as indicated by mean fluorescence intensity values (data not shown).

Phenotypic characterization of IFNγ-secreting CD4+ T cells from the arthritis patients and healthy donors was performed in parallel (Figure 3C). While PsA and AS patients showed a very similar phenotype, this time the RA patient phenotype was very different from that in healthy donors. In the patients, a significant reduction in naive IFNγ-secreting CD4+ T cells was observed as compared with cells from healthy donors. A higher proportion of effector memory and effector (CD45RA+CCR7–) Th1 cells was also detected in PBLs from the study patients, but this difference reached statistical significance only in the PsA patients (Figure 3C). Similar to the naive Th17 cells, IFNγ-secreting CD45RA+CCR7+ CD4+ T cells expressed both CD27 and CD28 molecules (Figure 3D). In contrast, CD27 and CD28 expression varied in central memory and effector/effector memory Th1 cells (Figure 3D), with a highly expanded population of effector memory/effector CD28 and CD27 double-negative Th1 cells in RA patients as compared with healthy donors, whereas PsA and AS patients demonstrated lower levels.

Overrepresentation of polyfunctional IL-17–secreting cells in PBLs from PsA and AS patients.

It has not yet been elucidated whether circulating Th17 cells in humans are able to secrete cytokines other than IL-17 (e.g., IL-2 or IFNγ). We could not detect significant proportions of cells secreting both IL-17 and IFNγ either in the healthy donors or in the arthritis patients (Figure 4A). In addition, none of the ex vivo–detected Th17 cells was able to secrete IL-22, IL-21, or granzyme B (data not shown). In contrast, a significant proportion of IL-17+ cells concomitantly produced IL-2, particularly in the arthritis patients (Figure 4A). Interestingly, for the PsA and AS patients, in whom the overall highest frequency of Th17 cells was observed (Figure 2C), up to 30% of the IL-17+ cells also secreted IL-2. In contrast, these IL-17/IL-2 double-positive cells represented only 5% and 19% of the total CD4+ T cells in healthy donors and RA patients, respectively (Figure 4B).

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Figure 4. Ex vivo detection of polyfunctional interleukin-17 (IL-17)–secreting cells. A, Representative dot plots showing the coproduction of IL-17 and interferon-γ (IFNγ), as well as IL-17 and IL-2, in CD4+ T cells from healthy donors (HD) and patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA). Dot plots are gated on CD4+ T cells. B, Proportion of IL-17 single-positive, IL-17/IL-2 double-positive, and IL-17/IFNγ double-positive cells in healthy donors and patients with PsA, AS, and RA. Total CD4+IL-17+ T cells represent 100%. C, Representative dot plots showing IL-17 and tumor necrosis factor α (TNFα) secretion in T cells from healthy donors and arthritis patients (top) and IL-2 secretion among CD4+IL-17+TNFα+ T cells from healthy donors and arthritis patients (bottom). Dot plots are gated on CD4+ T cells. D, Proportion of IL-17 single-positive, IL-17/TNFα double-positive, and IL-17/TNFα/IL-2 triple-positive cells in peripheral blood lymphocytes from healthy donors and arthritis patients. Total CD4+IL-17+ T cells represent 100%.

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Intracellular staining for TNFα revealed that in the arthritis patients, irrespective of the disease subtype, a high proportion of IL-17+ cells were TNFα+ after short-term polyclonal stimulation. Overall, 87% of Th17 cells from arthritis patients secreted TNFα, compared with only 47% of those from healthy donors (Figure 4C). In addition, a significant proportion of IL-17/TNFα double-positive cells from the arthritis patients also produced IL-2 (up to 20%), which was not seen in healthy donors (4%) (Figures 4C and D).

Heterogeneous expression of CCR6 in Th17 cells from PsA and AS patients.

Human Th17 cells may express a different pattern of chemokine receptors as compared with Th1 cells (7, 20). Reports of the overexpression of CCL20 and its receptor CCR6 in both RA (21) and PsA (22) patients prompted us to assess the expression of CCR6 in Th17 PBLs in the context of inflammatory diseases.

Ex vivo staining of PBLs with CCR6 revealed similar frequencies of CCR6+ cells among the CD4+ T cells in both healthy donors and arthritis patients (overall mean 10.7% CCR6+ cells among the total CD4+ T cells; n = 20 samples [5 from each study group]); a representative example is shown in Figure 5A. We successively performed ex vivo sorting of CCR6+ and CCR6− cells, followed by analysis of their cytokine secretion. Almost no CCR6− cells isolated from the peripheral blood of either the healthy donors or the RA patients secreted IL-17 (Figure 5B). In contrast, a non-negligible proportion of CCR6–CD4+ T cells from AS and PsA patients produced IL-17 (mean 0.13%). The frequency of IL-17–secreting CCR6–CD4+ T cells was significantly increased in SpA patients as compared with healthy donors and with RA patients, and represented up to 20% of the total IL-17+ CD4+ T cells in these patients (Figure 5C). However, in all of the samples tested, CCR6+ cells were more efficient in the production of IL-17 as compared with their CCR6− autologous counterparts, and no significant differences were observed in the frequency of Th17 cells among CCR6+ cells in healthy donors and arthritis patients. Further analysis of the phenotype of the CCR6+ and CCR6− IL-17+ cells from patients revealed the presence of both naive and memory Th17 cells in both the CCR6+ and the CCR6− subpopulations (data not shown).

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Figure 5. Th17 cells in the CCR6+ and CCR6− compartments. A, Representative dot plot for CCR6 and CD4 staining of peripheral blood lymphocytes ex vivo. B, Frequency of interleukin-17 (IL-17)–positive cells in sorted CCR6− and CCR6+ CD4+ T cells from healthy donors (HD) and patients with rheumatoid arthritis (RA) and the combined group of patients with psoriatic arthritis (PsA) and ankylosing spondylitis (AS). Bars show the mean. ∗∗ = P < 0.005; ∗∗∗ = P < 0.001 by Student's t-test. C, Percentage of CCR6− and CCR6+ cells among total IL-17–secreting CD4+ T cells from 5 healthy donors, 3 RA patients, and 4 patients in the combined group of PsA/AS. Values are the mean and SD.

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Normal numbers and function of Treg cells in PBLs from arthritis patients.

Recent studies in mice have suggested a mutually exclusive differentiation of Th17 cells and Treg cells (23). This prompted us to investigate whether FoxP3+CD4+ Treg cells were reduced in patients who had increased frequencies of circulating Th17 cells. Treg cell frequencies in RA and PsA patients were similar to those in healthy donors. However, a significant increase was observed in AS patients (6.4% of Treg cells among the total CD4+ T cells) (Figure 6A).

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Figure 6. Analysis of Treg cells in healthy donors (HD) and patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA). A, Frequency of forkhead box P3 (FoxP3)–positive cells in peripheral blood lymphocytes from healthy donors and patients with PsA, AS, and RA, shown as a representative dot plot for FoxP3 staining (left) and a graph summarizing the percentages of FoxP3+ cells in total CD4+ T cells (right). Bars show the mean. ∗∗∗ = P < 0.001 by Student's t-test. B, Phenotypic analysis of ex vivo–detected FoxP3+CD4+ T cells, using antibody conjugates specific for anti-CD25, anti-CD127, anti-CD27, and anti-CD39, shown as representative histograms for healthy donors and patients with PsA, AS, and RA. C, Inhibition of proliferation following coculture of purified CD25+CD127− Treg cells and CD25–CD127+ responder T cells (1:1 ratio) from 3 healthy donors and 3 arthritis patients (1 each with PsA, AS, and RA). Values are the mean and SD percentage inhibition of proliferation.

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We further characterized Treg cell differentiation and function, since it is possible that the Treg cell frequency could be normal but the function altered (24). No significant differences were observed in comparisons of the differentiation markers between the healthy donors and the arthritis patients (Figure 6B). Moreover, comparison of the proportion of CD39+ cells (25, 26) among the FoxP3+CD4+ T cells did not reveal any significant difference, despite a trend toward slightly lower frequencies of CD39+ cells in Treg cells in patients with AS (Figure 6B).

Finally, Treg cells (defined as CD4+CD25+CD127–) were sorted from selected patients and used for in vitro suppression assays. No major differences in the suppressive potential of Treg cells isolated from healthy donors (n = 3) and from arthritis patients (n = 3) was observed, although a slight decrease in suppression was observed in samples from the patients (Figure 6C).

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We identified increased frequencies of Th17 cells in PBLs from patients with seronegative spondylarthritides (AS and PsA), as compared with healthy donors and with RA patients. These cells exhibit a highly differentiated and polyfunctional phenotype in SpA, suggesting a specific role of Th17 cells in these diseases. Consistent with these results, increased levels of IL-17 in the sera of AS patients have been previously identified in studies using Quantikine ELISA (27). Unexpectedly, Th17 cell frequencies were comparable in RA patients and healthy donors, suggesting that Th17 cells may not be as central to the pathogenesis of RA as concluded from animal models (18). In this regard, an analysis of the levels of Th17 lymphocytes in the circulation of more than 100 RA patients was recently reported, and the results were very similar to ours. Moreover, the relative numbers of Th17 cells in synovial fluid were lower than those in the circulation (28).

Th17 cells may arise during immune responses against extracellular pathogens and fungal infections in mice as well as humans (6, 7, 10, 29, 30). The selective increase in the numbers of Th17 cells in patients with SpA is of special interest, knowing the strong association of this disease with HLA–B27 and infections (31, 32). Infections able to elicit Th17 responses may favor the development of autoimmune disease. Salmonella, Yersinia, Shigella, and Chlamydia have been implicated in reactive arthritis, another of the spondylarthritides (31, 32). Experiments in mice suggest a direct role of IL-17+ T cells in the host response to Klebsiella pneumoniae (33), a bacterium also associated with various autoimmune diseases, including SpA (34, 35). Finally, it has been speculated that elevated levels of serum antibodies to Saccharomyces cerevisiae reported in patients with AS, PsA, and undifferentiated SpA (36) might be related to Th17 cell involvement, as supported by their role in antifungal immune response in humans (7, 37).

Significantly higher frequencies of Th17 cells, both CD4+ and CD8+, have been reported in PBLs from patients with advanced ovarian carcinoma, as well as in tumor tissue from patients with various solid tumors (38). We detected no expansion of IL-17+ CD4+ or CD8+ T cells in PBLs or tumor-infiltrated lymph nodes from melanoma patients (data not shown) or in PBLs from 5 patients with vitiligo, a disorder thought to result from autoimmune destruction of melanocytes.

The presence of significantly elevated frequencies of both Th1 and Th17 CD4+ T cells in AS and PsA, but not in the other diseases studied, is an intriguing observation. Initial studies in mice suggested a common “pre-Th1” precursor that further differentiates into either Th1 or Th17 cells, depending on the cytokine milieu (39). Successive reports led to the proposition that Th17 cells represent a distinct subset of Th cells (1). Consistent with the findings of murine studies, we detected very low frequencies of IFNγ/IL-17 double-positive cells in healthy donors and in arthritis patients. This observation suggests that, in humans also, Th17 cells represent a unique and distinct CD4+ T cell population.

IFNγ seems to be able to interfere with the generation of Th17 at the level of naive precursors, but not once it is committed (1, 2), when IFNγ may even promote the expansion of Th17 cells through the generation of an inflammatory milieu. In this regard, the significantly elevated levels of both Th1 and Th17 subsets in SpA might be explained by an increased generation of Th17 cells prior to Th1 expansion. In addition to IFNγ+ cells, we found increased numbers of IL-2–secreting CD4+ T cells in the arthritis patients compared with the healthy donors. Published data concerning the role of IL-2 in Th17 homeostasis, predominantly suggesting an inhibitory role of IL-2 on Th17 survival (38, 40, 41), have been a subject of controversy. However, a recent study emphasizes the IL-2–mediated expansion of Th17 cells in patients with ocular inflammatory diseases (42), and our data might suggest a positive role of IL-2 in expanding and/or maintaining both Th1 and Th17 cells.

Little is known about the differentiation phenotype of IL-17+ cells in humans. Remarkably, we detected a significant proportion of circulating Th17 cells displaying a naive-like phenotype, in contrast with a recent study in which only memory Th17 cells were identified (7). This discrepancy is possibly attributable to the approach used to identify IL-17+ cells in the 2 studies. The expression of CD31 on a fair proportion of naive-like Th17 cells (data not shown), a marker that defines recent thymic emigrants (43), further supports the early generation of at least a proportion of Th17 cells as a distinct population directly in the thymus. The absence of detectable Th17 cells in fresh cell preparations from thymus and cord blood suggests that, while their frequency in these body compartments is too low to be detected by flow cytometry, peripheral expansion allows their direct ex vivo visualization in adult circulating lymphocytes. It is also possible that the state of differentiation in the thymus and cord blood does not allow a detectable production of IL-17 in these compartments.

As might be expected, the proportion of naive-like Th17 cells was decreased in AS and PsA patients, since the majority of the cells were differentiated, as compared with those in healthy donors and in RA patients. The observed expansion of memory Th17 cells was accompanied by a concomitant down-regulation of CD27, a pattern associated with cytotoxic terminally differentiated CD4+ T cells (44). Consistent with a hypothetical model in which Th1 and Th17 cells work in synergy to promote the development of autoimmune disease, we observed an expansion of effector memory cells as well as effector Th1 cells in SpA patients, with the simultaneous loss of CD28 and CD27 expression, a signature of a highly differentiated phenotype. Furthermore, Th17 cells from AS and PsA patients displayed concomitant secretion of multiple cytokines (i.e., IL-17, IL-2, and TNFα), and increased polyfunctionality may render those cells highly pathogenic. These observations are consistent with previous reports on virus-specific polyfunctional CD4+ and CD8+ T cells, which have been shown to correlate with effective antiviral immunity in humans (45–47). By analogy, polyfunctionality may account for disease severity and tissue damage in autoimmune diseases.

Recent studies have associated the absence of CCR5, as well as the expression of CCR6, with Th17 cells in healthy human PBLs (7, 20). While we confirmed the published data in healthy donors, we found significant frequencies of IL-17+ among sorted CCR6− CD4+ T cells in AS and PsA patients, but again, not in RA patients. CCR6 mediates T cell homing to skin and mucosal tissues (48), and its expression facilitates the recruitment of both dendritic cells and T cells in different diseases (49). The role of CCR6− Th17 cells in the pathogenesis of AS and PsA is far from obvious, knowing the importance of skin and mucosal tissues in SpA (31). They may represent a reservoir of IL-17+ T cells among PBLs; alternatively, they might be directly responsible for osteoarticular damage by reaching compartments such as enthesic sites via CCR6-independent migration.

Finally, we studied the relationship between IL-17+ and FoxP3+ CD4+ T cells in healthy donors and in arthritis patients. Evidence in mice suggests that Th17 cells and Treg cells differentiate in a mutually exclusive manner (23), while recent studies in tumor-bearing animals demonstrated a concomitant accumulation of both Th17 cells and Treg cells in the tumor stroma (38). To date, there is no clear evidence of a reciprocal Th17/Treg cell development in humans. In this regard, we analyzed the frequency, phenotype, and function of Treg cells in arthritis patients as compared with healthy donors. Our data indicate that Th17 and Treg cell populations can coexist in the circulation of healthy donors and arthritis patients. However, the increased frequencies of Th17 cells observed in patients with SpA may be sufficient to overrule any protective effect of Treg cells. Alternatively, different subtypes of Treg cells (e.g., natural versus induced Treg cells) might intervene at different time points during disease development and/or may be differentially susceptible to modulation by Th17.

Taken together, our findings suggest a role for Th17 cells in 2 types of SpA, but not in RA. Whether this observation holds true for all seronegative spondylarthritides remains to be determined. However, recent data on cytokine levels in sera and synovial fluid from patients with reactive arthritis seem to further support our observations (50). Numerous cytokines have been targeted in inflammatory rheumatic diseases, but with variable clinical benefit. In view of the recently reported data and our results, new approaches specifically targeting pathogenic Th17 cells may be of greater clinical benefit in patients with SpA.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Dr. Romero had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Jandus, Bioley, Dudler, Speiser, Romero.

Acquisition of data. Jandus, Bioley, Rivals, Dudler.

Analysis and interpretation of data. Jandus, Bioley, Rivals, Dudler, Romero.

Manuscript preparation. Jandus, Bioley, Rivals, Dudler, Speiser, Romero.

Statistical analysis. Jandus, Bioley.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We are thankful to the patients and healthy donors for their kind collaboration and blood donation. We would also like to thank Estelle Devêvre (Ludwig Institute for Cancer Research, Lausanne) for performing the flow cytometry–based cell sorting, Prof. L. Guilloud (Institute of Pathology, University Hospital, Lausanne) for providing synovial tissue sections, Prof. J. Plum and Dr. M. De Smedt (University Hospital of Ghent, Ghent, Belgium) for providing the thymus and cord blood samples, and Prof. A. So (Department of Rheumatology, University Hospital, Lausanne) for excellent collaboration and support for this project.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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