Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) constitutes a group of disorders characterized by autoimmune necrotizing inflammation of small blood vessels, which leads to systemic organ damage 1 . This group of systemic vasculitides includes Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), and Churg-Strauss syndrome (CSS). These disorders are predominantly associated with the presence of circulating ANCAs that are directed against proteins in the cytoplasmic granules of neutrophils. ANCAs with specificity for proteinase-3 (PR3-ANCA) are associated with WG to a high degree, whereas ANCAs with specificity for myeloperoxidase (MPO-ANCA) are predominant in MPA and to a lesser degree in CSS 2 . Although it remains unknown how these conditions develop, it has been postulated that ANCA in vivo bind to surface-expressed autoantigens (PR3 or MPO) on primed neutrophils, which enhances neutrophil degranulation and the release of toxic products that cause endothelial damage, ultimately leading to necrotizing vasculitis 2 .

In vivo experimental studies have clearly demonstrated that MPO-ANCAs are pathogenic factors. Xiao and colleagues have shown that immunization of MPO-deficient mice with mouse MPO results in an MPO-directed immune response, and transfer of splenocytes from these mice into immune-deficient mice leads to development of pauci-immune necrotizing crescentic glomerulonephritis and systemic necrotizing vasculitis reminiscent of MPA 3 . Further support for the pathogenicity of ANCA comes from a recent study by van Timmeren and coworkers 4 . They observed that administration of anti-MPO antibodies hydrolyzed by the bacterial enzyme endoglycosidase S, which abolishes IgG binding to Fcγ receptors, attenuated both neutrophil influx and formation of glomerular crescents in the above-described model of MPO-ANCA-induced glomerulonephritis. An immunopathogenic role for MPO-ANCA has also been strongly suggested by the occurrence of neonatal MPA in a child born to a mother with a history of MPO-ANCA-associated pulmonary renal syndrome 5 .

In contrast to MPO-ANCA, in vivo evidence is still lacking for a direct vasculitic pathogenicity of PR3-ANCA. So far only PR3-induced and PR3-ANCA-induced enhancement of inflammation has been demonstrated in an animal model 6 7 . Recent findings by Primo and colleagues suggest that, under certain conditions, anti-PR3 antibodies can be pathogenic in rodents 8 . They showed that adoptive transfer of splenocytes from PR3-immunized mice into NOD-SCID mice resulted in the appearance of circulating anti-PR3 antibodies and crescentic glomerulonephritis in the recipient mice. However, it is unclear whether glomerulonephritis in recipient mice is mediated by the humoral or the cellular arm of the anti-PR3 response.

Of note, infiltrating T cells in granulomatous lesions as well as persistent T-cell activation have been reported in AAV patients 9 10 11 12 . Interestingly, T-cell-depleting therapy with anti-CD52 antibodies (alemtuzumab) and anti-thymocyte globulin can induce remission in refractory AAV patients 13 14 . Moreover, the IgG subclass distribution of ANCA, predominantly consisting of IgG₁ and IgG₄, implies isotype switching of ANCA for which CD4 T-helper cells are required 15 . T-cell-mediated immunity is thus thought to contribute to the pathogenesis of ANCA-associated vasculitis. In the present review we will summarize the currently available data on the role of T cells in AAV. We shall first discuss current thoughts about the contribution of T cells to tissue injury in AAV. The main emphasis will then be on the plasticity of regulatory T cells (T_Regs), their transition into Th17 cells, and the involvement of Th17 cells in granuloma formation and disease progression.

Unlike other autoantibody-mediated diseases, AAV is characterized by an absence of deposited antibodies in affected tissue, in particular in glomeruli, designated as pauci-immune glomerular lesions 16 . Otherwise, immune effector cells such as CD4⁺ T cells, macrophages and granulocytes are enriched in granulomatous lesions 9 10 17 18 19 20 . This suggests a primary role of cell-mediated immunity in initiating granuloma formation. Studies in mice and humans have demonstrated a key role of CD4⁺ T cells in the generation of a granulomatous response. For instance, Saunders and colleagues have shown that CD4-deficient mice did not generate the typical mononuclear granulomatous lesions following Mycobacterium tuberculosis infection 21 . In humans, the extent of granuloma formation was correlated with peripheral CD4 T-cell counts in HIV patients with mycobacterial infection 22 23 . The important role of CD4 T cells in the expression of crescentic glomerulonephritis has been demonstrated by Ruth and colleagues 24 . They induced experimental anti-MPO-associated crescentic glomerulonephritis by immunizing C57BL/6 mice with human MPO followed by subsequent challenge with antiglomerular basement membrane antibodies. Mice depleted of CD4⁺ T cells at the time of administration of anti-mouse glomerular basement membrane developed significantly less glomerular crescent formation and less cell influx when compared with control mice. These data provide convincing evidence that CD4⁺ T cells are crucial in granuloma formation and glomerulonephritis.

Studies in AAV patients also support this notion. Proliferation of CD4⁺ T cells in response to the auto-antigens PR3 and MPO have been reported in patients with AAV, although CD4⁺ T cells from healthy controls also proliferated in response to PR3 and MPO, albeit to a lesser extent 25 . Persistent CD4 T-cell activation has also been observed in peripheral blood from AAV patients 26 27 28 . Importantly, Marinaki and colleagues observed an association between persistent CD4⁺ T-cell activation and disease severity in both WG patients and MPA patients 29 . Recently, Seta and colleagues evaluated the effect of depleting CD4 or CD8 T cells on the proliferative response to MPO fragments of peripheral blood mononuclear cells isolated from MPA patients 30 . Strikingly, proliferation was completely lost after the depletion of CD4⁺ T cells, but not after depletion of CD8⁺ T cells. In our studies in WG patients, we observed a persistent expansion of a subset of memory CD4⁺ T cells, termed effector memory T cells (T_EM), with a reciprocal decrease in naïve CD4⁺ T cells 11 . More-over, the CD8⁺ T-cell compartment also appears to be altered 31 32 . In accordance, infiltrating T cells in lung lesions and glomeruli were shown to consist mainly of CD4⁺ T cells with a memory phenotype 9 33 34 . Also in CSS patients, given the allergic background and hypereosinophilia in this disease, activated CD4⁺ T cells producing Th2 cytokines are believed to be the disease inducer 35 . Taken together, these findings indicate that CD4 T cells can serve as effector cells in the pathogenesis of AAV.

As mentioned above, several observations support the involvement of CD4⁺ T cells in the pathogenesis of AAV. Important evidence regarding their role in disease manifestations came also from the clinical observation that remission could be induced in WG patients by antibodies directed at T cells 36 . Indeed, an altered phenotype of CD4⁺ T cells has been found in AAV patients. An increased proportion of CD45RC ^LowCD4⁺ memory T cells was reported in peripheral blood of AAV patients 37 . In addition, an expanded population of CD4⁺ T cells lacking the co-stimulatory molecule CD28 was observed in peripheral blood and in granulomatous lesions of patients with WG 9 38 . These CD28^-CD4⁺ T cells display up-regulation of the T-cell differentiation marker CD57 and show intracytoplasmic perforin expression, indicating the cytotoxic potential of these cells 9 . Based on phenotype and functional characteristics, CD28^- T cells have been classified as a T_EM population that lacks the chemokine receptor CCR7 39 .

Consistent with these findings, we observed a significant increase in the frequency of circulating CD4⁺ T_EM (CD45RO⁺CCR7^-) in WG patients in remission compared with healthy individuals 11 . In addition, we have shown that the number of these circulating CD4⁺ T_EM decreases during active disease compared with that during complete remission, which is consistent with their migration towards inflamed tissues 11 . Indeed, our cross-sectional and follow-up studies confirmed migration of CD4⁺ T_EM during active renal disease into the diseased organs 40 . We observed a remarkable increase in CD4⁺ T_EM in the urinary sediment with a concomitant decrease of circulating CD4 ⁺ T_EM of WG patients with active renal involvement 40 . These urinary CD4⁺ T_EM decreased or disappeared from the urine during remission, which might reflect their role in renal injury. In accordance with these findings, Wilde and colleagues demonstrated that CD4⁺ T_EM expressing CD134 are expanded in peripheral blood of patients with WG 41 . CD134 is thought to contribute to T-cell migration and tissue infiltration through its interaction with OX40L on vascular endothelial cells 42 . Indeed, Wilde and coworkers have shown that CD134-expressing T cells were localized within the inflammatory lesions of WG patients, supporting our hypothesis on migration of this T-cell subset to inflamed sites 41 .

As mentioned, CD4⁺ T_EM display natural killer (NK)-like features such as cytotoxicity 39 . They also mimic NK cells by their surface expression of the NKG2D molecule. NKG2D is an activating C-type lectin-like homodimeric receptor, which differs from other NKG2 members as it apparently lacks an antagonist and substitutes for CD28-mediated co-stimulatory signaling in CD28^- T_EM 43 . One of the NKG2D ligands is the major histocompatibility complex class-I chain-related molecule A (MICA), which is expressed upon cellular injury and stress on target cells such as fibroblasts and epithelial cells 43 . Proof of concept for NKG2D-mediated tissue destruction was provided by Allez and colleagues 44 , who demonstrated that NKG2D⁺CD4⁺ T-cell clones from patients with Crohn's disease kill target cells that express MICA via NKG2D-MICA interaction. Importantly, MICA is upregulated in peritubular endothelium and glomerular epithelial cells in AAV patients during active renal disease 45 . Strikingly, Capraru and colleagues have shown that NKG2D is preferentially expressed on expanded CD28^-CD4⁺ T cells in the peripheral blood of WG patients 31 . Next, they showed that both NKG2D and MICA are expressed in granulomatous lesions in WG, but not in disease controls. Killing mechanisms via NKG2D-MICA interaction therefore probably contribute to vessel injury and disease progression in AAV patients (Figure 1). Accordingly, selective targeting of NKG2D⁺CD4⁺ T_EM or inhibiting MICA expression without impairing other parts of cellular immunity might have value in the treatment of AAV 46 .

Aberrant T-helper cell polarization has been described in AAV. Analysis of soluble markers for T-helper cell subsets in patient sera reveals a predominance of the Th1 pattern with expression of IFNγ and sCD26 in patients with localized WG and in patients with MPA, whereas a shift towards a Th2 pattern, with expression of IL-4, IL-5, IL-10, IL-13, sCD23, and sCD30, was observed in active generalized WG and CSS 47 48 . The same results were obtained from analysis of nasal granulomatous lesions in which abundant Th1-associated markers (IFNγ, sCD26, CCR5) were seen during localized WG, whereas Th1-associated and Th2-associated markers (IL-4 and CCR3) are found in generalized WG 20 49 . Besides the balance between Th1 and Th2, a recent breakthrough has revealed that IL-17-secreting T cells (Th17) are another major pathogenic effector subset involved in the induction of inflammation and autoimmunity 50 51 . It has been reported that induction of experimental autoimmune encephalomyelitis (EAE) was blocked in mice deficient in either IL-17 or the Th17 polarizing cytokine IL-23, whereas mice deficient in either IFNγ or the Th1 polarizing cytokine IL-12 show increased susceptibility to EAE 50 52 53 . Interestingly, Th17 cells in EAE infiltrate the brain prior to the onset of clinical symptoms, whereas Th1 cells dominate the cellular infiltrate thereafter when clinical disease develops 54 . It seems that T-cell-mediated disease manifestations are linked to Th17 cells and not primarily to Th1 responses.

The physiological role of Th17 cells lies in bacterial defense - for example, against Staphylococcus aureus - as shown in experimental pneumonia and the hyper-IgE syndrome 55 56 . Peptidoglycans as well as superantigens from S. aureus might have an immunomodulatory effect on dendritic cells by imprinting of a strong Th17 polarizing capacity 57 . In addition, S. aureus α-toxin was shown to induce IL-17A secretion in CD4 T cells 58 . Intriguingly, nasal S. aureus co-localization has been reported to be related to relapse and correlates with endonasal activity in WG 59 60 . Infection with S. aureus might therefore drive a Th17 response in AAV patients. Indeed, in patients with AAV we observed a skewing towards Th17 cells following in vitro stimulation of peripheral blood samples 61 . In line with this observation, Ordonez and coworkers have shown that the expanded CD4⁺ memory T cells in AAV patients are a source of IL-17 37 . Most importantly, we found a relative increase in autoantigen-specific Th17 cells in ANCA-positive patients in comparison with ANCA-negative patients and controls 61 . This observation suggests involvement of Th 17 cells in the process of autoantibody production in AAV. These results were corroborated by Nogueira and colleagues, who reported elevated levels of serum IL-17A and increased autoantigen-specific Th17 cells in AAV patients during disease convalescence compared with healthy controls 62 . In addition, Saito and colleagues observed an increased frequency of circulating Th17 cells in patients with active CSS compared with in patients with inactive disease and healthy controls 63 .

IL-17 has been reported to promote the release of pro-inflammatory cytokines, which are essential for triggering the expression of PR3 and MPO on the surface of neutrophils (priming of neutrophils), and also to induce CXC chemokine release and expression of adhesion molecules responsible for the recruitment of neutrophils to the site of inflammation 64 65 66 . Indeed, WG granulomata (site of inflammation) are rich in neutrophils 20 . On the other hand, IL-17 has been shown crucial for the formation of an autoreactive germinal center in autoimmune BXD2 mice 67 . IL-17-producing T cells and B cells expressing the IL-17 receptor have also been reported to localize together in germinal centers 67 . This observation suggests that IL-17 not only induces tissue inflammation but also could function on B cells to promote the germinal center reaction. The lymphocyte clusters in granuloma structures can resemble germinal center-like structures that might be induced by IL-17 and may provide a place for ANCA production. IL-17 thus seems to be an important player in disease development in AAV and in early granuloma formation in WG, whereas Th1 and Th2 cells might prevail in later stages 20 49 . Of note, Th17 cells have not so far been demon-strated at inflamed sites in AAV.

Natural T_Regs, a subset of thymus-derived CD4⁺ T cells expressing a high level of IL-2Rα (CD25) and a unique transcription factor FoxP3, have been shown critical for preventing autoimmune responses. Defects in T_Reg function or reduced numbers of T_Regs have been documented in several autoimmune diseases 68 . Indeed, we found that the suppressive function of T_Regs was defective in WG patients as compared with healthy controls 69 . In this group of patients, however, we observed a significant increase of memory FoxP3 ⁺CD25^High T_Regs. In line with these findings, Klapa and colleagues demonstrated an increased number of FoxP3 ⁺ T cells as well as phenotypical and functional alterations of T_Regs in WG patients 70 . They reported an increased number of interferon receptor I-positive T_Regs in the peripheral blood of WG patients 70 . In addition, they showed that IFNα exaggerates functional T_Reg impairment ex vivo in response to the autoantigen PR3 70 . T_Regs in WG patients might thus display functional anergy in the context of an inflammatory cytokine milieu.

Altered T_Reg function in WG patients has also been reported by Morgan and coworkers 71 . They observed that T_Regs from healthy controls and from ANCA-negative patients were able to suppress T-cell proliferation to PR3, whereas T_Regs from ANCA-positive patients failed to suppress this autoimmune response 71 . Dysfunction of T_Regs is thus believed to play a role in the development of WG. In contrast, T_Reg function in MPA patients was comparable with that in healthy controls, but FoxP3 levels were diminished in MPA patients 72 . MPA seems to be associated with a numerical deficiency rather than a functional deficiency of T_Regs. Moreover, studies in CSS patients showed that both patients and controls have a similar number of CD25⁺CD4⁺ T cells with an equal percentage of FoxP3- expressing cells. However, the suppressive function of T_Regs in CSS patients still needs to be investigated 63 73 .

A reciprocal relationship in the development of T_Regs and Th17 cells has recently been described. This may underlie the propensity of T_Regs to convert to Th17 cells in the context of proinflammatory stimuli, a phenomenon that has only recently been recognized 74 75 76 . Under neutral conditions in vitro, transforming growth factor beta can shift the balance towards functional FoxP3⁺ T_Regs - whereas in the context of an inflammatory cytokine milieu (IL-1β, IL-2, IL-6, IL-15, IL-21, IL-23), functional T_Regs convert towards IL-17-producing, nonfunctional T_Regs. The relatively novel notion of T-cell lineage plasticity is of interest in relation to many papers describing nonfunctional T_Regs in several autoimmune conditions, including AAV. Our hypothesis is that these nonfunctional FoxP3 ⁺ T cells have lost their suppressive function due to co-expression of a second Th17 lineage-associated transcription factor RORγt that interferes with Foxp3 activity 77 . Recently, different isoforms of FoxP3 have been investigated in human T_Regs that have been shown to impact T_Reg function and lineage commitment. More specifically, the full-length isoform (FoxP3fl) - but not the isoform lacking exon 2 (FoxP3Δ2) - interacts with RoRγt and inhibits the expression of genes that define Th17 cells 78 79 80 . Based on the aforementioned data, the putative nonfunctional T_Regs described in AAV may lack their suppressive function due to upregulation of the FoxP3Δ2 isoform that fails to inhibit RORγt-mediated IL-17A mRNA transcription. Upon stimulation in an inflamed context, these cells convert into IL-17-producing effector T cells.

Evidence from several groups of investigators, including our own, support this hypothesis of conversion of T_Regs into effector IL-17-secreting cells in AAV. As mentioned before, we found a significant increase in the percentage of FoxP3 ⁺CD25^High T_Regs with a defective regulatory function in AAV patients in remission as compared with healthy controls. Furthermore, we demonstrated a concurrent increase in the percentage of Th17 cells upon in vitro stimulation of peripheral blood samples from AAV patients. Consistent with this, patients with AAV had significantly higher serum levels of IL-17 compared with healthy controls 62 . Importantly, increased serum levels of IL-17 in AAV patients correlated significantly with increased levels of the cytokines that are involved in the conversion of T_Regs into Th17 cells; that is, IL-1β, IL-23 and IL-6 62 . In addition, patients with active CSS showed an increased frequency of Th17 cells with a decrease in the frequency of IL-10-producing T_Regs, whereas an inverse result was observed in CSS patients with inactive disease 63 . The aforementioned data appear to support a link between the conversion of T_Regs into Th17 cells and disease activity in AAV (Figure 1).

Because Th17 cells contribute to inflammation and granuloma formation, this T-cell subset could be a novel therapeutic target for AAV. Depletion of Th17 cells by targeting specific surface proteins may be difficult as Th17 cells share many surface markers with other T-cell subsets. A therapeutic approach targeting its cytokine (that is, IL-17) would therefore be more feasible. Indeed, neutralizing IL-17 by anti-IL-17 antibody or by soluble IL-17 receptors reduces inflammation and bone erosion in various animal arthritis models 81 . Interestingly, humanized anti-IL-17 mAbs - including AIN457 and LY2439821, which neutralize the biologic activity of IL-17 - are in clinical trials. These biologicals have been shown to induce clinically relevant responses in patients with psoriasis, rheumatoid arthritis, and non-infectious uveitis, compared with placebo without safety issues 82 83 . Neutralization of IL-17 could therefore represent a novel therapeutic approach for patients with AAV.

On the other hand, CD4⁺ T_EM - supposed to act as a key trigger of disease expression and relapse in AAV - may also serve as a therapeutic target. Selective targeting of CD4⁺ T_EM without impairing other parts of the humoral and cellular immune system could be a major step forward in the treatment of AAV. NKG2D blockade by anti-NKG2D antibodies has been reported to prevent autoimmune diabetes in NOD mice 84 . Blocking of NKG2D could be a new strategy in the treatment of AAV. Other studies have revealed that targeting of the voltagegated Kv1.3 channel, which is highly expressed on activated CD4⁺ T_EM, provides a specific immunomodulatory approach 85 86 . Blockade of the Kv1.3 channel by ShK(L5) amide effectively prevented autoimmune disease in the EAE model of multiple sclerosis and suppressed delayed-type hypersensitivity in rats 85 86 . The selective targeting of CD4⁺ T_EM using ShK(L5) amide and/or blocking the NKG2D-MICA interaction by anti-NKG2D antibodies may therefore hold therapeutic promise for AAV.

CD4⁺ T_EM seem to be involved in tissue damage and renal injury in patients with AAV. Besides CD4⁺ T_EM, impaired T_Reg function and an increased Th17 response are also reported in AAV patients. During the past 2 years, multiple studies indicate a link between T_Regs and Th17 cells. Indeed, in the context of an inflammatory cytokine milieu, conversion of T_Regs into IL-17-producing cells has been demonstrated. Evidence from several studies supports this conversion in AAV patients. Defective T_Reg function in AAV patients can thus be explained by their conversion into effector Th17 cells. Instead of suppressing autoreactive responses, these converted T_Regs - through production of IL-17 - can participate in granuloma formation and tissue injury, which contribute to necrotizing granulomatous vasculitis in AAV patients. The mechanisms underlying the conversion of suppressive T_Regs into nonfunctional T_Regs in AAV await further investigation. This novel view into the role of converted T_Regs in the pathophysiology of vasculitis will improve our understanding of AAV pathogenesis, which may lead to the identification of new biomarkers and targets for therapeutic intervention.

AAV: anti-neutrophil cytoplasmic autoantibody-associated vasculitides; ANCA: anti-neutrophil cytoplasmic autoantibody; CSS: Churg-Strauss syndrome; EAE: experimental autoimmune encephalomyelitis; FoxP3: transcription factor forkhead box P3; IFN: interferon; IL: interleukin; mAb: monoclonal antibody; MICA: major histocompatibility complex class-I chain-related molecule A; MPA: microscopic polyangiitis; MPO: myeloperoxidase; NK: natural killer; PR3: proteinase-3; T_EM: effector memory T cells; Th: T-helper type; TNF: tumor necrosis factor; T_Reg: regulatory T cell; WG: Wegener's granulomatosis.

The authors declare they have no competing interests.

Autoimmune Basis of Rheumatic Diseases

This article is part of a review series on Vasculitis, edited by Cees Kallenberg, which can be found online at http://arthritis-research.com/series/vasculitis

This series forms part of a special collection of reviews covering major autoimmune rheumatic diseases, available at: http://arthritis-research.com/series/abrd