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Clin Exp Immunol. Apr 2005; 140(1): 181–191.
PMCID: PMC1809336

Abnormalities of CD4+ T cell subpopulations in ANCA-associated vasculitis

Abstract

In patients with ANCA-associated vasculitis (AAV), CD25 expression is increased on circulating T cells. Although in animal experiments the role of CD4+ CD25+ T-regulatory-cells (Treg) in protection against autoimmunity is well established, the role of these cells in AAV is unknown. To investigate the hypothesis that an increased expression of CD25 on T cells is related to persistent T cell activation and not to disturbances in Treg cells in AAV (34 patients, six of them after renal transplantation), we investigated CD25 expression in different subpopulations of CD4+ cells and FOXP3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). In addition, T cell proliferation and cytokine secretion after stimulation with anti-CD3 and anti-CD28 and intracellular cytokine production after stimulation with phorbol myristate acetate (PMA)-ionomycin was determined. Controls were non-vasculitic renal transplant patients (n = 9) and healthy controls (HC) (n = 13). In AAV the total number of lymphocytes, CD4+ lymphocytes and the percentage of naive T cells are lower than in HC and RTX. An increased percentage of CD25+ cells was found in AAV and AAV/RTX, irrespective of disease activity, but not in HC or RTX. This was confined to the naive (CD4+ CD45RBhigh) population only. FOXP3 mRNA expression in CD4+ T cells did not differ between AAV patients and healthy controls. In vitro T cell proliferation was enhanced in AAV patients compared to HC (P < 0·01). PBMC of AAV patients produced significantly less interleukin (IL)-10 and interferon (IFN)-γ after anti-CD3/CD28 stimulation. The percentage of IL-10 and IL-12, but not IFN-γ, IL-4 or tumour necrosis factor (TNF)-α-producing cells was significantly higher in patients compared to HC. These findings were confined to the memory population of CD4+ cells. We conclude that AAV patients are lymphopenic and have low numbers of CD4+ T cells, which seem to be in a persistent state of activation.

Keywords: ANCA, CD4 subsets, pathogenesis, T cells, vasculitis

Introduction

Although there is increasing evidence of a direct pathogenetic role of antiproteinase 3 (PR3) and antimyeloperoxidase (MPO) ANCA, which are associated strongly with AAV [1,2], evidence also accumulates for a pathogenetic significance of T cells in these autoimmune disorders. Several studies have highlighted the importance of regulatory T cells (Treg) in the pathogenesis of autoimmunity [3,4]. It is now generally believed that autoreactive T lymphocytes leave the thymus and can be found within the population of naive CD4+ CD45RBhigh T cells [5,6]. These cells are controlled or rendered inactive by CD4+ CD25+ Treg cells found in the memory or CD45RBlow population [58]. Interleukin (IL)-10 produced by the Treg cells plays an important role as mediator of suppression of T cell proliferation. Although the protective role of Treg cells in the development of autoimmunity has been demonstrated consistently in animal models, evidence is lacking that an imbalance of Treg cells also plays a role in human autoimmune diseases. Because Treg cells are important inhibitors of T cell activation and might have a functional relevance in maintenance of peripheral tolerance, a malfunction in these cells could theoretically be associated with the increased CD25 expression on lymphocytes, which is known to occur in AAV patients [8].

There have been several reports on abnormal T cell phenotype and function in patients with AAV. First, both monocytes and lymphocytes are found in inflammatory lesions as well as in granulomas of patients with Wegener's granulomatosis [9,10]. Secondly, the expression of CD25 is increased on circulating T lymphocytes in patients with AAV and plasma levels of soluble CD23, CD25, CD26 and CD30 are elevated, suggesting activation of the cellular immune response [1114]. Moreover, an increased expression of the co-stimulatory molecules CTLA-4, CD80 and CD86 on T lymphocytes has been reported in AAV patients, while the expression of CD28 in such patients was reported to be down-regulated [15,16]. Interesting in this respect is the finding that neutrophils from Wegener's granulomatosis patients express CD80 and CD86, hence acquiring characteristics of antigen presenting cells [17]. Thirdly, AAV patients benefit from therapeutic approaches that remove T lymphocytes [18,19], again indicating that the cellular immune response does play a role in the onset or perpetuation of this disease.

Inasmuch as persistent T cell activation has become an attractive hypothesis in vasculitis research to explain phenotypic alterations in peripheral blood mononuclear cells (PBMC) of AAV patients [1114], the cause of persistent T cell activation is poorly understood. It is also not known if the increased expression of CD25 on T cells in AAV is related to disturbances in Treg cells [13]. Therefore, in this study we first investigated in which subpopulation of CD4+ cells of AAV patients an increased CD25 expression can be found and whether expression of FOXP3, a transcription factor specific for Treg, in CD4+ cells, can be demonstrated. Secondly, we investigated whether T cell proliferation and cytokine production within these CD4+ subsets are different from healthy controls and thirdly, we investigated the role of accessory cells in T cell proliferation and cytokine production, especially interleukin 10 production, by anti-CD3/anti-CD28 stimulated PBMC.

Materials and methods

Patients

To establish functional and phenotypic differences between T cell populations we investigated 29 patients with AAV, six patients who received a kidney transplant with AAV as underlying disease (AAV + RTX), nine renal transplant recipients without underlying AAV (RTX) and 13 healthy controls (HC). In addition, two PR3-ANCA+ patients without evidence of AAV were studied (two male, 46 and 58 years, infectious endocarditis, treatment: ceftriaxone and gentamicin).

All patients with AAV [17 male, 12 female; mean age 54 ± 5 years (range 34–81)] had histologically proven disease and were defined according to the Chapel Hill Classification Criteria [20] as Wegeners's granulomatosis (n = 22) and microscopic polyangiitis (n = 7); 25 were cANCA positive with antigen specificity for PR3 and four were pANCA positive with antigen specificity for MPO. Four patients were analysed two times, once in remission and once during active disease. Thus, a total of 33 analyses of PBMC were performed in patients who were either in remission, with (n = 23) or without (n = 5) immunosuppression (IS), and in five patients with recently detected active disease (n = 5). Disease activity was defined by the Birmingham vasculitis activity score (BVAS) [21]. IS in patients with remission consisted of mycophenolate mofetil (n = 7), azathioprine (n = 7), 15-deoxyspergualin (n = 1) (22), cyclophosphamide (n = 4) and/or corticosteroids (n = 11). In patients with active disease oral cyclophosphamide and corticosteroids had been started only a few days (two patients were treated already for 2 days and three patients treated already for 3 days) before the analysis.

The AAV/RTX group [one male, five female; mean age 55 years (range 28–75)] consisted of patients with underlying AAV. Two had Wegener's granulomatosis (both cANCA positive and anti-PR3 positive) and four had microscopic polyangiitis (all pANCA positive and anti-MPO positive), and all were in remission. IS in this group consisted of cyclosporin, azathioprine or mycophenolate mofetil and corticosteroids (n = 2), cyclosporin mono-therapy (n = 3), cyclosporin and azathioprine (n = 1).

The RTX group (n = 9) consisted of stable renal transplant recipients [four male, five female; mean age 56 years (range 45–71), diagnosis: two with cystic kidney disease, two with benign nephrosclerosis, one with diabetic nephropathy, two with chronic interstitial nephritis, one with chronic pyelonephritis and one with end-stage renal failure of unknown cause] and were chosen to document effects of IS. All these patients received cyclosporin, mycophenolate mofetil or azathioprine and corticosteroids. Thirteen volunteers, recruited from our department [six male, seven female, mean age 41 years (range 21–50)] served as HC. The study was approved by the local ethic committee and all patients gave informed consent.

Media, reagents and antibodies

Ficoll-Hypaque was purchased from Amersham Biosciences (Freiburg, Germany). All antibodies were directly conjugated and obtained from Dako (Hamburg, Germany) with the exception of CD45RB (BD Biosciences, Heidelberg, Germany). FACS lysis solution and cell wash solution were also from BD Biosciences. Anti-CD3 stimulatory monoclonal antibodies (UCHT-1) and anti-CD28 (clone 37407·11) were both from R&D systems (Wiesbaden, Germany). OKT3 ascites was from Eurogentec (Seraing, Belgium). Carboxyfluorescein diacetate, succinimidyl ester (CFSE) was purchased from Molecular Probes (Leiden, the Netherlands). CD4+ T cells were isolated by means of negative selection using the CD4 T cell isolation kit was from Miltenyi Biotec (Bergisch Gladbach, Germany). Penicillin and streptomycin as well as IL-2 were purchased from Sigma-Aldrich (München, Germany). Iscove's modified Dulbecco's medium (IMDM) and fetal calf serum (FCS) were purchased from PAN Biotec (Aidenbach, Germany). Cell culture plates were obtained from Greiner Bio-One (Solingen, Germany).

Analysis of lymphocyte subsets

Both FACS analysis and a differential leucocyte cell count, using an automatic cell counter and/or microscopic evaluation, were performed to obtain absolute and relative numbers of the different leucocyte subsets.

Whole blood (100 µl) was incubated with saturating amounts (10 µl) of conjugated monoclonal antibodies (CD4-RPE-Cy5, CD45RB-RPE, CD25-FITC) for 30 min at 8°C. Subsequently, erythrocytes were lysed by addition of 3 ml FACS lysing solution for 30 min. Hereafter leucocytes were collected by centrifugation, washed twice and resuspended in cell wash.

Three-colour analysis was performed by FACSCalibur, BD Biosciences using WinMDI 2·8 software.

Immunohistological staining of renal biopsies

Immunohistochemical analysis for CD4 and CD25 was performed on four renal biopsies of focal necrotizing crescentic glomerulonephritis due to AAV and three renal biopsies of RTX patients with interstitial rejection. Acetone fixed 4-µm-thick cryostat sections of snap-frozen tissue were washed in Tris-buffered saline (TBS) and incubated overnight with anti-CD4 or anti-CD25 monoclonal antibody (Dako) in a dilution of 1 : 50. After incubation with rabbit antimouse immunoglobulin antibody, sections were incubated with an alkaline phosphatase–anti-alkaline phosphatase (APAAP) complex. A positive immune reaction was visualized with fast red substrate solution followed by counterstaining with haemaloun.

RNA isolation and real time polymerase chain reaction (PCR)-analysis

Total RNA was isolated from CD4+ cells of AAV patients (n = 24) and controls (n = 10) using Trizol reagent according to the manufacturer's instructions. Hereafter, DNAse digestion was performed prior to reverse transcription to exclude amplification of genomic DNA. Five hundred ng of total RNA was reversed transcribed into cDNA using the first strand cDNA synthesis kit (Roche, Mannheim, Germany). Serial dilutions of cDNA were amplified in 20 µl containing 2 µl of FastStart DNA Master SYBR green I, 10 pmol of each gene specific primer and 4 m m MgCl2. The oligonucleotide used for PCR were as follows: FOXP3 forward: 5′-ACA GGC CAC ATT TCA TGC ACC-3′, FOXP3 reverse: 5′-ATT GTG CCC TGC CCT TCT CAT-3′, G6PDH forward: 5′-CCG TCG AAT AAT CCG TAC CAT-3′, G6PDH reverse: 5′-AAT CCG GGC TTA TTA CCG-3′. PCR reactions were performed by Lightcycler® using the following profile: 2 min at 50°C, 5 min at 95°C followed by 45 cycles of amplification, each consisting of denaturation at 95°C for 15 s, annealing at 55°C for 1 min and extension at 72°C for 1 min. Specific DNA standards were generated by PCR amplification of cDNA, purification of the amplified product and quantification by spectrophotometry. In each experiment for each PCR reaction standard curves were generated by serial dilution of the purified product. PCR efficiency was assessed from the slopes of the standard curves and was found to be >90%. Linearity of the assay could be demonstrated for all standards and all samples. All samples were normalized for an equal expression of G6PDH.

Cell culture

PBMC were isolated from whole blood of HC and patients using Ficoll-Hypaque. Primary T cells were isolated from PBMC by magnetic depletion of non-CD4+ T cells using Midi MACS (Miltenyi Biotec). The purity ranged typically from 97% to 99%. Labelling of PBMC with CFSE was performed according to the manufacturer's instructions.

CFSE-labelled PBMC (1 × 106 per well) were cultured for 5 days in six-well plates (1 ml), plus OKT3 (1 : 100) with or without IL-2 (50 U/ml). In additional experiments, T cell stimulation was performed by using plate-bound anti-CD3 (UCHT-1), with or without anti-CD28 (clone 37407·11), in microlon 96-well plates. The optimal concentrations of anti-CD3 and anti-CD28 antibodies were determined by dose–response experiments. CFSE-labelled PBMC or CD4+ T cells (2 × 105 per well) were cultured in 200 µl of IMDM containing 10% FCS and penicillin/streptomycin (10 000 U/ml/10 mg/ml) at 37°C/5% CO2. After 5 days cells were collected and analysed by FACS. Supernatants were collected for assessment of interferon (IFN)-γ and IL-10 production by enzyme-linked immunosorbent assay (ELISA) (R&D, Wiesbaden, Germany). ELISAs were performed according to the manufacturer's instructions.

Intracellular cytokine production

In 27 of the AAV patients and 10 healthy controls, intracellular cytokine production in PBMCs was measured. To this end, PBMCs were stimulated for 4 h with phorbol myristate acetate (PMA)/iononomycin (50 ng/ml and 1 µg/ml, respectively) and hereafter stained for intracellular cytokines. 1 × 106 PBMC were first incubated with 10 µl of the conjugated monoclonal antibodies CD45RO FITC, CD8-PercP and CD4-APC for 30 min at 4°C. After the cells were washed twice with PBS, the cell membrane was permeabilized by adding 600 µl of 1/10 diluted perm-wash buffer (BD Biosciences) and RPE-labelled monoclonal anti-IL-2, anti-IL-4, anti-IL-8, anti-IL-10, anti-IL-12, anti-IFN-γ or anti-tumour necrosis factor (TNF)-α antibodies (all from BD) were added. After incubation of 30 min at 4°C, the cells were washed twice more with 600 µl perm-wash buffer, resuspended in 300 µl cell wash at 4°C and four-colour analysis was performed within the next 24 h.

Statistical analysis

All data are given as means ± standard deviation. For comparison of means between different groups anova for multiple comparisons was applied (statsdirect 2·2.2). A P-value <0·05 was considered to be significant.

Results

Number and phenotypic characteristics of T cells

The absolute number of lymphocytes was significantly decreased in AAV and in RTX compared to HC (Table 1). Similarly, the absolute number of CD4+ cells was decreased in AAV and RTX (470 and 670 in AAV and RTX, respectively, versus 1070 cells/µl in HC; P = 0·0001 for AAV versus HC and P = 0·0252 for RTX versus HC). In contrast to the absolute number of lymphocytes, the percentage of CD4+ cells was decreased only in AAV but not in AAV/RTX or RTX (Table 1).

Table 1
Lymphocyte populations in AAV, AAV/RTX, RTX and HC.

CD25 expression on CD4+ cells

Within the CD4+ population the percentage of CD25-expressing cells was clearly increased in AAV and AAV/RTX compared to HC but not in RTX patients, while the absolute number of CD4+ CD25+ cells was decreased in AAV and RTX but not in AAV/RTX patients. Both in AAV and AAV/RTX the relative and absolute number of naive CD4+ CD45RBhigh+ cells was decreased (Table 2).

Table 2
Lymphocyte populations in AAV, AAV/RTX, RTX and HC.

We investigated further if the increased CD25 expression was confined to the naive or memory CD4+ cells and if this correlated with disease activity. The percentage of CD25+ cells in the naive CD4+ CD45RBhigh population was significantly higher in the AAV group compared to HC (Table 3; Fig. 1a). This was observed both in patients with active disease and in patients in remission who did not receive IS therapy. The number of patients in these groups was, however, relatively low (n = 5). In AAV patients in remission but under IS therapy, CD25 expression was lower than in those without IS. When compared to healthy controls, the expression of CD25 was still higher in both AAV patient groups (48 versus 30%, P = 0·0395, AAV remission + IS versus HC). It thus seems that the increased expression of CD25 on naive CD4+ cells was associated with AAV, but not necessarily with disease activity or immunosuppressive therapy. This notion was supported by the observation of a significant increase in CD25+ cells in the CD4+ CD45RBhigh population in the AAV/RTX, but not in the RTX group (Table 3). Although in the CD4+ CD45RBlow population no differences in the percentage of CD25+ cells were found between all groups, there was a significant increase in the mean fluorescence intensity (MFI) of CD25 in the AAV group irrespective of disease activity, while in the AAV/RTX group the MFI of CD25 in the CD4+ CD45RBlow cells was reduced significantly (Table 3). To see if the presence of ANCA per se could induce these T cell abnormalities, we studied two PR3-ANCA positive patients with endocarditis without AAV. In these two patients no abnormalities were found in CD25 expression in the CD4+ CD45RBlow or CD45RBhigh populations (Fig. 1b).

Fig. 1
Differences in CD25 expression in CD45RB subsets of CD4+ T cells in healthy controls and AAV patients. (a) Peripheral blood of healthy controls (HC) and vasculitis patients (AAV) were stained for CD4, CD45RB and CD25 using directly conjugated antibodies. ...
Table 3
CD25 expression in naive and memory CD4+ cells in AAV, AAV/RTX, RTX and HC.

It is unknown if the abnormal CD25+ CD4+ T cells in the peripheral blood of patients with AAV are of aetiological importance for tissue lesions in AAV. Because, in AAV, inflammatory lesions are characterized by dense mononuclear infiltrates, we investigated CD4 and CD25 expression in active renal lesions of these patients. To this end, renal biopsies of AAV patients (n = 4) with necrotizing crescentic glomerulonephritis were studied. Biopsies of RTX patients with acute interstitial rejection (n = 3) were investigated as a positive control and analysed for the expression of CD4 and CD25 in serial cryostat sections. Whereas high numbers of both CD4+ and CD25+ cells were found in biopsies of RTX patients, CD25+ cells were present to a much lesser extent than CD4+ cells in biopsies of AAV patients, suggesting that in these patients the majority of infiltrating CD4+ cells did not express CD25 (Table 4, Fig. 2).

Fig. 2
Immunohistochemical analysis of CD4+ and CD25+ cells in renal lesions of AAV and RTX patients. Cryostat sections were stained either with monoclonal antibodies directed against CD4 (upper panels) or CD25 (lower panels). In RTX patients both CD4+ and CD25 ...
Table 4
Semi-quantitative representation of CD4+ and CD25+ cells in renal lesions of AAV and RTX patients.

FOXP3 mRNA expression

To better determine the nature of the increased CD4+ CD25+ subpopulation of T cells we measured the FOXP3 mRNA expression in purified CD4+ T cells in 24 AAV patients and 10 HC. There was no difference in the FOXP3 expression between the two groups (Fig. 3), suggesting that the increased CD4+ CD25+ T cell subpopulation probably does not consist of Tregs, compatible with the finding that Treg cells are usually located in the memory CD4+ CD45RO+ or CD4+ CD45RBlow compartment.

Fig. 3
FOXP3 expression in CD4+ T cells. CD4+ T cells were isolated from 24 AAV patients (filled circles) and 10 healthy controls (open circles). FOXP3 expression was determined using LightCycler RT-PCR. The results are expressed as quotient of FOXP3 and G6PDH. ...

T cell proliferation

CD25 identifies the alpha chain of the interleukin 2 receptor, which is expressed on naive cells upon T cell activation. Although the percentage of CD25+ cells was increased in AAV patients, neither T cells from AAV patients (n = 10) nor from HC (n = 8) proliferated when cultured in normal medium supplemented with IL-2 (data not shown). Stimulation of PBMC with soluble anti-CD3 monoclonal antibody (OKT3) resulted in a significant higher proliferation of the memory CD4+ CD45RBlow population of AAV patients compared to HC, while no differences were found in the proliferation of naive CD4+ CD45RBhigh cells (Fig. 4, upper panel). In the presence of both OKT3 and IL-2, however, proliferation of both CD45RBlow and CD45RBhigh cells was significantly higher in AAV patients compared to HC (Fig. 4, lower panel).

Fig. 4
Proliferation of anti-CD3 and IL-2 stimulated CD4+ CD45RB subsets of AAV patients and HC. PBMC were labelled with CSFE and subsequently stimulated for 5 days with anti-CD3 in the absence (upper graph) or presence (lower graph) of IL-2 (50 U/ml). The cells ...

To test if the increased T cell proliferation in AAV patients was dependent on the presence of co-stimulatory molecules on antigen-presenting cells (APCs) in the cell culture system, proliferation assays were performed either with PBMC or purified CD4+ cells using plate-bound anti-CD3 or plate-bound anti-CD3+ anti-CD28. Dose–response experiments indicated that stimulation with a concentration of 0·5 µg anti-CD3 alone resulted in half maximal proliferation, while a concentration of 0·1 µg of anti-CD3 did not stimulate T cell proliferation. Under the latter condition, T cell proliferation was strictly dependent on co-stimulation with plate-bound anti-CD28 monoclonal antibody (data not shown).

When 0·1 µg of anti-CD3 antibody alone was used, no proliferation was observed in the AAV and in the HC group (data not shown). In contrast, 0·5 µg of anti-CD3 antibody resulted in half-maximal proliferation but no differences in T cell proliferation between AAV and HC were observed, neither when PBMC nor purified CD4+ cells were used (Fig. 5, upper panel). Using a combination of anti-CD3 (0·1 µg) and anti-CD28 antibody, T cell proliferation in PBMC of HC was more profound compared to purified CD4+ cells (P < 0·01). This was not observed in AAV patients where T cell proliferation in both PBMC and purified CD4+ cells was equal. Only in purified CD4+ T cells was there a significant increase in T cell proliferation when comparing AAV patients with HC (P = 0·0014 for CD4 AAV versus HC) (Fig. 5, lower panel).

Fig. 5
Proliferation of PBMC and purified CD4+ cells from AAV patients (n = 9) and HC (n = 9). PBMC (filled circles) and purified CD4+ (open circles) were labelled with CSFE and stimulated with either 0·5 µg of cross-linked anti-CD3 (upper graph) ...

Cytokine production

To characterize further the T cell phenotype in patients and controls, we studied differences in cytokine production (IFN-γ and IL-10) between PBMC and purified CD4+ cells. Both PBMC and CD4+ cells from AAV patients produced significantly less IFN-γ. Whereas PBMC of HC produced significantly more IFN-γ than purified CD4+ cells when stimulated with plate-bound anti-CD3+ anti-CD28, this was not seen in AAV patients (Fig. 6, upper panel). IL-10 production was less in PBMC, but not in purified CD4+ cells, of AAV patients compared to HC (Fig. 6, lower panel).

Fig. 6
Cytokine production in PBMC (filled circles) and purified CD4+ cells (open circles) of AAV patients and HC (n = 8 for both). PBMC or purified CD4+ cells were stimulated for 5 days using plate-bound anti-CD3 and anti-CD28 (1 µg for both). Hereafter ...

Intracellular cytokine production

To gain more insight into the functional differences of T cells from AAV patients and HC, i.e. whether there is a prevalence for Th1 or Th2 cytokines in AAV patients compared to controls, we determined further the percentage of IL-2, IL-4, IL-8, IL-10, IL-12, IFN-γ and TNF-α-producing lymphocytes in PBMC of AAV patients (n = 27) and HC (n = 10). The percentage of IL-10-producing CD4+ T cells was significantly higher in AAV patients compared to HC. This was found predominantly in the memory population (CD4+ CD45RO+) (Fig. 7a). Similar findings were obtained for intracellular IL-12 (Fig. 7b); for the other cytokines no differences between patients and controls were found (data not shown).

Fig. 7
(a) Percentage of IL-10-producing cells. PBMC were isolated from 27 AAV patients (filled circles) and 10 healthy controls (open circles). After 4 h stimulation with PMA/ionomycin cells were harvested and fixed. IL-10 production was analysed by intracellular ...

Discussion

In the present study we found profound abnormalities in the number and phenotype of peripheral blood lymphocytes in patients with AAV. These findings confirm and extend earlier studies [1316,2325]. The most characteristic finding was an increase in the percentage of CD25 positive cells in the CD4+ CD45 RBhigh subpopulation. The increase in CD25 positivity in the population of naive CD4+ T cells does not suggest primarily that there is an increased number of Treg cells in AAV, as Treg cells are usually located within the CD4+ CD45RBlow compartment.

This was substantiated further by the finding that the expression of FOXP3, a specific marker for Treg cells, did not differ between AAV patients and HC. Persistent T cell stimulation is therefore probably a better explanation for the increase in the number of CD4+ CD25+ cells in AAV. Although the mean age between AAV patients and HC was different, this was probably not underlying the increased percentage of CD25 in AAV patients, as in older patients CD25 expression was not always higher than in younger patients. Moreover, persistent T cell activation has been found in other studies independently of the patient's age [1316].

The fact that persistent T cell activation was not observed in two PR3-ANCA positive patients without AAV suggests that the mere presence of ANCA is in itself not sufficient to elicit this phenomenon in patients. In addition, we found that the proportion of CD4+ cells was diminished in AAV patients, compatible with findings previously reported by others [23,24]. Within the CD4+ cells, the naive CD45RBhigh subset was significantly decreased in AAV patients, suggesting a skewing towards the memory population. This was not found in the RTX group, but also occurred in AAV/RTX patients, indicating a disease-related entity rather than an effect of IS. Skewing of T lymphocytes towards the memory population might also occur as a consequence of immune activation. Persistent T cell activation has also been reported to occur in systemic lupus erythematodes (SLE) [25]. Is this a general phenomenon in autoimmunity? There are marked differences between the findings reported in SLE and our own findings in AAV patients. First, in SLE patients CD25 expression in CD4+ cells was increased only in patients with active disease, but did not differ between patients in remission and healthy controls [25]. This is in sharp contrast to our study, where no association between disease activity and CD25 expression was noted, although other groups have claimed that in active AAV patients CD25 is increased in comparison to patients in remission [13]. It must be stressed, however, that in the latter study [13] in AAV patients in general an increased CD25 expression was found when compared to healthy controls, a finding compatible with our data.

It can be argued that IS might have influenced our results. By looking at the patients in remission and comparing those on IS with those without IS there are indeed small differences in the number of CD4+ CD25+ cells. Patients without IS tend to have a lower percentage of these cells, but this was not statistically different. Similarly, the difference in the percentage of CD4+ CD45RBhigh was not significant between both groups (patients in remission, with IS versus without IS Table 2), although in patients without IS the absolute number of these cells was increased. This was due most probably to the fact that in these patients the total number of lymphocytes was much higher (Table 2).

It remains to be elucidated what the actual trigger for T cell activation in AAV patients is. Although several groups have demonstrated clearly that T cells from AAV patients are able to proliferate in response to PR3 and MPO [2628], the enormous expression of CD25 in the naive CD4 population of AAV patients, which sometimes lies between 80 and 90%, makes it unlikely that this is due exclusively to the activation by these ANCA antigens.

The results of the proliferation experiments also seem to support the notion of a persistent T cell stimulation in AAV. We found that purified CD4+ cells of AAV patients proliferate more strongly than CD4+ cells of healthy controls and that the presence of APC did not lead to a stronger proliferation of lymphocytes of AAV patients. In contrast, in healthy controls the presence of APC increased T cell proliferation significantly (Fig. 4). It thus seems that the increased T cell proliferation observed in AAV patients was intrinsic to the T cells. Our data are in agreement with a previous study from Lúdvíksson et al. [29], who also showed that T cell proliferation was more pronounced in AAV patients, particularly in patients with active disease. In the study of Lúdvíksson et al. T cell proliferation was induced by the combination of an anti-CD2 with an anti-CD28 monoclonal antibody, which makes their and our results not completely comparable.

The abnormal T cell response in AAV was also reflected in an abnormal production of cytokines. Although in AAV patients less IL-10 was found in the supernatant of anti-CD3/CD28-stimulated PBMC, this was not seen when purified T cells were tested. The fact that less IL-10 was produced in stimulated PBMC of AAV patients might therefore have been caused by the lower number of CD4+ cells in these patients. In fact, based on intracellular cytokine staining by FACS analysis, we observed that the percentage of IL-10 producing CD4+ cells in AAV patients was increased compared to healthy controls, suggesting that IL-10 production per T cell was lower in AAV patients than in healthy controls.

Impaired IFN-γ production was also observed in anti-CD3/CD28-stimulated PBMC as well as in CD4+ cells of AAV patients. Interestingly, in healthy controls, PBMC produced significantly more IFN-γ than purified CD4+ cells.

Based on the percentage of cytokine-producing CD4+ cells, no evidence for a Th1 or Th2 prevalence was observed.

Furthermore, we found that the abnormally high CD25 expression on CD4+ cells in the blood of AAV patients was not present on CD4+ cells of renal lesions of AAV patients. This is in contrast to the observations in active lesions of RA patients [30]. These findings raise the question of whether in AAV patients CD4+ CD25 cells migrate from the blood into the vascular lesions or whether recruitment of these cells triggers the inflammatory lesion. Experimental data show that autoimmune diseases can be induced by transfer of CD4+ CD25 cells, while induction can be inhibited by the co-transfer of CD4+ CD25+ cells [3,3133]. It thus appears that a balance between both subsets might be a pivotal factor that determines autoimmunity [3,3133]. Thus, the results in this study do not suggest an imbalance between CD4+ CD25+ and CD4+ CD25 cells or between Th1 and Th2 cells. The presence of lymphopenia, T cell activation and increased proliferation bears resemblance to the model of autoimmunity in NOD mice, where lymphopenia and homeostatic-type proliferation of T cells generate autoimmunity [34].

In conclusion, we have demonstrated profound and reproducible abnormalities of CD4+ T cells in patients with AAV. Patients with AAV are lymphopenic and have low numbers of naive CD4+ cells, which are CD25 positive. Because there were no differences in the expression of the transcription factor FOXP3, the CD4+ CD25+ T cell subpopulation probably consists of persistently activated rather than of regulatory T cells. In line with this, purified CD4+ T cells of AAV patients showed increased proliferation in vitro, but defective IFN-γ production, after stimulation with the combination of anti-CD3 and anti-CD28 monoclonal antibody. We found no evidence for an altered Th1/Th2 balance.

As lymphopenia and persistent T cell activation have been shown to be crucial for the induction of autoimmunity in animal models [34], the T cell abnormalities described in this paper may reflect proximal events in the pathogenetic cascade leading to AAV.

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