Persistent T-cell activation and clinical correlations in patients with ANCA-associated systemic vasculitis

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1 NDT Advance Access published March 26, 2006 Nephrol Dial Transplant (2006) 1 of 8 doi:10.109/ndt/gfl097 Original Article Persistent T-cell activation and clinical correlations in patients with ANCA-associated systemic vasculitis Smaragdi Marinaki*, Anna-Isabelle Kälsch*, Peter Grimminger, Annette Breedijk, Rainer Birck, Wilhelm H Schmitt, Christel Weiss 1, Fokko J van der Woude and Benito A Yard Fifth Department of Medicine and 1 Department of Medical Statistics University Hospital Mannheim, University of Heidelberg, Germany. *S.M. and A.-I.K. contributed equally to this work Abstract Background. Although in antineutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitis (AASV) patients, activation of T-cells has been described, persistence of these alterations has not been well characterized. This study was conducted to define persistent T-cell activation (PTA) in AASV patients and to assess whether this correlates with disease activity, disease severity, age or therapy. Methods. The expression of CD4, CD45RO, CD25, CD26, CD28, CCR7 and HLA-DR was examined longitudinally in 8 consecutive AASV patients. Clinical parameters were compared by univariate and multiple analysis and Kaplan Meier curves for relapsefree survival were calculated. Results. PTA could be defined as either of two activation phenotypes, i.e. a low percentage of CD4 þ CD45RO T-cells or a high percentage of CD25 in the naı ve CD4 þ population (n ¼ 26), since only these phenotypes were stable over time and were not associated with active disease. In patients with PTA, major organ involvement was significantly more often found than in patients without PTA. Moreover, the cumulative cyclophosphamide dose (26.86 vs 8.5 P<0.01) was significantly increased in these patients, suggesting that PTA was associated with disease severity. In general, patients with PTA were older than those without (62.92±9.4 years vs 48.42±16.9 years respectively, P<0.01). PTA was independent of disease duration. Interestingly, patients with a low percentage of CD4 þ CD45RO T-cells were significantly more often diagnosed as microscopic polyangiitis (P<0.01). Correspondence and offprint requests to: Anna-Isabelle Ka lsch, MD, Fifth Department of Medicine, (Nephrology/Endocrinology/ Rheumatology), University Hospital Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-, D-6815 Mannheim, Germany. anna-isabelle.kaelsch@med5.ma.uni-heidelberg.de Conclusion. We identified two independent phenotypes of T-cell activation in AASV patients. These phenotypes are persistent and do not reflect disease activity. PTA predominantly occurs in patients with severe disease. This might explain the higher cumulative cyclophosphamide dose found in these patients. Keywords: ANCA-associated vasculitis; clinical correlation; persistence; T-cell activation Introduction Antineutrophil cytoplasmic autoantibodies (ANCA)- associated systemic vasculitis (AASV) is, according to the Chapel Hill classification, characterized by necrotizing inflammation of the small blood vessels, particularly of the respiratory tract, nerves and skin, and by pauci-immune crescentic glomerulonephritis. Since AASV is strongly associated with ANCA, detection of anti-proteinase- (PR-) or antimyeloperoxidase (MPO) antibodies by ELISA and indirect immune-fluorescence (IIF) techniques have been widely recognized as valuable tools in laboratory diagnostics [1]. A number of experimental studies in vitro and in animals [2 4] have, over the past decade, pointed towards a direct role for ANCA in the pathogenesis of AASV nevertheless, antibody titres seem to correlate with clinical disease activity only in some and not all patients (meta-analysis of Birck et al. [19]). But apart from humoral immunity, T-cells are likely to be involved in the pathogenesis of AASV as well. Brouwer et al. [5] reported on an increased PR--specific proliferation of T-cells in patients with Wegener s granulomatosis (WG). The presence of activated T-cells in the granulomatous lesions of WG is also compatible with a pathogenetic role of T-cells in AASV [6]. Some studies have also demonstrated an ß The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 2of8 association between elevated soluble T-cell-activation markers, e.g. soluble interleukin-2 receptor or soluble CD0 and disease activity in patients with WG [7,8]. Additionally, therapy with monoclonal or polyclonal antibodies directed against T-cell antigens like antithymocyte globulin has been reported to be effective in patients with frequent relapses resistant to conventional treatment [9,10]. Signs of T-cell abnormalities in AASV patients have been reported in many studies [11,12]. Recently, we were able to demonstrate that the altered T-cell phenotype encountered in AASV fits best to a state of T-cell activation [1]. In most of these studies, this phenomenon has been referred to as persistent T-cell-activation (PTA) [11,14]. Nevertheless, PTA is poorly defined thus far since in most studies different T-cell-activation markers have been investigated, e.g. CD25, CD28, CD26, HLA-DR and CD29, and most of these studies were not performed in a longitudinal fashion. At present, it is therefore unclear which of the activation markers are persistently increased or decreased over time in AASV patients. In addition, it seems that PTA does not occur in all AASV patients [1]. The present study was conducted to examine persistency of T-cell activation in AAVS patients. Our working hypothesis is that persistent T-cell activation is associated with disease severity and/or activity. Materials and methods Patients We investigated 8 patients with AASV from our outpatient clinic and 10 healthy controls (HCs). Peripheral blood was obtained from all patients periodically at 8 week intervals for 12 months at their regular ambulatory visits in addition to their routine laboratory control. Two patients were examined six times and more, four patients five times, three patients four times, 11 patients three times and 18 patients were examined two times. Clinical characteristics of all patients are given in Table 1. All patients with AASV were diagnosed according to the Chapel Hill nomenclature as WG (n ¼ 26) or microscopic polyangiitis (MPA, n ¼ 12). All but two patients were ANCA positive as specified in Table 1. The activity of disease was evaluated according to clinical and radiological parameters and assessed according to the Birmingham Vasculitis Activity Score (BVAS). Adopted from the criteria used within the therapeutic trials of the European Vasculitis Study Group (EUVAS), complete remission was defined as the absence of pathological findings in these variables attributable to active vasculitis, irrespective of ANCA titres. The absence of clinical disease activity was indicated by a BVAS of zero. Five patients had active disease during the study period, as indicated by a mean BVAS score of 9.67 (range 29). Thirty-three patients were in complete clinical remission (BVAS score ¼ 0). The definition of relapse required the recurrence of active disease threatening the function of major organs (examples: glomerulonephritis, pulmonary infiltrates or granulomata). Major organ involvement was defined as the involvement of organs of vital importance (heart, lung, central nervous system and kidney). All patients with active Table 1. Clinical characteristics Characteristic disease were treated with cyclophosphamide (CYC) and corticosteroids until they reached remission at least for months and were then switched to a less toxic immunosuppressive regimen (azathioprine or mycophenolate mofetil). Although in remission, the majority of patients were still on immunosuppressive therapy (n ¼ 2), while only six patients had no immunosuppressive therapy at all. From these six patients, four could be studied at disease onset before the beginning of the immunosuppressive treatment. The immunosuppressive treatment in patients in remission consisted of mycophenolate mofetil (n ¼ 14), azathioprine (n ¼ 10), 15-deoxyspergualin (n ¼ 5), methotrexat (n ¼ 1), rituximab (n ¼ 1), etanercept (n ¼ 1) and/or corticosteroids (n ¼ 25). None of the studied patients were receiving CYC when being entered into the study. This was possible because the patients were either in remission or could be investigated before the initiation of CYC treatment. To calculate Kaplan Meier survival estimates for relapsefree survival, patients were included at the time of diagnosis and followed until 1 January 2005 or until the occurrence of the first relapse. Ten healthy volunteers, recruited from our department (4 male, 6 female, mean age 48.5±10 years, range 7 66 years) served as HC. The study was approved by the local ethic committee and all patients gave informed consent. Isolation of peripheral blood mononuclear cells (PBMCs) and CD4 þ T-cells PBMCs were prepared by gradient centrifugation using Ficoll Hypaque (Amersham Biosciences, Freiburg, Germany). CD4 þ T-cells were isolated from PBMC by negative selection (Miltenyi Biotec, Bergisch-Gladbach, Germany). Overall purity of the isolated CD4 þ T-cells was above 95%. Flow cytometry Frequency S. Marinaki et al. Gender (M/F) 17/21 Age (years) 58.0±1.87 Diagnosis 12 MPA, 26 WG ANCA-IIF pattern 27 C-ANCA, 9 P-ANCA, 2 ANCA neg ANCA-IIF pattern 22 C-ANCA, 9 P-ANCA, 7 ANCA neg at study entry ANCA-ELISA 18 anti-pr-, 5 anti-mpo, 15 neg BVAS 5 active, inactive Therapy 2 with, 6 without immunosuppression (4 could be studied at the time of diagnosis before any treatment) MPA ¼ microscopic polyangiitis, WG ¼ Wegener s granulomatosis, IIF ¼ indirect immunofluorescence, BVAS ¼ Birmingham Vasculitis Activity Score. Antigen expression on T-lymphocyte subsets was determined by quadruple immunofluorescence staining using directly conjugated antibodies. Therefore, PBMCs were incubated for 0 min at 4 C with saturating amounts (1 ml) of conjugated monoclonal antibodies directed against CD4, CD8, CD45RO, CD25, CCR7, CD28 and HLA-DR (all from

3 Persistent T-cell activation in patients with ANCA-associated vasculitis of 8 BD Biosciences, Heidelberg, Germany). The antibodies were either conjugated to FITC, RPE, PercP or APC depending on the combination of antibodies used. The cells were washed twice to remove unbound antibodies and were finally resuspended in 0 ml of Cell Wash (BD Biosciences, Heidelberg, Germany). Four-colour analysis was performed on a FACS Calibur flow cytometer (BD Biosciences, Heidelberg, Germany) and the data were analysed using WinMDI 2.8 software. Statistical analysis Quantitative data are given as mean±standard deviation. Differences in continuous variables were compared by means of Student s t-test in case of normal distribution or otherwise by Mann Whitney test. Proportions between two groups were compared with Fisher s exact test. Multiple logistic regression with backward selection was used to test for possible confounding factors. Relapse-free survival was compared by Kaplan Meier survival curves and log-rank test. Spearmen correlation coefficient was calculated to evaluate the correlation between age and cumulative CYC dose. A two-sided P<0.05 was considered to indicate statistical significance. Table. Absolute numbers of cells in cell per microlitre Results Markers of persistent T-cell activation in AASV patients In order to investigate which activation markers were persistently changed over time, the expression of HLA-DR, CD26, CD25, CCR7 and CD28 was studied in memory CD45RO þ and naı ve CD45RO lymphocytes. As previously shown, in this population there was also a significant decrease in naïve CD45RO CD4 þ T-cells as well as an increase in the percentage of CD25-positive cells within the CD4 þ CD45RO subpopulation (Table 2). In addition, the expression of HLA-DR in memory T-cells was significantly increased and there was no difference between patients and HCs concerning the other tested surface markers (Table 2). Comparing absolute numbers of cells, AASV patients were lymphopenic and had less CD4 þ T-cells, less naı ve CD45RO CD4 þ and less memory CD45RO þ CD4 þ cells. Additionally, AASV patients had also less CD8 þ T-cells, less naı ve CD45RO CD8 þ and less CD45RO þ CD8 þ compared with HCs (Table ). Table 2. Cross-sectional analysis of the amount of naı ve T-cells and of T-cell-activation markers in naïve CD45RO and memory CD45RO þ CD4 þ T-cells of patients and HCs. Data are given as mean±standard deviation of the percentage of positive cells Patients (n ¼ 8) Controls (n ¼ 10) P-value Naı ve T-cells (%) 22.72± ± CD25 CD45 RO 6.05± ± CD45 RO þ 78.02± ±12.95 NS HLA-DR CD45 RO 7.9± ±2.21 NS CD45 RO þ 26.25± ± CCR7 CD45 RO 84.19± ±7.01 NS CD45 RO þ 58.78± ±15.94 NS CD26 CD45 RO 92.41± ±4.64 NS CD45 RO þ 84.22± ±9.17 NS CD28 CD45 RO 9.70± ±.40 NS CD45 RO þ 9.14± ±8.59 NS NS ¼ non-significant. Patients Controls P-value CD ± ± CD45RO 128.4± ± CD45RO þ 17.2± ± CD ± ± CD45RO 14.2± ±6.2 NS CD45RO þ 120.9± ±100.1 NS CD4 CD45RO CD ± ± Patients with activated T-cells Patients with normal T-cells P-value CD4 94.5± ± CD45RO 74.8± ± CD45RO þ 19.7± ±228.0 NS CD8 25.2± ±151.9 NS CD45RO 12.2± ±101.0 NS CD45RO þ 120.9± ±86.6 NS CD4 CD45RO CD ± ± NS ¼ non-significant.

4 4of8 Interestingly, AASV patients also had significantly less CD25 þ naı ve CD45RO CD4 þ T-cells, although patients had a significantly higher percentage of CD25 þ cells in the naı ve CD45RO CD4 þ T-cells (Tables 2 and ). Similarly, when comparing the absolute numbers of patients with PTA and those with normal T-cells, patients with PTA also had significantly less naïve CD4 þ T-cells (Table ). In contrast, the absolute numbers in CD8 þ T-cells was not significantly different between patients with and without PTA (Table ). Then the changed parameters were studied longitudinally. However, the expression of HLA-DR strongly varied in some patients (range 25 72%, data not shown). In contrast, the percentage of CD25 þ cells in naı ve CD4 þ lymphocytes and the overall percentage of naı ve CD4 þ lymphocytes, appeared to be persistently increased or decreased, respectively. We therefore defined PTA in AASV patients as follows: (1) a high percentage of CD25 in naı ve (CD45RO CD4 þ ) cells or (2) a low percentage of naı ve (CD45RO ) CD4 þ cells. To distinguish between patients with high or normal expression of CD25 or patients with a low or normal overall percentage of naı ve CD4 þ cells, the cut-off for Healthy control S. Marinaki et al. each of these markers was chosen by measuring the mean%±2 SD for CD4 þ CD45RO CD25 þ (cut-off >61%) and CD4 þ CD45RO (cut-off <11%) lymphocytes in an HC population (n ¼ 10; Table 2). By using these criteria, patients could be split into four groups, i.e. patients with and without high CD25 expression, and patients with and without a low percentage of naı ve CD4 þ cells (Figure 1). In general, we found that 20 out of 8 patients had a high percentage of CD25 þ cells in the naïve population, and 18 out of 8 were normal in this regard. For the overall percentage of naı ve CD4 þ cells, 14 out of 8 showed a low percentage of naïve CD4 þ cells and 24 patients were normal. These phenotypes were independent from each other as suggested by chi-square testing (Table 4). PTA was therefore defined as having either of the two activated phenotypes, i.e. a high percentage of CD4 þ CD45RO CD25 þ (CD25 high : n ¼ 20) or a low percentage of CD4 þ CD45RO (CD45RO low : n ¼ 14). Thus, in our study, PTA was observed in 26 out of 8 patients (Table 4). The stability of the T-cell phenotypes was further supported since two patients were studied intermittently during active disease and remission. Both CD25 and CD45RO expression did not change during periods of different clinical disease activity, AASV patient Fig. 1. A representative example of the activation phenotypes found in AASV patients. Above: dot plot of naı ve CD45RO and memory CD45RO þ in CD4 þ T-cells. Below: histogram of CD25 expression in the naı ve CD45RO CD4 þ T-cells.

5 Persistent T-cell activation in patients with ANCA-associated vasculitis 5 of 8 confirming the stability of these T-cell phenotypes. Addionally, there was no difference concerning disease activity (active disease vs remission) in patients with and without PTA (Table 5). Clinical correlations Since an activated phenotype was not present in all patients, we subsequently studied whether patients with PTA differ clinically from those without. By univariate analysis, patients with PTA were significantly older compared with those without (Table 5; vs 48.42, P ¼ ). In addition, they had received a significantly higher cumulative dose of CYC (Table 5; 8.5 vs 26.8 g, P ¼ 0.006). Importantly, in patients with PTA, major organ involvement was significantly more often observed (Table 5, P ¼ ). The type of AASV (WG vs MPA), antibody specificity (PR- vs MPO vs negative) or ANCA immunfluorescence pattern (C-ANCA vs P-ANCA vs negative), disease activity (remission vs active disease) and disease duration were not significantly different between the groups (Table 5). Table 4. Association between the two persistently activated phenotypes, i.e. high expression of CD25 in CD45RO CD4 þ T-cells and low expression of CD45RO in CD4 þ T-cells CD25 in CD45RO High Normal CD45RO Normal NS Low NS NS ¼ non-significant. ¼ Sum. We subsequently investigated whether the different phenotypes of PTA correlated with clinical or demographic parameters (Tables 6 and 7). In the CD25 high group, the mean age and cumulative dose of CYC was also significantly higher compared with the CD25 norm group (Table 6; age: 5.1 vs 6.1 years, P ¼ 0.028, dose of CYC: 11.5 vs 29.7 g, P ¼ 0.044). Similar findings were also observed in the CD45RO low group (Table 7; age: 6.7 vs 55.2, P ¼ 0.029, CYC dose: vs.10 g, P ¼ ). Interestingly, CD45RO low was significantly more frequently found in MPA than in WG patients (9/12 vs 5/21, P ¼ ), independent of age, gender, CD25-positivity and cumulative CYC dosage as demonstrated by multiple logistic regression (backward selection) (Table 8). From six patients who had never received CYC, four could be studied at the onset of disease and two had never received CYC since they had no major organ involvement. The patients without major organ involvement had no altered T-cell phenotype as described in Tables 5 7, and from the other four patients, two belonged to the activated group and the other two to the non-activated group. Using Kaplan Meier curves and log-rank test, no significant difference in relapse-free survival was seen in patients with PTA (Figure 2), or in patients with either of the activated phenotypes CD25 high or CD45RO low (data not shown). Discussion In this study, we describe two independent phenotypes of PTA, i.e. patients with an increased percentage of CD25 in naı ve CD4 þ T-cells and patients with a decreased percentage of naı ve CD4 þ T-cells. Both activation phenotypes, i.e. CD25 high and CD45RO low, reflect persistent and not recent T-cell Table 5. Univariate analysis of clinical parameters between patients with persistently activated phenotype and patients with a normal T-cell phenotype Activated (n ¼ 26) Normal (n ¼ 12) P-value Age 62.92± ±16.9 P ¼ Gender (M/F) 11/15 6/6 NS Diagnosis WG NS MPA 10 2 Disease activity Active 2 NS Remission 2 10 Disease duration (month) 54.04± ±80.67 NS Relapse/month/patient WG 0.018± ±0.017 NS MPA 0.00± ±0.014 NS Cum. dose of CYC (g) 26.86± ±1.24 P ¼ ANCA-type (IIF pattern) C-ANCA 18 9 NS P-ANCA 7 2 Negative 1 1 Antibody specificity Anti-PR NS Anti-MPO 7 2 Negative 1 1 Major organ involvement Yes 26 9 P ¼ No 0 WG¼ Wegener s granulomatosis, MPA ¼ microscopic polyangiitis, IIF ¼ indirect immunofluorescence, PR- ¼ Proteinase-, MPO¼ myeloperoxidase, cum ¼ cumulative, CYC ¼ cyclophosphamide.

6 6of8 S. Marinaki et al. Table 6. Univariate analysis of clinical parameters of patients with a high percentage of CD25 (CD25 high ) and patients with a normal expression of CD25 (CD25 norm ) in the CD45RO CD4 þ T-cells CD25 norm (n ¼ 18) CD25 high (n ¼ 20) P-value Age (years) 5.1± ±10. P ¼ Gender (M/F) 9/9 8/12 NS Diagnosis WG NS MPA 7 5 Disease activity Active 2 NS Remission Disease duration (month) 60.± ±46.1 NS Relapse/month/patient WG 0.009± ±0.022 NS MPA 0.005± ±0.007 NS Cum dose of CYC (g) 11.5 (11.7) 29.7 (.) P ¼ ANCA-type c-anca NS p-anca 5 4 Negative 1 1 Antibody specificity Anti-PR NS Anti-MPO 5 4 Negative 1 1 Major organ involvement Yes P ¼ No 0 WG ¼ Wegener s granulomatosis, MPA ¼ microscopic polyangiitis, IIF ¼ indirect immunofluorescence, PR- ¼ Proteinase-, MPO ¼ myeloperoxidase, cum ¼ cumulative, CYC ¼ cyclophosphamide, NS ¼ non-significant. Table 7. Univariate analysis of clinical parameters of patients with a low percentage of CD45RO (CD45RO low ) and patients with a normal expression of CD45RO (CD45RO norm ) in CD4 þ T-cells CD45RO low (n ¼ 14) CD45RO norm (n ¼ 24) P-value Age 6.7± ±15.9 P ¼ Gender (M/F) 6/8 11/1 NS Diagnosis WG 5 21 P ¼ MPA 9 Disease activity Active 1 4 NS Remission 1 20 Disease duration (month) 56.4± ±62.8 NS Relapse/month WG 0.010± ±0.021 NS MPA 0.00± ±0.012 NS Cum dose of CYC (g) 14.05± ±7.14 P ¼ ANCA-type c-anca 8 19 P ¼ p-anca 6 Negative 0 2 Antibody specificity Anti-PR P ¼ Anti-MPO 6 Negative 0 2 Major organ involvement Yes NS No 0 WG ¼ Wegener s granulomatosis, MPA ¼ microscopic polyangiitis, IIF ¼ indirect immunofluorescence, PR- ¼ Proteinase-, MPO ¼ myeloperoxidase, cum ¼ cumulative, CYC ¼ cyclophosphamide, NS ¼ non-significant. Table 8. Multiple logistic regression (backward selection) of the association between diagnosis, CD45RO low and other parameters Step Effect Wald chi-square P-value 1 Disease duration Age Cumulative dose of CYC Gender CD25 high ANCA-type (IIF pattern) CD45RO CYC ¼ cyclophosphamide, IIF ¼ indirect immunofluorescence. activation for the following reasons. First, in both phenotypes a relatively large proportion of T-cells were affected. In some patients, the percentage of CD25- positivity in the naı ve CD4 þ population was as high as 90% while in other patients almost no naı ve CD4 þ T-cells could be found. This would suggest a strong polyclonal stimulation, not affecting memory T-cells. Second, one would expect a certain dynamic in the activation phenotypes as T-cell markers tend to change upon activation. We do not know the reason for the persistency of these phenotypes, nor do we know the trigger that initiates these phenotypes. It also remains to be elucidated whether PTA reflects proliferation of

7 Persistent T-cell activation in patients with ANCA-associated vasculitis 7 of Non-activated Activated 0.00 Patients at risk 0 Patients with PTA 0 Patients without PTA Time Fig. 2. Kaplan Meier estimates of relapse-free survival. Relapse-free survival in the PTA vs the normal group. Dashed line represents patients with a normal phenotype. No significant difference in relapse-free survival between the groups was found. T-cells in vivo. Both, an altered effector memory T-cell population [15] and shortened telomeres [14] have been described in AASV and are compatible with active proliferation and in vivo expansion of T-cells in these patients. It must be stressed that the increased percentage of CD25 in the naı ve CD4 population unlikely reflects regulatory T-cells for the following reasons. First, regulatory T-cells have a memory phenotype and second, FOXP expression was not increased in our collective of patients [1]. Neither of the two activation phenotypes correlated with disease activity (active vs remission). Nevertheless, in patients with PTA, disease seems to be more severe as reflected by a significantly larger proportion of patients with major organ involvement. Our findings confirm and extend previous studies of Schlesier et al. [16] who also reported on an association between the degree of organ involvement and T-cell activation in AASV patients. PTA in their study was, however, only defined by an increased percentage of CD25 in CD4 þ cells. In contrast, Giscombe et al. [17] have reported that an increased CD25 expression is associated with disease activity but not with major organ involvement. It must be stressed that in the studies of Schlesier et al. [16] and Giscombe et al. [17] only T-cells from patients with WG, but not T-cells from patients with MPA, were investigated. Since PTA in our study was defined as the presence of either of the two activation phenotypes, and since a decreased percentage of naïve CD4 þ cells was particularly found in MPA patients, this might, to some extent, explain the different results in these studies. It is not clear what event is causally related to the T-cell activation phenotypes described in this article. One possibility might be the influence of corticosteroids. There was no significant difference in the number of patients with and without PTA receiving corticosteroids at the time of analysis (P ¼ 0.15). This study was not designed to investigate whether corticosteroid treatment leads to T-cell activation. Nevertheless, as is clear from our results, long-term corticosteroid treatment is not a single cause for PTA, since the majority of patients without PTA were also treated with corticosteroids for a long time. Therefore, it is not likely that corticosteroids or other immunosuppresants per se influence lymphocyte phenotype distribution, although we cannot formally exclude this. To our knowledge, this is the first time an association between MPA and a low percentage of naı ve CD4 þ cells in AASV patients has been described. The association was independent of age and cumulative CYC dose, but was weakly related to MPO ANCA specificity. Thus, it seems that the CD45RO low T-cell phenotype may represent a T-cell alteration predominantly occurring in MPA patients. Further studies with a larger number of MPA patients are required to confirm this finding. In our study, patients with PTA were significantly older than those without. While a decrease in naı ve CD4 þ cells was never observed in healthy individuals, there was no correlation between CD25 expression in naïve CD4 þ cells and age in HCs (data not shown). It thus seems rather unlikely that PTA is solely attributed to age. Interestingly, The Wegener s Granulomatosis Etanercept Trial Research Group has reported that severity of disease is associated with age [18]. These data are compatible with ours since PTA was not only associated with age but also with severity of disease. Severity of disease was not only reflected by a larger proportion of patients with major organ involvement but also by a higher cumulative CYC dose. Due to the small number of patients we were not able to perform multivariate analysis to investigate whether major organ involvement and cumulative CYC dose were independent variables. In conclusion, we describe two different T-cell phenotypes in AASV patients, which are persistently present in most but not all cases. These T-cell phenotypes are not associated with disease activity, but correlate with severity of disease. Whereas the CD45RO low phenotype was associated with MPA, the CD25 þhigh phenotype was found in both MPA and WG patients.

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