Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2015 Apr 14;180(2):178–188. doi: 10.1111/cei.12483

Reduced CD5+CD24hiCD38hi and interleukin-10+ regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies

L T Aybar *,†,✉, J G McGregor †, S L Hogan †, Y Hu †, C E Mendoza †, E J Brant †, C J Poulton †, C D Henderson †, R J Falk †, D O Bunch †
PMCID: PMC4408152  PMID: 25376552

Abstract

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs were evaluated in addition to their CD5+ subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine–phosphate–guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5+CD24hiCD38hi B cells and IL-10+ B cells compared to patients in remission and healthy controls (HCs). As IL-10+ and CD5+CD24hiCD38hi B cells normalized in remission within an individual, ANCA titres decreased. The CD5+ subset of CD24hiCD38hi B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5+ B cells are enriched in the ability to produce IL-10 compared to CD5neg B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs. The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production.

Keywords: AAV, ANCA, CD5, interleukin-10, regulatory B cells

Introduction

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease where pathogenesis is dependent upon autoantibodies that target the self-antigens myeloperoxidase (MPO) and/or proteinase 3 (PR3) 1,2. The importance of B cells in the immunopathogenesis of AAV is underscored by the fact that these autoantibodies cause disease in mice 3,4 and by the effectiveness of rituximab, a B cell-depleting therapy 5,6. However, a key subset of B cells, referred to as regulatory B cells (Bregs), also play a role in the maintenance of immunological tolerance. Bregs, which control immunological homeostasis via the hallmark immunosuppressive cytokine interleukin (IL)-10 7–11, have been reported in mice and humans, but are not well characterized in humans.

We recently demonstrated that B cells from patients with active AAV express low levels of CD5, a surface molecule which negatively regulates B cell signalling through the B cell receptor (BCR) to maintain immunological tolerance 12,13. In contrast, patients who are in remission have CD5+ B cell levels comparable to those in healthy individuals 13. Moreover, we found that CD5+ B cells are a harbinger of relapse following rituximab therapy when low or in decline. In mice, CD5+CD1dhi B cells secrete IL-10 and have a regulatory function evidenced by their inhibition of interferon (IFN)-γ and tumour necrosis factor (TNF)-α expression in T cells 14. We surmised that CD5 might serve as a marker of regulatory B cells. Two phenotypes for IL-10-producing regulatory B cells in humans have been described, CD24hiCD38hi and CD24hiCD27+ 7,11.

To explore the role of Bregs in patients with AAV, we (i) measured the reported phenotypes, CD24hiCD38hi and CD24hiCD27+ as well as CD5+ subsets of these populations (ii), determined B cell IL-10 production and (iii) correlated these B cell populations with changes in ANCA titre. Herein, we show that the CD5+ subset of CD24hiCD38hi B cells (CD5+CD24hiCD38hi) is reduced in patients with active AAV compared with healthy controls (HCs) and patients in remission. Moreover, IL-10-producing B cells also decrease during active disease. As patients go into remission, both CD5+CD24hiCD38hi and IL-10-producing B cells are present at levels similar to HCs. In contrast, the CD24hiCD38hi B cell population does not decrease significantly during active disease, but expands during disease remission. Longitudinal analysis of patients' B cells reveals that CD5+CD24hiCD38hi B cells and IL-10+ B cells normalize upon disease remission. Our data are consistent with the hypothesis that functionally competent regulatory B cells characterized as CD5+CD24hiCD38hi or IL-10+ support long-term clinical remission and that absence of functional regulatory B cells may be associated with disease onset and relapse in patients with AAV.

Study population and methods

Patient and healthy control samples

Peripheral blood mononuclear cell (PBMC) samples were collected from 30 HCs and 69 patients with AAV (see Table 1) after informed consent was obtained in accordance with the University of North Carolina's Institutional Review Board. Patient inclusion required diagnosis of AAV in accordance with criteria established by the Chapel Hill Consensus Conference 15. Diagnosis of microscopic polyangiitis (MPA) or granulomatosis with polyangiitis and/or crescentic glomerulonephritis without overt signs of systemic vasculitis were based on previously established criteria 16,17. Individuals with anti-glomerular basement membrane disease, immunoglobulin A nephropathy, eosinophilic granulomatosis with polyangiitis or any other glomerular disease process in addition to AAV were excluded. Patients who had reached end-stage renal disease (on dialysis or a renal transplant recipient) were excluded. Clinical and serological data were gathered during routine clinic visits at the time of blood draw for B cell analysis.

Table 1.

Patient and healthy control demographic characteristics

Characteristic Active (n = 28) Remission (n = 41) Healthy controls (n = 30) P-value*
Age <0·0001
 Median (IQR) 60 (42, 69) 59 (54, 72) 32 (26, 48)
Sex 0·43
 Female 16 (57%) 26 (63%) 22 (73%)
Ethnicity 0·47
 Asian 0 (0%) 1 (2%) 2 (7%)
 Black 4 (15%) 8 (20%) 2 (7%)
 Hispanic 1 (4%) 2 (5%) 0 (0%)
 White 21 (81%) 30 (73%) 25 (86%)
ANCA n.a. 0·34
 PR3 15 (54%) 17 (41%)
 MPO 13 (46%) 24 (59%)
Disease n.a. 0·18
 GPA 9 (39%) 14 (34%)
 MPA 13 (57%) 18 (44%)
 GN 1 (4%) 9 (22%)
Organ involvement n.a.
 Upper respiratory 11 (48%) 24 (60%) 0·43
 Pulmonary 13 (57%) 18 (44%) 0·43
 Renal limited 1 (4%) 9 (22%) 0·04
Peak creatinine at disease onset n.a. 0·12
 n/N 20/28 38/41
 Median (IQR) 2·1 (1·4, 3·0) 1·4 (0·9, 2·8)
BVAS n.a. <0·0001
 n/N 26/28 41/41
 Median (IQR) 6·5 (2, 12) 0 (0, 0)
Open in a new tab
*

P-values were calculated by Fisher's exact test for categorical variables and Wilcoxon's two-sample tests and the Kruskal–Wallis test for continuous variables. Data are summarized as number (n) and percentage (%) or median with interquartile range (IQR). n = number of observations; N = total number of patients. ANCA = anti-neutrophil cytoplasmic autoantibody; BVAS = Birmingham Vasculitis Activity Score; GN = glomerulonephritis; GPA = granulomatosis with polyangiitis; MPA = microscopic polyangiitis; MPO = myeloperoxidase; PR3 = proteinase 3.

Disease activity was classified based, in part, on the Birmingham Vasculitis Activity Score (BVAS) 18. Charts were reviewed extensively to distinguish persistent or recurrent disease from disease quiescence or non-vasculitic symptoms. Patients were classified as being in remission at the time of the sample only if they had a BVAS of 0 and had no clinical evidence of active disease for at least 3 months before and 3 months after the sample date. Patients were classified as having active disease if they had a BVAS greater than 0 and had clear clinical evidence of active disease (e.g. haematuria). In those patients for whom BVAS and clinical presentation were discordant (BVAS = 0 with clinical signs and/or symptoms), clear clinical evidence of disease activity superseded BVAS, and these patients were classified as ‘active’. Any patients designated as having ‘unclear’ disease activity by clinical evaluation, regardless of BVAS, were not used in this study.

The protocol for treatment has been described extensively 19,20. All patients included in this study received immunosuppressive therapy with corticosteroids and cyclophosphamide or rituximab at disease onset or at time of relapse. Remission maintenance therapy included azathioprine (AZA), mycophenolate mofetil (MMF) or rituximab. Patients with ≥1% B cells at the time of sample collection were included in our analysis. Patients not requiring remission maintenance treatment were followed at close intervals during periods of remission off therapy. ANCA titres were determined by the McLendon Clinical Laboratories at the University of North Carolina using enzyme-linked immunosorbent assay (ELISA) kits specific for either MPO or PR3 (Inova Diagnostics, San Diego, CA, USA).

Blood collection

Whole blood was collected in sodium heparin tubes (BD Biosciences, Franklin Lakes, NJ, USA). To facilitate erythrocyte removal, one part HetaSep (Stemcell Technologies Inc., Vancouver, Canada) was added per five parts heparinized whole blood and centrifuged at room temperature at 90 g with the brake off for 6 min. The leucocyte-rich supernatant was harvested and layered onto 5 ml of Histopaque®-1077 (Sigma-Aldrich, St Louis, MO, USA) and centrifuged at room temperature at 400 g with no brake for 30 min. The buffy coat was washed twice and resuspended in Hanks's balanced salt solution (HBSS, Life Technologies, Grand Island, NY, USA) supplemented with 2% fetal bovine serum (FBS).

Flow cytometric analysis

The expression of cell surface molecules reported to designate Bregs was examined by flow cytometry at the time of blood collection. First, cells were stained with Human TruStain FcX™ Fc receptor blocking solution (Biolegend, San Diego, CA, USA) to prevent non-specific antibody binding to Fc receptors. Next, cells were stained with the following fluorochrome-labelled anti-human antibodies: CD19 Pacific Blue (clone HIB19; Biolegend, San Diego, CA, USA), CD38 peridinin chlorophyll-cyanin 5·5 (PerCP-CY5·5) (clone HIT2; Biolegend), CD24 PE-CY7 (clone ML5; Biolegend), CD27 Alexa Fluor-647 (clone O323; Biolegend) and CD5 phycoerythrin (PE) (clone UCHT2; Biolegend) and then fixed with 1% paraformaldehyde. Cells were analysed using a LSRII (BD Biosciences) flow cytometer. Data were analysed with FlowJo software (TreeStar, Ashland, OR, USA). After selection of the lymphocyte population based on forward- and side-scatter, B cells were gated based on CD19+ staining and categorized according to their expression of CD38 and CD24, CD24 and CD27 and CD5+ subsets of these populations. The gating strategy for each B cell phenotype examined is provided in Supporting information, Fig. S1.

Cell culture

Human PBMCs were cultured in Iscove's modified Dulbecco's medium (IMDM; Gibco® Life Technologies, Carlsbad, CA, USA) supplemented with 100 U/μg/ml penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (FBS) (Gibco® Life Technologies). To ascertain B cell ability to produce IL-10, PBMCs were stimulated with 1 μg/ml recombinant human CD40 ligand (CD40L) (R&D Systems, Inc., Minneapolis, MN, USA) and 1 μg/ml cytosine–phosphate–guanosine (CpG) oligodeoxynucleotide (ODN) 2006 (Invivogen, San Diego, CA, USA) for 96 h. PBMCs were cultured for the final 6 h with 1 μl/ml GolgiPlug (BD Biosciences), 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich), and 1 μg/ml ionomycin (Sigma-Aldrich). CD19+IL-10+ B cells were measured by intracellular cytokine staining. To exclude dead cells from our analysis, cells were labelled using the Live/Dead® Fixable Blue dead cell stain kit (Life Technologies). To prevent non-specific antibody binding, cells were incubated with human TruStain FcX™ Fc receptor blocking solution (Biolegend) and stained with CD19 Pacific Blue (clone HIB19; Biolegend). Post-surface staining, cells were fixed and permeabilized using the Fix & Perm® cell fixation and cell permeabilization kit (Life Technologies). Permeabilized cells were stained with anti-IL-10 antibody (PE, clone JES3-9D7; Biolegend). IL-10 expression in CD19+ B cells was assessed relative to a fluorescence minus one (FMO) control where the IL-10 antibody was omitted 21.

Sorting of B cell populations

Leucocytes were obtained from healthy controls (Gulf Coast Regional Blood Center, Houston, TX, USA) and processed as described above to obtain a buffy coat containing lymphocytes. Cells were stained with antibodies to CD19 and CD5 and sorted into CD19+CD5+ and CD19+CD5neg populations using a fluorescence activated cell sorter (FACS)Aria II flow cytometer (BD Biosciences). Cells were collected into IMDM containing 50% FBS (unless specified otherwise, all culture reagents from Life Technologies). Sorted populations were washed twice and then cultured in IMDM containing 5% human AB serum, 1 μg/ml CpG, 0·1 μg/ml CD40L and PenStrep in U-bottomed 96-well plates (Falcon, Corning Inc., Corning, NY, USA) at 2·5 × 106 cells per ml. After 72–96 h, cells were processed for IL-10 intracellular staining as described above.

Statistical and graphical analysis

Demographic and clinical characteristics were summarized by descriptive statistics. P-values were calculated by Fisher's exact test for categorical variables and Wilcoxon's two-sample tests and Kruskal–Wallis test for continuous variables. A paired signed-rank test was used to test the paired difference of B cell phenotypes in the subgroups. P-values reported with a two-sided P-value of ≤0·05 indicate a significant difference. A Bonferroni correction was used in all analyses that compared more than two groups, making values ≤0·0056 significant. Analyses were conducted using sas version 9·1 (SAS Institute, Cary, NC, USA). Graphs were created using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Cohort description

To investigate Bregs in patients with AAV, we analysed PBMC samples from 69 patients with AAV and 30 HCs by flow cytometry (Table 1). Patient samples were evaluated at the time of either active disease or remission. There were no significant differences between active disease patients compared to those in remission with respect to age, sex, ethnicity, ANCA serotype, disease diagnosis or peak creatinine at disease onset (Table 1). A higher proportion of patients with renal limited organ involvement were in remission compared with active disease (22 versus 4%; P = 0·04). HCs were significantly younger than patients; however, B cell phenotype and IL-10 production did not correlate with age in patients or healthy individuals (data not shown). Patient therapy is summarized in Table 2. Standard induction therapy was high-dose prednisolone and cyclophosphamide. Maintenance therapy was comprised of AZA or MMF in combination with low-dose prednisolone. More patients with active disease were treated with cyclophosamide and corticosteroids as well as prednisone than patients in remission (Table 2); however, median values of CD24hiCD38hi and CD5+CD24hiCD38hi B cells did not differ between active patients on medication compared to patients prior to initiation of therapy. A greater percentage of patients in remission had been treated previously with rituximab than patients in active disease; however, median values for B cell populations did not differ from those for rituximab-naive patients with comparable disease activity (data not shown).

Table 2.

Medications

Active Remission P-values**
n = 27* n = 39
Cyclophosamide and corticosteroids 12 (44%) 1 (3%) 0·000034
Mycophenolate mofetil (MMF) 6 (22%) 8 (21%) 1·0000
Azathioprine 3 (11%) 5 (13%) 1·0000
Prednisone 11 (46%) 6 (15%) 0·0176
Off therapy 6 (22%) 17 (44%) 0·1144
Prior rituximab therapy (>1% B cells) 9 (33%) 29 (74%) 0·0012
Open in a new tab
**

P-values were calculated by Fisher's exact test.

*

n = 1 active and n = 3 remission patients had no medications documented in clinic records.

CD24hiCD38hi B cells do not correlate with disease activity

For our initial examination of putative regulatory B cells in patients with AAV, we analysed B cells with the CD24hiCD38hi and CD24hiCD27+ phenotypes (Supporting information, Fig. S1). When compared to healthy individuals [median = 11 (IQR = 8,12)], CD24hiCD38hi B cells did not differ in patients with AAV during active disease [median = 8 (IQR = 3,22); Fig. 1a; Table 3]. Patients in remission, however, had elevated percentages of CD24hiCD38hi B cells [median = 17 (IQR = 10, 32)] compared to HCs. The trend of increased percentages of CD24hiCD38hi B cells during disease remission when compared to HCs was observed in patients with both MPO and PR3 serotypes, but reached significance only in patients with PR3-AAV (P = 0·002, Fig. 1a). In patients with an MPO-ANCA serotype, CD24hiCD38hi B cells were increased significantly in remission [median = 15 (IQR = 10, 27)] when compared to patients in active disease [median = 5 (IQR = 2,11); P = 0·002; Fig. 1a], but not HCs (P = 0·03). In patients who were in remission and had been treated previously with rituximab, there was a modest expansion of CD24hiCD38hi B cells (median = 22%) compared to patients with no prior rituximab treatment (median = 12%). In contrast, there was no difference in the median CD24hiCD38hi Bregs in patients with active disease whether they had been treated with rituximab (median = 13%) or not (7%). Regardless of whether or not patients had prior treatment with rituximab, there was no difference in CD24hiCD38hi Bregs during active disease compared to disease remission. No differences were observed in CD24hiCD27+ B cells when patients with active or quiescent AAV were compared to HCs or when patients with active disease were compared to patients in remission (Supporting information, Fig. S2a).

Fig 1.

Fig 1

Open in a new tab

CD5+CD24hiCD38hi and IL-10+ B cells decrease during active disease and rebound during remission. Population analysis of the percentage of CD24hiCD38hi B cells (a), the CD5+ subset of CD24hiCD38hi B cells (b) and interleukin (IL)-10+ B cells (c) is shown for healthy controls (HCs) (HC, n = 14–30), patients with active anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) (ACT, n = 10–29), remitting AAV (ANCA REM, n = 23–43), active myeloperoxidase (MPO)-ANCA (MPO ACT, n = 5–13), remitting MPO-ANCA (MPO REM, n = 15–25), active proteinase 3 (PR3)-ANCA (PR3 ACT, n = 5–16) and remitting PR3-ANCA (PR3 REM, n = 7–18). IL-10 producing B cells (c) were analysed after 96 h of peripheral blood mononuclear cells (PBMC) stimulation with CD40 ligand and cytosine–phosphate–guanosine (CpG) DNA. Aggregate data show increased CD24hiCD38hi B cells during disease remission (a) but no significant change during active disease. Percentages of both the CD5+ subset of CD24hiCD38hi B cells and IL-10+ B cells decrease during active disease and rebound during remission. Lines indicate median values. *P ≤ 0·005; **P ≤ 0·001 and ***P ≤ 0·0001.

Table 3.

Regulatory B cell phenotypes in healthy controls and patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis

B cell population Healthy controls All ANCA active All ANCA remission MPO active MPO remission PR3 active PR3 remission
CD24hiCD38hi (%CD19+ B cells) (IQR) 11 (8, 12) 8 (3, 22) 17† (10, 32) 5§ (2, 11) 15 (10, 27) 14 (4, 35) 23† (12, 33)
Number n = 30 n = 29 n = 43 n = 13 n = 25 n = 16 n = 18
CD5+CD24hiCD38hi (%CD24hi38hi B cells) 74 (50, 92) 28†‡ (17, 41) 54 (31, 65) 24† (0, 50) 54 (39, 59) 29† (20, 39) 48 (30, 76)
n = 23 n = 24 n = 35 n = 10 n = 18 n = 14 n = 17
IL-10+ B cells (%CD19+ B cells) 25 (22, 34) 13†‡ (4, 19) 24 (17, 34) 7 (4, 14) 24 (23, 32) 19 (12, 19) 34 (13, 41)
n = 14 n = 10 n = 23 n = 5 n = 15 n = 5 n = 7
Open in a new tab

P-values were calculated by a Wilcoxon two-sample test. Due to multiple comparisons between groups, a Bonferroni correction was applied resulting in a P-value ≤ 0·006 being considered statistically significant. Data are presented as the median and interquartile range (IQR). The number of samples examined (n) is also given.

†

Statistically different from healthy control.

‡

Statistically different from all ANCA remission.

§

Statistically different from myeloperoxidase (MPO)-ANCA remission. PR3 = proteinase 3.

CD5+CD24hiCD38hi B cells are reduced during active disease and rebound upon disease remission

CD5 is a negative regulator of B cell receptor signalling, and in concert with CD1d 14, CD5 is known to be a marker of B cells that secrete IL-10 in mice. We previously investigated CD5 on total B cells and established its importance as an indicator of AAV disease activity and future relapse when decreasing or low 13. To examine the CD24hiCD38hi B cell subpopulation expressing this inhibitory protein, we analysed the CD5 marker on CD24hiCD38hi B cells in patients with AAV and healthy individuals. Patients with active disease have significantly lower percentages of the CD5+ subset of CD24hiCD38hi B cells (CD5+CD24hiCD38hi) [median = 28 (IQR = 17,40)] than HCs [median = 74 (IQR = 50,92), P ≤ 0·0001, Fig. 1b; Table 3]. This decrease in CD5+CD24hiCD38hi B cells during active disease was observed in patients of both MPO [median = 24 (IQR = 0,50), P ≤ 0·003] and PR3 [median = 29 (IQR = 20,39), P ≤ 0·0001] serotypes compared to HCs (Fig. 1b). Further, the reduced percentage of CD5+CD24hiCD38hi B cells during active disease increased to an intermediate level not different from HCs during remission for all AAV patients together [median = 54 (IQR = 31,65), P = 0·0064] as well as both MPO [median = 54 (IQR = 39,59), P = 0·05] and PR3 ANCA [median = 48 (IQR = 30,76), P = 0·02] serotypes (Fig. 1b). The percentage of CD5+CD24hiCD38hi B cells did not differ in patients with active disease whether they had prior treatment with rituximab (median = 31%) or not (28%). Similarly, patients in remission had comparable levels of CD5+CD24hiCD38hi B cells whether they had been treated with rituximab (52%) or not (55%). Moreover, the percentage of CD5+CD24hiCD38hi B cells remained significantly lower (median = 28%, P = 0·006) in patients with active disease compared to patients in remission (median = 55%) when only patients who had not been treated with rituximab were considered. In contrast, examination of the CD5+ subset of CD24hiCD27+ B cells did not reveal a B cell population that correlated with disease activity. No significant differences were observed in CD5+CD24hiCD27+ B cells when patients with AAV were compared to HCs or when patients with active disease were compared to patients in remission (Supporting information, Fig. S2b).

B cells in patients with active disease have reduced production of IL-10

To determine the competency of B cells to produce IL-10, 33 patients with AAV and 14 HCs were examined for IL-10-producing B cells after 96 h of stimulation with CD40 ligand and CpG DNA. Percentages of IL-10-producing B cells in patients with AAV during remission [median = 24 (IQR = 17,34)] of either MPO [median = 24 (IQR = 23,32)] or PR3 [median = 34 (IQR = 13,41)] serotype were similar to healthy individuals [median = 25 (IQR = 22,34)] with regard to IL-10-producing B cells (Fig. 1c; Table 3). In contrast, B cells from patients with active disease [median = 13 (IQR = 4,19)] produced significantly less IL-10 than patients in remission (P = 0·005) and HCs (P = 0·001).

CD5+CD24hiCD38hi and IL-10+ B cells normalize as individual patients transition from active disease to remission

To eliminate any interpatient variation, B cells of individual patients with AAV were analysed during times of disease activity and remission. Although levels during active disease did not differ from the baseline level of HCs, paired active disease and remission samples from nine patients exhibited a significant expansion (P = 0·004) of CD24hiCD38hi B cells as individuals transitioned from active disease into remission (Fig. 2a). Inclusion of CD5 as a marker denoted a B cell population that was decreased significantly in our population studies. This finding was substantiated in our paired analysis. When CD5 was included as a marker, six of seven paired active and remission disease samples demonstrated an increase of CD5+CD24hiCD38hi B cells as individuals transitioned from active disease to remission (P = 0·05, Fig. 2b). Similarly, paired active and remission disease samples from six patients demonstrated a significant increase (P = 0·02) in IL-10-producing B cells as individuals transitioned from active disease to remission (Fig. 2c) in all six cases.

Fig 2.

Fig 2

Open in a new tab

CD24hiCD38hi, CD5+CD24hiCD38hi and interleukin (IL)-10+ B cells increase during remission in paired active and remission samples. Paired active and remission disease samples from nine patients demonstrate an increase in CD24hiCD38hi B cells (a) as an individual transition from active disease to remission. Paired active and remission disease samples from seven patients demonstrate that the CD5+ subset of CD24hiCD38hi B cells (b) decreased in patients with active disease and increased during remission. Paired active and remission disease samples from six patients demonstrate a similar increase in CD19+IL-10+ B cells (c) as all individuals transition from active disease to remission. The line indicates the median value for healthy controls (HCs).

CD5+ B cells are enriched in B cells capable of producing IL-10

Given the similar decrease of CD5+CD24hiCD38hi and IL-10+ B cells during active AAV and similar rebound during disease remission, we tested whether CD5+ B cells contained a population of B cells capable of producing IL-10. Enriched B cells, CD5+ B cells and CD5neg B cells were evaluated for IL-10-producing B cells after 72–96 h of stimulation with CD40 ligand and CpG DNA. CD5+ B cells are enriched in IL-10 producing B cells (median = 19%) when compared to CD5neg B cells (11%) or total B cells (16%, n = 2). Representative flow histograms depicting the percentage of IL-10+ B cells in cultured populations of total B cells (Fig. 3a), CD5neg B cells (Fig. 3b) and CD5+ B cells (Fig. 3c) demonstrate that CD5+ B cells are enriched in IL-10-producing B cells compared to CD5neg and total B cells.

Fig 3.

Fig 3

Open in a new tab

CD5+ B cells are enriched in B cells capable of producing interleukin (IL)-10. Total B cells, CD5+ B cells and CD5neg B cells were evaluated for IL-10-producing B cells after 72–96 h of stimulation with CD40 ligand and (CpG) DNA. Shown are representative flow histograms depicting the percentage of IL-10+ B cells in cultured populations of total B cells (a), CD5neg B cells (b) and CD5+ B cells (c) demonstrating that CD5+ B cells are enriched in IL-10-producing B cells compared to CD5neg and total B cells.

As CD24hiCD38hi, CD5+CD24hiCD38hi or IL-10+ Bregs increase, circulating ANCA titres decrease

To determine if the increase in CD24hiCD38hi, CD5+CD24hiCD38hi and IL-10+ B cells during disease remission had a suppressive effect, the influence on circulating ANCA titres was calculated. ANCA titres are reported from either MPO-ANCA or PR3-ANCA tests, as appropriate. In paired active and remission samples from the same patient, the percentage of each B cell phenotype during active disease (A) was subtracted from the value during remission (R) to generate the change or delta (Δ; R-A = Δ). Similarly, the ANCA titre during active disease was subtracted from the ANCA titre during remission for each patient. The change in each B cell phenotype is a positive integer when the percentage of B cells at the remission time-point is greater than the percentage of B cells in active disease. Conversely, the ANCA titre values are negative integers when the ANCA titre is higher in active disease than in disease remission. Figure 4 contains graphic representations of how the B cell phenotype relates to the change in ANCA titre during remission compared to active disease for CD24hiCD38hi (Fig. 4a), CD5+CD24hiCD38hi (Fig. 4b) and IL-10+ B cells (Fig. 4c). In all three analyses, the trend indicates that as CD24hiCD38hi, CD5+CD24hiCD38hi and IL-10+ B cells increase, ANCA titre decreases (P = 0·02, 0·02 and 0·03, respectively).

Fig 4.

Fig 4

Open in a new tab

As CD24hiCD38hi, CD5+ CD24hiCD38hi or IL-10+ B cells increase, circulating anti-neutrophil cytoplasmic autoantibody (ANCA) titres decrease. Paired active and remission sample data were used to generate the change (Δ) in each B cell subset CD24hiCD38hi (a) CD5+CD24hiCD38hi (b) or interleukin (IL)-10+ (c) by the subtraction of the active value from the remission value (R–A = Δ). A positive value for the ΔB cell subset indicates a greater percentage of B cells present during remission than during active disease. The change in ANCA titre was generated by subtracting the ANCA titre during active disease from the ANCA titre at remission, utilizing the same time-points used for B cell subset data. A negative value for ΔANCA titre indicates a greater titre during active disease than remission. Statistical significance was determined with a signed rank test; P ≤ 0·05 is considered significant.

Discussion

In this study, we demonstrate that the CD5+ subset of CD24hiCD38hi B cells and IL-10+ B cells are reduced in patients with active AAV compared with HCs and patients in remission. As patients go into remission, both CD5+CD24hiCD38hi and IL-10-producing B cells are present at levels similar to HCs. These data suggest that CD5+CD24hiCD38hi B cells may infer IL-10-producing B cells. In our population-based examinations, two of the phenotypes reported for human Bregs, CD19+CD24hiCD38hi and CD19+CD24hiCD27+, did not correlate with disease activity. Although patients with active disease do not show a significant decrease in CD24hiCD38hi B cells, we observed an expansion of this population as patients went into remission. In paired, longitudinal analysis of the same patient, CD5+CD24hiCD38hi B cells and IL-10+ B cells are similarly decreased during active disease and increased upon disease remission. Moreover, we show that CD5+ B cells are enriched in IL-10-producing cells compared to CD5neg B cells. Importantly, for the first time, we demonstrate that an increase in CD24hiCD38hi B cells, CD5+CD24hiCD38hi B cells and IL-10+ B cells correlates with a decrease in autoantibody titre, specifically ANCA titre.

In humans, Bregs have not been distinguished from their reported CD5+CD1dhi 14,22, CD19hiFcγRIIb+ 23, FASL+ 24 or IL-10-producing counterparts in mice. Human B cells with the phenotype CD24hi and either CD38hi 7 or CD27+ 11 have been described as IL-10+ Bregs. These multiple immunophenotypes reported for regulatory B cells may simply indicate multiple Breg subsets with different functions. In a chronic inflammatory environment, a murine antigen-specific CD1dhiCD5+ B cell subset has been shown in vitro and in vivo to differentiate and suppress T cells via IL-10, IL-1β and signal transducer and activator of transcription-3 (STAT-3) activation and secretion of TGF-β, IFN-γ and IL-12 10,25. CD5 is one of the surface molecules that defines most murine Breg subsets 14. Although not included in the reported definitions of human Bregs, a subset of both of these phenotypes also expressed CD5 in healthy individuals. We have shown recently that CD5 marks B cells that portended active disease when low or decreasing 13, a pattern expected for B cells with a regulatory function. Moreover, CD5 is reported to induce IL-10 expression and promote cell survival in a human Daudi B cell line 26, human chronic lymphocytic leukaemia B cells 27 and mice 28. Our own data confirm that CD5+ B cells are enriched in IL-10-producing B cells when compared to CD5neg B cells.

In several autoimmune diseases, including type 1 diabetes 29, systemic lupus erythematosus (SLE) and AAV 30, regulatory T cells (Treg) are present but lack suppressive ability 7,31. Bregs and type 1 regulatory T cells (Tr1) 32,33 exert suppressive effects through IL-10, a cytokine which can drive a change in immunological response from T helper type 1 (Th1) to Th2. A lack of B cell IL-10 is common to several relapsing and remitting inflammatory autoimmune diseases characterized by pathogenic B cells such as multiple sclerosis (MS) 34 and SLE 7. IL-10 deficiency infers regulatory B cell malfunction. IL-10-producing B cells have been proven to diminish clinical symptoms in MS 34. An IL-10-dependent increase in forkhead box protein 2 (FoxP3) expression, a Treg indicator, was shown in the central nervous system after B cell transfer in the experimental autoimmune encephalitis (EAE) mouse model 35. IL-10-secreting B cells are essential for recovery in arthritis and EAE murine models of human inflammatory autoimmune disease and MS 9,36. Interestingly, a high capacity to produce IL-10 protects from metabolic syndrome and diabetes mellitus in geriatric adults 37. Much evidence implicates IL-10 as a protective agent in a spectrum of chronic inflammatory diseases. Our findings indicate that IL-10-producing B cells are decreased during active disease and reappear in disease remission at levels similar to healthy individuals. These findings are in line with those of Hruskova and colleagues, who showed that patients with AAV in remission who relapsed produced significantly less circulating IL-10 than those without relapse; however, these investigators did not determine the source of IL-10 38. Patients with AAV have an increased frequency of the IL-10-1082AA genotype that is associated with decreased IL-10 production 39. Our results confirm and extend those of Wilde et al., who showed that B cells from 11 patients with active AAV produced less IL-10 40. In contrast, these investigators also reported a significant decrease in IL-10+ B cells during disease remission, whereas we observed an increase in IL-10+ B cells during remission to a level that did not differ from that observed in HCs. Todd 41 and Lepse et al. 42 also investigated Breg subsets in AAV. Todd et al. found that CD19+CD24hiCD38hi cells are more decreased in remission than during active disease; conversely, the ‘tolerant’ patient population (defined as: ‘those with a history of active AAV who subsequently became negative for ANCA by ELISA, remaining free from pathology after withdrawal of treatment for a minimum of 2 years’ 41) had the highest values of CD24hiCD38hi B cells that were indistinguishable from the HC population. In their study of patients with PR3-AAV, Lepse et al. reported the frequency of CD19+CD24hiCD38hi cells was not different in patients in remission compared with HCs, but was decreased in patients with active disease compared to either HCs or patients in remission. Our findings with regard to CD24hiCD38hi B cells differed from both of these groups which also differed from each other. Our results greatly extend the analysis of this putative Breg phenotype by investigating the CD5+ subset of CD24hiCD38hi B cells and demonstrating that CD5+CD24hiCD38hi B cells are decreased during disease activity and normalize upon remission, as expected for a Breg population. Furthermore, whereas Lepse's group found that CD24hiCD27+ B cells were decreased significantly in both remission and active patients when compared with HCs, we found no significant differences in either CD24hiCD27+ or CD5+CD24hiCD27+ B cell populations (Supporting information, Fig. S2).

The differences in our observations could be due to our strict definition of remission that disallowed inclusion of patients with clinically active disease within 3 months of remission time-points or our inclusion of CD40L in B cell cultures for IL-10 stimulation. Although culture conditions were different (2 versus 4 days and slight concentration differences in CpG and CD40L), our findings are in concert with those of Todd et al. 41, where IL-10+ B cells in ANCA remission patients do not differ in frequency from HCs. Of note, this is the first demonstration that patients in active disease have fewer IL-10+ B cells that rebound to HC levels when the patient goes into remission.

One strength of this study is the inclusion of patients with both MPO- and PR3-ANCA, as we now realize these are genetically and serologically distinct diseases with different risks for relapse 16,43. CD5+CD24hiCD38hi B cells and IL-10-producing B cells were decreased in patients with active disease and were similar to healthy individuals when patients were in remission, regardless of ANCA serotype. The strongest evidence presented is our analysis of paired samples from the same patient over time, demonstrating that a significant increase in CD24hiCD38hi, CD5+CD24hiCD38hi and IL-10+ B cells was observed when patients transitioned from active disease to remission. Our results suggest that CD5+CD24hiCD38hi and IL-10+ B cell phenotypes can be used as indicators of disease activity, as these B cell populations are decreased during active disease and rebound to levels similar to HCs during remission. Furthermore, it could logically be proposed that therapeutic up-regulation of these Bregs in patients with humoral autoimmune disease could promote disease quiescence.

Rituximab treatment eradicates all peripheral CD20+ B cells indiscriminately and is a prominent, effective therapy for AAV 5,6. The high values of CD24hiCD38hi B cells (>40% of total B cells) observed in some patients with AAV may be influenced by the repopulation of B cells post-rituximab treatment; however, only patients who had repopulated to ≥1% B cells in their lymphocytes were included in our studies. Additionally, there was no correlation between the percentage of total B cells present and the percentages of CD24hiCD38hi, CD5+CD24hiCD38hi or IL-10+ B cells detected (data not shown). Moreover, prior rituximab therapy did not alter median values significantly for B cell populations relative to those for rituximab-naive patients with comparable disease activity. Regardless of whether patients had been treated or not with rituximab, there was no difference in CD24hiCD38hi B cells during active disease compared to disease remission. In contrast, the percent CD5+CD24hiCD38hi B cells remained significantly lower in patients with active disease compared to patients in remission when only rituximab-naive patients were considered.

Patients with SLE who repopulated with CD24hiCD38hi B cells, which have been described as both transitional and regulatory, had a longer time to relapse post-rituximab therapy, suggesting that the phenotype of repopulating B cells may be important to follow with respect to disease outcome 44. Our own previous findings demonstrate that repopulation with a low percentage of CD5+ B cells portends a shorter time to relapse after B cell depletion with rituximab 13. Addition of CD5 to this CD24hiCD38hi B cell phenotype denotes a crucial B cell subpopulation that not only correlates inversely with active disease but also parallels IL-10 production and suppressive function.

Elucidation of crucial molecules that define and orchestrate the regulatory functions of B cells, including the suppression of pathogenic autoantibodies, is crucial to the development of more directed and safer therapies for individuals who suffer from AAV and, conceivably, autoimmune diseases as a whole. Our data are consistent with the hypothesis that functionally competent Bregs characterized as CD5+CD24hiCD38hi and IL-10+ support long-term clinical remission by inhibiting production of autoantibodies that drive disease pathogenesis. Whether the CD5+CD24hiCD38hi or IL-10-producing B cells can guide immunosuppressive therapy prospectively in patients to prevent unnecessary treatment and ensure treatment when appropriate is an open question that would be best answered in a clinical trial.

Acknowledgments

Portions of this work were presented previously in poster form at the 2012 and 2013 American Society of Nephrology meetings and the 2013 ANCA Vasculitis Workshop. This work was supported by a Program Project Grant number 5P01DK058335-14 from NIH/NIDDK and the Vasculitis Foundation. L. A. was supported by a NIH/NIDDK Minority Supplement number 3P01DK058335-14S1. The UNC Flow Cytometry Center is supported in part by an NCI Center Core Support Grant (P30CA06086) to the UNC Lineberger Comprehensive Cancer Center. We thank Dr William Pendergraft III for his critical review of the manuscript and helpful discussions.

Disclosure

The authors have no financial or commercial conflicts of interest to disclose.

Author contributions

L. A., C. M. and D. B. performed the experiments; L. A., R. F., S. H. and D. B. designed the study; S. H. and Y. H. provided expert statistical analysis; R. F., J. M., E. B. and C. P. provided patient clinical assessment information; C. P. and C. H. consented patients and acquired research blood samples; L. A., J. M., E. B., C. P., R. F., S. H. and D. B. wrote the paper.

Supporting Information

Additional Supporting information may be found in the online version of this article at the publisher's web-site:

Fig. S1. Gating strategies for B cell subpopulations. Shown are the gating strategies for CD24hiCD38hi (a–c), CD5+CD24hiCD38hi (d–f), CD24hiCD27+ (g–i), CD5+CD24hiCD27+ (j–l) and interleukin (IL)-10+ B cells (m–o). Typical examples of these B cell subpopulations observed in healthy controls (HCs) (a, d, g, j and m), patients with active anti-neutrophil cytoplasmic autoantibody-associated vasculitis (AAV) (b, e, h, k and n) and patients in remission (c, f, i, l and o) are provided.

cei0180-0178-sd1.pptx (307.4KB, pptx)

Fig. S2. CD24hiCD27+ and CD5+CD24hiCD27+ B cells do not correlate with disease activity. Percentages of CD24hiCD27+ B cells in healthy controls (HCs) (n = 11), patients with active anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) (n = 18), ANCA patients in remission (n = 19), active myeloperoxidase (MPO)-ANCA (n = 9), remission MPO-ANCA (n = 9), active proteinase 3 (PR3)-ANCA (n = 8) and remission PR3-ANCA (n = 9) are shown (a). Percentages of the CD5+ subset of CD19+CD24hiCD27+ B cells are depicted for HCs (HC, n = 9), patients with active ANCA (n = 13), ANCA patients in remission (n = 15), active MPO-ANCA (n = 5), remission MPO-ANCA (n = 7), active PR3-ANCA (n = 8) and remission PR3-ANCA (n = 7) are presented (b).

cei0180-0178-sd2.tif (614.9KB, tif)

References

  1. Jennette JC, Falk RJ, Hu P, Xiao H. Pathogenesis of antineutrophil cytoplasmic autoantibody associated small-vessel vasculitis. Annu Rev Pathol. 2013;8:139–160. doi: 10.1146/annurev-pathol-011811-132453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Kallenberg CGM. Pathogenesis of ANCA-associated vasculitides. Ann Rheum Dis. 2011;70(Suppl. 1):i59–i63. doi: 10.1136/ard.2010.138024. [DOI] [PubMed] [Google Scholar]
  3. Xiao H, Heeringa P, Hu P, et al. Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest. 2002;110:955–963. doi: 10.1172/JCI15918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Little MA, Al-Ani B, Ren S, et al. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system. PLOS ONE. 2012;7:e28626. doi: 10.1371/journal.pone.0028626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jones RB, Cohen Tervaert JW, Hauser T, et al. Rituximab versus cyclophosphamide in ANCA-associated renal vasculitis. N Engl J Med. 2012;363:211–220. doi: 10.1056/NEJMoa0909169. [DOI] [PubMed] [Google Scholar]
  6. Stone JH, Merkel PA, Spiera R, et al. Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med. 2010;363:221–232. doi: 10.1056/NEJMoa0909905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blair PA, Norena LY, Flores-Borja F, et al. CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic lupus erythematosus patients. Immunity. 2010;32:129–140. doi: 10.1016/j.immuni.2009.11.009. [DOI] [PubMed] [Google Scholar]
  8. Bouaziz JD, Le Buanec H, Saussine A, Bensussan A, Bagot M. IL-10 producing regulatory B cells in mice and humans: state of the art. Curr Mol Med. 2012;12:519–527. doi: 10.2174/156652412800620057. [DOI] [PubMed] [Google Scholar]
  9. Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells regulate autoimmunity by provision of IL-10. Nat Immunol. 2002;3:944–950. doi: 10.1038/ni833. [DOI] [PubMed] [Google Scholar]
  10. Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg R, Bhan A. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity. 2002;16:219–230. doi: 10.1016/s1074-7613(02)00274-1. [DOI] [PubMed] [Google Scholar]
  11. Iwata Y, Matsushita T, Horikawa M, et al. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood. 2011;117:530–541. doi: 10.1182/blood-2010-07-294249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Berland R, Wortis HH. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol. 2002;20:253–300. doi: 10.1146/annurev.immunol.20.100301.064833. [DOI] [PubMed] [Google Scholar]
  13. Bunch DO, McGregor JAG, Khandoobhai NB, et al. Decreased CD5+ B cells in active ANCA vasculitis and relapse after rituximab. Clin J Am Soc Nephrol. 2013;8:382–391. doi: 10.2215/CJN.03950412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yanaba K, Bouaziz J, Haas K, Poe J, Fujimoto M, Tedder T. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity. 2008;28:639–650. doi: 10.1016/j.immuni.2008.03.017. [DOI] [PubMed] [Google Scholar]
  15. Jennette JC, Falk RJ, Bacon PA, et al. 2012 revised international chapel hill consensus conference nomenclature of vasculitides. Arthritis Rheum. 2013;65:1–11. doi: 10.1002/art.37715. [DOI] [PubMed] [Google Scholar]
  16. Lionaki S, Blyth ER, Hogan SL, et al. Classification of antineutrophil cytoplasmic autoantibody vasculitides: the role of antineutrophil cytoplasmic autoantibody specificity for myeloperoxidase or proteinase 3 in disease recognition and prognosis. Arthritis Rheum. 2012;64:3452–3462. doi: 10.1002/art.34562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides. Arthritis Rheum. 1994;37:187–192. doi: 10.1002/art.1780370206. [DOI] [PubMed] [Google Scholar]
  18. Luqmani R, Bacon P, Moots R, et al. Birmingham vasculitis activity score (BVAS) Dim system necrotizing vasculitis. Q J Med. 1994;87:671–678. [PubMed] [Google Scholar]
  19. Jennette JC, Wilkman AS, Falk R. Anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and vasculitis. Am J Pathol. 1989;135:921–930. [PMC free article] [PubMed] [Google Scholar]
  20. Hogan SL, Falk RJ, Chin H, et al. Predictors of relapse and treatment resistance in antineutrophil cytoplasmic antibody associated small-vessel vasculitis. Ann Intern Med. 2005;143:621–631. doi: 10.7326/0003-4819-143-9-200511010-00005. [DOI] [PubMed] [Google Scholar]
  21. Herzenberg LA, Tung J, Moore WA, Herzenberg LA, Parks DR. Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol. 2006;7:681–685. doi: 10.1038/ni0706-681. [DOI] [PubMed] [Google Scholar]
  22. Matsushita T, Yanaba K, Bouaziz JD, Fujimoto M, Tedder TF. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest. 2008;118:3420–3430. doi: 10.1172/JCI36030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Qian L, Qian C, Chen Y, et al. Regulatory dendritic cells program B cells to differentiate into CD19hiFcγIIbhi regulatory B cells through IFN-β and CD40L. Blood. 2012;120:581–591. doi: 10.1182/blood-2011-08-377242. [DOI] [PubMed] [Google Scholar]
  24. Klinker MW, Lundy SK. Multiple mechanisms of immune suppression by B lymphocytes. Mol Med. 2012;18:123–137. doi: 10.2119/molmed.2011.00333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mauri C, Gray D, Mushtaq N, Londei M. Prevention of arthritis by interleukin 10-producing B cells. J Exp Med. 2003;197:489–501. doi: 10.1084/jem.20021293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gary-Gouy H, Harriague J, Bismuth G, Platzer C, Schmitt C, Dalloul AH. Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production. Blood. 2002;100:4537–4543. doi: 10.1182/blood-2002-05-1525. [DOI] [PubMed] [Google Scholar]
  27. Garaud S, Morva A, Lemoine S, et al. CD5 promotes IL-10 production in chronic lymphocytic leukemia B cells through STAT3 and NFAT2 activation. J Immunol. 2011;186:4835–4844. doi: 10.4049/jimmunol.1003050. [DOI] [PubMed] [Google Scholar]
  28. O'Garra A, Chang R, Go N, Hastings R, Haughton G, Howard M. Ly-1 B (B-1) cells are the main source of B cell-derived interleukin 10. Eur J Immunol. 2005;22:711–717. doi: 10.1002/eji.1830220314. [DOI] [PubMed] [Google Scholar]
  29. von Herrath M, Sanda S, Herold K. Type 1 diabetes as a relapsing–remitting disease? Nat Rev Immunol. 2007;7:988–994. doi: 10.1038/nri2192. [DOI] [PubMed] [Google Scholar]
  30. Abdulahad WH, Stegeman CA, van der Geld YM, Doornbos-van der Meer B, Limburg PC, Kallenberg CGM. Functional defect of circulating regulatory CD4+ T cells in patients with Wegener's granulomatosis in remission. Arthritis Rheum. 2007;56:2080–2091. doi: 10.1002/art.22692. [DOI] [PubMed] [Google Scholar]
  31. Lindley S, Dayan C, Bishop A, Roep B, Peakman M, Tree T. Defective suppressor function in CD4+CD25+ T-cells from patients with type 1 diabetes. Diabetes. 2005;54:92–99. doi: 10.2337/diabetes.54.1.92. [DOI] [PubMed] [Google Scholar]
  32. Roncarolo M, Bacchetta R, Bordignon C, Narula S, Levings M. Type 1 T regulatory cells. Immunol Rev. 2002;182:68–79. doi: 10.1034/j.1600-065x.2001.1820105.x. [DOI] [PubMed] [Google Scholar]
  33. Roncarolo M, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings M. Interleukin 10 secreting type 1 regulatory T cells in rodents and humans. Immunol Rev. 2006;212:28–50. doi: 10.1111/j.0105-2896.2006.00420.x. [DOI] [PubMed] [Google Scholar]
  34. Duddy M, Niino M, Adatia F, et al. Distinct effector cytokine profiles of memory and naive human B cell subsets and implication in multiple sclerosis. J Immunol. 2007;178:6092. doi: 10.4049/jimmunol.178.10.6092. [DOI] [PubMed] [Google Scholar]
  35. Mann M, Maresz K, Shriver L, Tan Y, Dittel B. B cell regulation of CD4+CD25+ T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis. J Immunol. 2007;178:3447. doi: 10.4049/jimmunol.178.6.3447. [DOI] [PubMed] [Google Scholar]
  36. Mauri C, Feldmann M, Williams R. Down-regulation of Th1-mediated pathology in experimental arthritis by stimulation of the Th2 arm of the immune response. Arthritis Rheum. 2003;48:839–845. doi: 10.1002/art.10832. [DOI] [PubMed] [Google Scholar]
  37. van Exel E, Gussekloo J, de Craen A, Frölich M, Bootsma-van der Wiel A, Westendorp R. Low production capacity of interleukin-10 associates with the metabolic syndrome and type 2 diabetes. Diabetes. 2002;51:1088–1092. doi: 10.2337/diabetes.51.4.1088. [DOI] [PubMed] [Google Scholar]
  38. Hruskova Z, Rihova Z, Mareckova H, et al. Intracellular cytokine production in ANCA-associated vasculitis: low levels of interleukin-10 in remission are associated with a higher relapse rate in the long-term follow-up. Arch Med Res. 2009;40:276–284. doi: 10.1016/j.arcmed.2009.04.001. [DOI] [PubMed] [Google Scholar]
  39. Bártfai ZGK, Russell KA, Muraközy G, Müller-Quernheim J, Specks U. Different gender-associated genotype risks of Wegener's granulomatosis and microscopic polyangiitis. Clin Immunol. 2003;109:330–337. doi: 10.1016/s1521-6616(03)00211-0. [DOI] [PubMed] [Google Scholar]
  40. Wilde B, Thewissen M, Damoiseaux J, et al. Regulatory B cells in ANCA-associated vasculitis. Ann Rheum Dis. 2013;72:1416–1419. doi: 10.1136/annrheumdis-2012-202986. [DOI] [PubMed] [Google Scholar]
  41. Todd SK, Pepper RJ, Draibe J, et al. Regulatory B cells are numerically but not functionally deficient in anti-neutrophil cytoplasm antibody-associated vasculitis. Rheumatology. 2014;53:1693–1703. doi: 10.1093/rheumatology/keu136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lepse N, Abdulahad WH, Rutgers A, Kallenberg CG, Stegeman CA, Heeringa P. Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission. Rheumatology. 2014;53:1683–1692. doi: 10.1093/rheumatology/keu149. [DOI] [PubMed] [Google Scholar]
  43. Lyons PA, Rayner TF, Trivedi S, et al. Genetically distinct subsets within ANCA-associated vasculitis. N Engl J Med. 2012;367:214–223. doi: 10.1056/NEJMoa1108735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Palanichamy A, Barnard J, Zheng B, et al. Novel human transitional B cell populations revealed by B cell depletion therapy. J Immunol. 2009;182:5982–5993. doi: 10.4049/jimmunol.0801859. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Gating strategies for B cell subpopulations. Shown are the gating strategies for CD24hiCD38hi (a–c), CD5+CD24hiCD38hi (d–f), CD24hiCD27+ (g–i), CD5+CD24hiCD27+ (j–l) and interleukin (IL)-10+ B cells (m–o). Typical examples of these B cell subpopulations observed in healthy controls (HCs) (a, d, g, j and m), patients with active anti-neutrophil cytoplasmic autoantibody-associated vasculitis (AAV) (b, e, h, k and n) and patients in remission (c, f, i, l and o) are provided.

cei0180-0178-sd1.pptx (307.4KB, pptx)

Fig. S2. CD24hiCD27+ and CD5+CD24hiCD27+ B cells do not correlate with disease activity. Percentages of CD24hiCD27+ B cells in healthy controls (HCs) (n = 11), patients with active anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) (n = 18), ANCA patients in remission (n = 19), active myeloperoxidase (MPO)-ANCA (n = 9), remission MPO-ANCA (n = 9), active proteinase 3 (PR3)-ANCA (n = 8) and remission PR3-ANCA (n = 9) are shown (a). Percentages of the CD5+ subset of CD19+CD24hiCD27+ B cells are depicted for HCs (HC, n = 9), patients with active ANCA (n = 13), ANCA patients in remission (n = 15), active MPO-ANCA (n = 5), remission MPO-ANCA (n = 7), active PR3-ANCA (n = 8) and remission PR3-ANCA (n = 7) are presented (b).

cei0180-0178-sd2.tif (614.9KB, tif)

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES