Increased circulating PD-1^hiCXCR5⁻ peripheral T helper cells are associated with disease activity of ANCA-associated vasculitis

Abstract

Newly identified PD-1^hiCXCR5^–CD4⁺ T-cells, termed as peripheral helper T-cells (Tph), have been found elevated and playing a pathogenic role in some autoimmune diseases like systemic lupus erythematosus (SLE) and rheumatic arthritis (RA). However, the potential role of Tph-cells in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) remains unclear. Here, we explored the potential clinical significance of circulating Tph-cells in the pathogenesis of AAV. Comparing 32 active AAV patients and 18 age- and sex-matched healthy controls (HCs), we found that the frequency of circulating Tph-cells was significantly expanded in active AAV patients. Besides, programmed death 1 (PD-1) expression on the surface of Tph-cells was significantly up-regulated in active AAV patients. Importantly, the frequency of circulating Tph-cells was greatly decreased in AAV patients after receiving treatment. Tph-cells frequency was positively correlated with the Birmingham Vasculitis Activity Score (BVAS), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), neutrophil lymphocyte ratio (NLR), and cellular crescent in active AAV patients, but negatively correlated with fibrosus crescent. Tph-cells frequency was also positively correlated with naïve B-cells, serum concentration of MPO-ANCAs, serum tumor necrosis factor-α (TNF-α), IL-4, IL-21, and IL-12. However, serum IL-10 exhibited a negative correlation with circulating Tph-cells in active AAV patients. These results demonstrate that circulating Tph-cells are greatly expanded in active AAV patients and are positively associated with serum MPO-ANCAs and disease activity, thus contributing to the pathogenesis of AAV.

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Introduction

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small-vessel vasculitis characterized by excessive activation of the immune response [1, 2]. In the pathogenesis of AAV, plenty of pathogenic ANCAs targeting serine proteinase 3 (PR3) and/or myeloperoxidase (MPO) are secreted by activated plasma cells differentiated from B-cell subsets [3]. ANCAs are thought to be major pathogenic factors in AAV development, contributing to multiorgan injury [4–6]. Even though ANCAs are produced by autoreactive B-cells, T-cells undoubtedly are involved in regulating the differentiation and maturation of the ANCA-producing B cell population [7]. Follicular helper T (Tfh) cell, a unique subset of CD4⁺ T-cell population, possesses the capacity to migrate to germinal center (GC) and promote B-cell proliferation, differentiation into plasma cells, and isotype switching and affinity maturation [8]. Besides secondary lymphoid tissue, PD-1^hiCXCR5⁺CD4⁺ Tfh-cells exist in peripheral blood as well [9]. Some studies on systemic lupus erythematosus (SLE) have revealed Tfh-cell population was increased in peripheral blood both in animal models and patients [10–12]. A recent study on AAV has also discovered that the frequency of circulating Tfh-cell subsets of AAV patients was elevated compared with HCs and this subset was positively associated with disease activity [13].

Peripheral T helper (Tph) cell is a newly identified subset of CD4⁺ T-cells with a pathogenic role in rheumatoid arthritis (RA) [14]. This subset was firstly discovered by mass cytometry in joint tissue from patients with RA and characterized by PD-1^hiCXCR5^– in CD4⁺ T-cells. Although high expression of PD-1 is often considered indicative of an exhausted condition, Tph cells sorted from RA synovial fluid showed higher interleukin 21 (IL-21), IL-10, interferon γ(IFN- γ), and CXC chemokine ligand 13 (CXCL13) expression, but lower IL-2 expression, indicating Tph cells possess B-cell helper function [14]. CXCL13, a ligand of CXC chemokine receptor 5 (CXCR5), is able to recruit CXCR5⁺ B-cells and CXCR5⁺ Tfh-cells. In contrast to PD-1^hiCXCR5⁺CD4⁺ Tfh-cells, PD-1^hiCXCR5^–CD4⁺-Tph cells in RA synovial fluid show upregulated expression of transcription factor B lymphocyte-induced maturation protein 1 (BLIMP1) but downregulated expression of B-cell lymphoma 6 (BCL6), a master transcription factor in Tfh-cells [14]. In accordance with Tph-cells in synovial fluid, PD-1^hiCXCR5^–CD4⁺ Tph-cells in peripheral blood of RA patients also show increased levels of IL-21, IL-10, IFN-γ, CXCL13, and BLIMP1, but decreased levels of IL-2 and BCL6 [15]. Compared with Tfh-cells, Tph-cells show lower chemokine CC receptor 7 (CCR7) expression, indicating a potentially distinct migratory capacity. Tph-cells show higher expression of the inducible costimulatory molecule (ICOS), being able to bind to ICOS ligand (ICOSL) expressed on the surface of B-cells, an indicative of helper function to B-cell differentiation and antibody production. Tph-cells sorted from peripheral blood, synovial fluid, and synovial tissue all could induce co-cultured memory B-cells differentiation into plasma cells and enhance immunoglobulin G (IgG) production in vitro. This B-cell-helper function may be mediated by cytokine IL-21, due to inhibited plasma cells differentiation after neutralizing IL-21 in co-culture systems [14, 16]. Besides RA, circulating PD-1^hiCXCR5^–CD4⁺ Tph-cells were also found increased in IgG4-related disease, characterized by high serum IgG4 and chronic inflammation [17]. A more recent study on systemic lupus erythematosus (SLE) has revealed circulating PD-1^hiCXCR5^–CD4⁺ Tph-cells are highly expanded and have pathologic potential in this autoimmune disease [16]. Besides, Tph-cells were recently reported to be elevated and play a pathologic role in patients with type 1 diabetes [18], IgA nephropathy [19], psoriasis vulgaris [20], and active ulcerative colitis [21]. However, it is unclear whether this newly identified subset of CD4⁺ T-cells play a pathogenic role in AAV.

Therefore, defining the pathogenic T-cell populations that drive B-cell responses in AAV may contribute to new therapies that specifically target pathogenic cell subsets. In this study, we evaluated the phenotypes of CD4⁺ T-cells in peripheral blood of 32 active AAV patients and identified a highly expanded PD-1^hiCXCR5⁻CD4⁺ Tph-cell population compared with HCs. Besides, we investigated the relationship between this newly identified Tph-cell population and disease activity in patients with AAV.

Materials and methods

Subjects

In this cross-sectional study, we enrolled 32 active AAV patients without any treatment and 18 HCs. Thirty-two patients with active AAV from the Xinqiao (second affiliated) Hospital of Army Medical University were recruited from December 2019 to May 2021. Active AAV means the Birmingham Vasculitis Activity Score (BVAS) is more than 1 score and higher score indicates increasingly active disease [22, 23]. AAV patients were diagnosed according to the revised 2012 Chapel Hill Consensus Conference definitions [24] and eligible patients were subjected to the prescription of induction therapy after first blood collection. The length of induction therapy (from first blood collection to second blood collection) of follow-up AAV patients is 10–382 days. The detailed characteristics of included active AAV patients and HCs are indicated in Table 1. This study was in compliance with the Declaration of Helsinki and was approved by the Medical Ethical Committee of the Xinqiao Hospital, Army Medical University (No: yandi2015018). All patients were given written informed consent as mandated by the Declaration of Helsinki. Patients with current infection, malignancy, coexisting other autoimmune disorders, and maintaining dialysis were excluded. Patients who had previously received Rituximab or other biological therapies were excluded as well.

Antibodies and flow cytometry

Peripheral blood mononuclear cells (PBMCs) were isolated from heparin anticoagulant-treated peripheral venous blood of AAV patients or HCs by density gradient centrifugation with Lymphprep^TM (STEMCELL). For Tph-cells analysis, isolated PBMCs were stained with the following antibodies: anti-CD3-PE/Cy7 (HIT3a; Biolegend), anti-CD4-PerCP/Cy5.5 (OKT4; Biolegend), anti-CD45RO-FITC (UCHL1; Biolegend), anti-PD1-APC (EH12.2H7; Biolegend), anti-CXCR5 (J252D4; Biolegend), and LIVE/DEAD fixable near-IR dead cell dye (Thermofisher). PBMCs were stained with anti-CD3-FITC (HIT3a; Biolegend), anti-CD14-FITC (HCD14; Biolegend), anti-CD16-FITC (3G8; Biolegend), anti-CD56-FITC (5.2H11; Biolegend), anti-CD19-PerCP/Cy5.5 (HIB19; Biolegend), anti-CD27-PE/Cy7 (M-T271; Biolegend), anti-IgD-Brilliant Violet 421 (IA6-2; Biolegend), anti-CD38-Brilliant Violet 510 (HB-7; Biolegend), anti-CD70-APC (113-116; Biolegend), and LIVE/DEAD fixable near-IR dead cell dye (Thermofisher), for B-cell subpopulations analysis. Samples were acquired by FACSCanto II (BD Biosciences, San Jose, CA, USA) flow cytometer and analyzed by FlowJo software v10.

Enzyme-linked immunosorbent assay (ELISA)

Serums of all AAV patients and HCs were collected with a coagulation tube, and serum concentrations of IL-4, IL-10, IL-21 were detected by ELISA kits from Invitrogen (Carlsbad, CA, USA) following the manufacturer’s instructions. Serum concentrations of IL-12 and MPO-ANCAs were determined by using an ELISA kit from Abcam (Cambridge, England) and Shanghai Keshun Science and Technology Co., Ltd (Shanghai, China). The standards were measured with a duplicate to generate a standard curve, thereafter calculating cytokines concentrations. The analytical sensitivity of IL-4, IL-10, IL-12, IL-21, and MPO-ANCAs are 1.3 pg/ml, 0.66 pg/ml, 5.8 pg/ml, 20.0 pg/ml, and 0.25 ng/ml, respectively.

Statistical analysis

Comparisons between two groups were analyzed with the nonparametric Mann–Whitney test. Tph-cells frequencies of AAV patients before and after treatment were compared by the Wilcoxon matched-pairs signed-rank test. Spearman’s correlation test was used to analyze correlations between Tph-cells and clinical parameters, B-cell subpopulations, serum cytokines, or serum MPO-ANCAs concentrations. All results were shown as the median (interquartile range). P values less than 0.05 were considered to be statistically significant. All data analysis was conducted with Prism software (GraphPad Software, San Diego, CA, USA).

Results

Baseline characteristics of active AAV patients and HCs

The clinical characteristics of 32 cases of active AAV patients and 18 HCs were included in this study (Table 1). Of the 32 patients, the median (interquartile range, IQR) age was 63 (56–70). There are 18 males and 14 females out of these patients. The median (IQR) of BVAS score for AAV patients was 11.5 (6.0–14.8).

Tph cells were significantly expanded in active AAV patients

In this study, we analyzed the frequency of Tph cells in peripheral blood of active AAV patients and HCs. We gated PD-1^hiCXCR5⁻ Tph cells in the CD4⁺ T-cell population and calculated the frequency of this subset in circulating lymphocytes (Fig. 1A). The proportion of circulating PD-1^hiCXCR5⁻ Tph cells in circulating lymphocytes was significantly expanded in active AAV patients compared with HCs (Fig. 1B and C), as expected. To clearly describe the significance of Tph-cells in active AAV patients, we also gated PD-1^hiCXCR5⁺ Tfh-cells in the CD4⁺ T-cell subsets and compared their frequency among circulating lymphocytes. However, on the contrary to previous reports [25], the frequency of Tfh-cells from AAV patients was similar to HCs (Fig. 1D and E).

PD-1 expression was significantly upregulated on circulating Tph-cells in active AAV patients

While PD-1 has been reported as an exhausted marker, the function of PD-1 in circulating Tph-cells remains elusive. Studies on Tfh-cells revealed that PD-1 might be necessary for optimal IL-21 production by Tfh-cells [26, 27]. Besides, PD-1 has been reported to increase the stringency of GC affinity selection through interacting with PD-L1 expressed on the surface of B-cells [28]. Therefore, PD-1 expressed on Tph-cells might play a significant role in assisting B-cells through PD-1–PD-L1 interaction. In return, PD-1 interacting with PD-L1 on B-cells might contribute to optimal IL-21 production of circulating Tph-cells to aggravate disease activity of AAV. Thus, we analyzed the expression of PD-1 on circulating Tph-cells. As shown in Fig. 2, PD-1 expression on Tph-cells from active AAV patients was significantly upregulated, compared with HCs.

The frequency of Tph cells was significantly decreased after treatment in active AAV patients

We further investigated the change of Tph-cells in patients with active AAV before and after treatment. As shown in Fig. 3, the percentage of Tph-cells was significantly decreased in 11 followed-up active AAV patients after treatment with methylprednisolone and cyclophosphamide. However, the mean fluorescence intensity (MFI) of PD-1 among Tph-cells was hardly altered after treatment (Fig. 3C).

The percentage of Tph-cells was positively correlated with disease activity index in active AAV patients

To explore the clinical significance of circulating Tph-cell subsets in patients with active AAV, we investigated the correlation between the percentage of Tph-cells and clinical parameters of AAV including the Birmingham Vasculitis Activity Score (BVAS). BVAS has been validated for the assessment of disease activity in AAV [23]. Therefore, we calculated the BVAS of included AAV patients and analyzed the association between BVAS and circulating PD-1^hiCXCR5⁻ Tph-cells to validate whether circulating PD-1^hiCXCR5⁻ Tph-cells could partially indicate the disease activity of patients with AAV. We found that frequency of circulating PD-1^hiCXCR5⁻ Tph-cells was significantly positively correlated with BVAS (ρ = 0.431, P = 0.014; Fig. 4A). Meanwhile, we analyzed the association between circulating Tph-cells and serum CRP, ESR, NLR, serum C3, cellular crescent, cellular fibrosus crescent, or fibrosus crescent. As shown in Fig. 4, Tph-cell subset exhibited a positive relationship with serum CRP (ρ = 0.647, P = 0.004; Fig. 4B) and ESR (ρ = 0.505, P = 0.004; Fig. 4C). Tph-cell subset was also positively correlated with NLR (ρ = 0.447, P = 0.013; Fig. 4D) and cellular crescent (ρ = 0.386, P = 0.032; Fig. 4F). While Tph-cell subset was negatively correlated with fibrosus crescent (ρ = −0.393, P = 0.029; Fig. 4H). However, no significant correlation was demonstrated between Tph-cells and serum C3(ρ = −0.015, P = 0.944; Fig. 4E) or cellular fibrosus crescent (ρ = −0.090, P = 0.630; Fig. 4G).

Tph-cells were partially positively correlated with subpopulations of circulating B lymphocytes

The most important function of Tph-cells is to assist B-cells [14, 29]. To investigate the B-cell-helper function of Tph-cells in AAV patients, we analyzed the ­association between Tph-cells and subpopulations of circulating B-cells. According to the surface marker expression, including CD19, CD70, CD27, CD38, and IgD, B-cell subsets could be divided into: CD19⁺CD70⁻IgD⁺CD27⁻ naïve B-cells, CD19⁺CD70⁻IgD⁺CD27⁺ unswitched memory B-cells, CD19⁺CD70⁻IgD^–CD27⁺ class-switched memory B-cells, CD19⁺CD70⁻IgD⁻CD27⁻ double-negative memory B-cells, CD19⁻CD27⁺CD38⁺ potent plasmablasts [30] and CD19⁺CD70⁻CD38⁺ plasmablasts. These subpopulations of B-cells were quantified in lymphocyte populations. A remarkable positive correlation was found between the frequency of Tph-cells and serum MPO-ANCAs in active AAV patients (ρ = 0.711, P < 0.001; Fig. 5H). Furthermore, the frequency of Tph-cells was positively correlated with naïve B-cells (ρ = 0.400, P = 0.023; Fig. 5D). However, no significant correlation was found between the percentage of Tph-cells and potent plasmablasts (ρ = 0.273, P = 0.137; Fig. 5B) or plasmablasts (ρ = 0.074, P = 0.686; Fig. 5C). Besides, we measured the relationship between Tph-cells and other B-cell subsets including unswitched memory B-cells, class-switched memory B-cells, and double-negative memory B-cells. The results showed no correlation between Tph-cells and unswitched memory B-cells (ρ = −0.016, P = 0.930; Fig. 5E). Besides, no significant correlation was demonstrated between Tph-cells and other subpopulations of B-cells including class-switched memory B-cells (ρ = 0.073, P = 0.690; Fig. 5F) and double-negative memory B-cells (ρ = 0.002, P = 0.987; Fig. 5G).

The correlation between Tph-cells and serum cytokines

Several cytokines including TNF-α, IL-4, IL-21, IL-10, and IL-12 play an important role in the progression of AAV, as well as the development and B-cell-helper function of Tph-cells [14, 16, 17, 20], thus we detected these cytokines and analyzed their correlation with Tph-cells. From our data, Tph-cells were positively correlated with the levels of TNF-α (ρ = 0.449, P = 0.005; Fig. 6A), IL-4 (ρ = 0.688, P < 0.001; Fig. 6B), IL-21 (ρ = 0.709, P < 0.001; Fig. 6C) and IL-12 (ρ = 0.540, P = 0.001; Fig. 6E). However, circulating Tph-cells in active AAV patients were negatively correlated with IL-10 (ρ = −0.684, P < 0.001; Fig. 6D).

Discussion

In this study, we firstly demonstrated that circulating Tph-cells were significantly elevated in patients with AAV, compared with HCs. PD-1, a common activation molecule providing help to B-cells, was significantly upregulated on circulating Tph-cells from active AAV patients, compared with HCs. Furthermore, we found that Tph-cells were positively correlated with BVAS, CRP, ESR, NLR, and cellular crescent, but negatively correlated with fibrosus crescent. These results indicated that Tph-cells might play a pathogenic role in AAV and could have the potential to act as an indicator of disease activity. In addition, we discovered a positive correlation between Tph-cells and serum MPO-ANCAs or naïve B-cells. Tph-cells were positively correlated with serum IL-4 and IL-21 as well. From these data, we speculate that Tph-cells may promote severity of AAV by contributing to plasma cells activation, autoantibody secreting, and up-regulation of serum IL-4 and IL-21. Our research provides a new mechanism of AAV and may contribute to discovering new biomarkers and effective treatments for this severe autoimmune disease.

Tph-cells were reported to play a severe pathogenic role in several autoimmune diseases [14, 16–21], thus whether Tph-cells play a pathogenic role in AAV was analyzed and it proved that Tph-cells were positively correlated with BVAS, CRP, ESR, NLR, and cellular crescent. Therefore, we conclude that circulating Tph-cells frequency may partially be an alternative parameter for disease activity of AAV. The plausible reasons are as follows: (i) BVAS is a specific parameter for disease activity in AAV; (ii) CRP and ESR are two universal parameters reflecting disease activity of inflammatory disease; and (iii) NLR has been proved to be a promising inflammatory marker to assess systemic inflammation in AAV [31]. Cellular crescent is a hallmark of AAV, relevant with renal prognosis, and might be partially responsible for the severity of AAV [32]. Therefore, Tph-cells may act as a substitute of the cellular crescent for predicting renal prognosis thanks to its significant relationship with the cellular crescent. Cellular fibrosus crescent and fibrosus crescent usually act as an index for nephropathy in the late stage. Tph-cells might play its pathogenic role in the early stage of AAV; thus, it exhibits no significant correlation with cellular fibrosus crescent.

Tph-cells appear to be well equipped to mediate B-cell–T-cell interaction, particularly localizing in chronically inflamed peripheral tissues like peripheral blood, where they may function to promote and maintain accumulation of antibody-secreting cells like plasma cells [33]. The positive correlation between Tph-cells and serum MPO-ANCAs in active AAV patients is consistent with the results in SLE [16], indicating the ability of these cells to stimulating activation and maintenance of antibody-secreting cells in most autoimmune diseases. Although kidney involvement of AAV is known as the pauci-immune glomerulonephritis in their pathology, the pathogenic role of MPO-ANCAs is reported in many papers. On the one hand, studies in several animal models of AAV have shown that ANCAs possess pathogenic potential themselves, including passive immune models [34, 35], active immune models [36, 37], drug-induced models [38, 39], molecular mimicry models [40], and spontaneous models [41]. On the other hand, MPO-ANCAs could bind to MPO antigen expressed on the surface of neutrophils. At the same time, the crystallizable fragment (Fc) portion of these MPO-ANCAs binds to Fcγ receptors on neutrophils, inducing excessive activation of these cells. This hyperactivation leads to neutrophil extracellular traps (NETs) formation and abnormal cytokine production accompanied by the release of lytic enzymes and reactive oxygen species (ROS), which could injure vascular endothelial cells of involved kidneys [1]. Furthermore, the disappearance of immunoglobulins from AAV lesions could be attributed to the digestion of NETs from neutrophils, as evidenced by disappeared ANCAs when TNF-primed neutrophils were exposed to ANCAs in vitro [42]. While a positive correlation was discovered between Tph-cells and plasmablasts in active ulcerative colitis patients, no significant correlations between Tph-cells and these B-cell subsets were found in active AAV patients. Besides, Tph-cells were not correlated with other subpopulations of B-cells as well, including unswitched memory B-cells, class-switched memory B-cells, and double negative memory B-cells. These negative results might indicate that Tph-cells are not able to promote differentiation of any subpopulation of memory B-cells. Potent plasmablasts with cytoplasmic functional immunoglobulins were found to be largely expanded in the acute phase of hantavirus pulmonary syndrome [30]. There is a positive correlation trend between Tph-cells and potent plasmablasts, although it is with low ρ value and without significance. These might be attributed to the small sample size. These novel potent plasmablasts possess plenty of cytoplasmic functional immunoglobulins, indicating their MPO-ANCAs producing ability in AAV, which need to be further verified. Our results demonstrated a positive correlation between Tph-cells and peripheral naïve B-cells, indicating Tph-cells might promote naïve B-cells migrating from bone marrow to peripheral blood. Increased peripheral naïve B-cells are ready to differentiate into plasma cells or memory B-cells producing autoreactive pathogenic antibodies. According to these results, we boldly speculate that circulating Tph-cells may promote activation of plasma cells, secreting plenty of pathogenic autoantibodies in active AAV patients.

Cytokines play a significant role in many biological procedures including progression of AAV and development and function of Tph-cells [43]. IL-10 exhibits suppressive function in Tfh-cells to reduce autoantibody production in many autoimmune diseases [44], but IL-12 is considered to play an important role in promoting the development of Tfh-cells [45]. Our results demonstrate Tph-cells are negatively correlated with IL-10 and positively correlated with IL-12, indicating their role in promoting Tph-cells development and B-cell-helper function. This is in line with the consensus that IL-10 plays a protective role in AAV and IL-12 is a scathing factor. These results may provide an explanation for the role of IL-10 and IL-12 in AAV. IL-21 is a cytokine produced by circulating Tph-cells to regulate B-cells proliferation and differentiation and promote autoantibody production [45]. Besides, serum IL-21 may be a biomarker to indicate the disease activity of AAV [46]. IL-4 and IL-21 are two vital cytokines for Tph-cells promoting differentiation and activation of plasma cells; thus, we detected their serum levels and analyzed their relationship with Tph-cells in active AAV patients [14]. We observed that Tph-cells were positively correlated with IL-4 and IL-21, and showed a positive correlation with serum MPO-ANCAs. Therefore, we speculate Tph-cells promote activation of plasma cells through secreting IL-4 and IL-21, although more function experiments remain to be conducted to prove this suppose.

Despite these strengths, our study also has several certain limitations. First, we have not detected ICOS expressions on Tph-cells. ICOS is one of the mostly reported regulators of Tfh-cells, and ICOS deficiency in humans could result in decreased Tfh-cells and less germinal center formation [47]. Tph-cells were discovered similar phenotypes with Tfh cells, including increased expression of ICOS [14]. ICOS might play an important role in Tph-cells providing help to B-cells via ICOS–ICOSL interaction. Second, our cohort only includes AAV patients with MPO-ANCA positive, excluding those populations with PR3-ANCA positive and ANCA negative. This issue is caused by low frequency of PR3-ANCA positive patients in the Chinese Han population [48]. Third, functional studies of Tph-cells remain to be conducted to search basic mechanisms of these peripheral helper T-cells in providing B-cell-helper function.

In conclusion, circulating Tph-cells are greatly expanded in active AAV patients and are positively associated with the disease activity and serum MPO-ANCAs, suggesting that Tph-cells may play a vital role in AAV disease activity and may affect the severity of AAV through promoting B-cell activation by up-regulating serum IL-4 and IL-21. Our results also implied that Tph-cells frequency might be one potential index to reflect the inflammatory status of AAV patients and partially evaluate the disease activity of this autoimmune disease.

Abbreviations

Abbreviations
 
• AAV

  ANCA-associated vasculitis

 
• ANCA

  Anti-neutrophil cytoplasmic antibody

 
• BCL6

  B cell lymphoma 6

 
• BLIMP1

  B lymphocyte-induced maturation protein 1

 
• BVAS

  Birmingham Vasculitis Activity Score

 
• CCR7

  CC receptor 7

 
• CRP

  C-reactive protein

 
• CXCL13

  CXC chemokine ligand 13

 
• CXCR5

  CXC chemokine receptor 5

 
• ELISA

  Enzyme-linked immunosorbent assay

 
• ESR

  Erythrocyte sedimentation rate

 
• GC

  Germinal center

 
• HCs

  Healthy controls

 
• ICOS

  Inducible costimulatory molecule

 
• ICOSL

  ICOS ligand

 
• IFN-γ

  Interferon γ

 
• IgG

  immunoglobulin G

 
• IL

  Interleukin

 
• MPO

  Myeloperoxidase

 
• MFI

  Mean fluorescence intensity

 
• NETs

  Neutrophil extracellular traps

 
• NLR

  Neutrophil lymphocyte ratio

 
• PBMCs

  Peripheral blood mononuclear cells

 
• PD-1

  Programmed death 1

 
• PR3

  Proteinase 3

 
• RA

  Rheumatic arthritis

 
• ROS

  Reactive oxygen species

 
• SLE

  Systemic lupus erythematosus

 
• Tfh

  Follicular helper T cell

 
• TNF-α

  Tumor necrosis factor-α

 
• Tph

  Peripheral T helper cells

Acknowledgements

We are very grateful to Miss. Qi Han, Miss. Mengyuan Li, and Mr. Shining Wang for their technical support. We would also like to recognize and thank all of our patients with AAV from Xinqiao hospital for donating blood samples. Our thanks also go to the healthy control volunteers.

Funding

This work is supported by the National Natural Science Foundation of China (No. 81571604), Social Science and Technology Innovation Foundation of Chongqing Science & Technology Commission (cstc2019 jscx-msxm0318), Personal Training Program for Clinical Medicine Research of Army Medical University (2019XLC1006) to J.Z. and the Postgraduate Research and Innovation Project of Chongqing (No. CYB20196) to Z.L.

Disclosures

The authors declare no conflicts of interest.

Author contributions

Z.L., X.L., and N.F. were responsible for patient recruitment and data collection; Z.L. analyzed the data with the help of H.W., W.X., W.L., and S.T., J.Z., and Z.L. designed the study and wrote the paper; X.Z., and Y.W. provided several grants; J.Z., L.Z., and J.L. supervised the study. All authors contributed to the article and approved the submitted version.

Ethical approval

This study was in compliance with the Declaration of Helsinki and was approved by the Medical Ethical Committee of the Xinqiao Hospital, Army Medical University (No: yandi2015018). All patients gave written informed consent as mandated by the Declaration of Helsinki.

Data availability

All data used to support the findings are included in the article and are available from the corresponding author upon request.

References

Author notes