University of Groningen B cells in ANCA-associated vasculitides von Borstel, Anouk

B cells in ANCA-associated vasculitides

von Borstel, Anouk

DOI:

10.33612/diss.93537940

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2019

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von Borstel, A. (2019). B cells in ANCA-associated vasculitides: from pathogenic players to biomarkers.

University of Groningen. https://doi.org/10.33612/diss.93537940

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and Future Perspectives

7

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) is

a rare systemic autoimmune disease characterized by necrotizing inflammation of

small- to medium-sized blood vessels

_{. AAV comprises different clinicopathological}

phenotypes including granulomatosis with polyangiitis (GPA), microscopic polyangiitis

(MPA), eosinophilic GPA and renal limited necrotizing crescentic glomerulonephritis

_.

ANCA are considered to play an important role in the pathogenesis of AAV. These

autoantibodies (autoAbs) are primarily directed against the neutrophil- and

monocyte-derived enzymes proteinase (PR) 3 or myeloperoxidase (MPO)

_.

Although the disease etiology is unknown, the pathogenesis of AAV is generally

considered to be multifaceted involving genetic predisposition, environmental

exposure (e.g. infections) and a complex interplay between multiple immune cells.

Particularly T- and B cells belonging to the cellular adaptive immune response play an

important role in AAV disease mechanisms. An altered distribution of CD4

_{T helper (Th)}

cell subsets reflected by an expansion of a subset of circulating memory Th cells, termed

effector memory T cells (T

), has been observed in AAV patients in remission

_{. Also, in}

these patients a disbalance has been demonstrated in circulating Th cell responses with

a relative skewing towards Th17 cells as evidenced by increased circulating frequencies

of T

17 cells

_{, increased serum interleukin (IL) 17A levels}

_{, and an expanded}

population of pathogenic PR3-specific Th17 cells

_{. Importantly, regulatory T cells}

(Tregs), characterized by their immune modulating capabilities, are defective in AAV

patients

_{. Besides Th cells, B cells are well known to be involved in the pathogenesis of}

GPA as ANCA-producing cells. Next to their capacity to produce (auto)Abs, B cells are

also considered to contribute to autoimmune responses by Ab-independent functions

such as antigen (Ag) presentation

_{and cytokine production}

_{. Recently, an additional}

subset of B cells with immunomodulatory capability, termed regulatory B cells (Bregs),

has been identified

_{. However, their role in AAV remains largely unexplored.}

To suppress the effector cells of the cellular immune compartment, patients with AAV

receive standard remission induction and subsequent maintenance immunosuppressive

therapies. Two of the commonly used immunosuppressive agents in AAV used as

maintenance treatment include mycophenolate mofetil (MMF) and azathioprine (AZA).

AZA acts by inhibiting enzymes of the iosine-5’-monophosphate dehydrogenase (IMDP)

family thereby inhibiting leucocyte proliferation whereas MMF inhibits IMDP type 2

resulting in inhibition of lymphocyte proliferation

_{. Although it is widely acknowledged}

these drugs block immune cell proliferation, the effects of MMF and AZA on the

distribution and function of B cell subsets is poorly understood. Of note, it is currently

unknown whether differential immunomodulatory effects of MMF and AZA are related

to the fact that MMF-treated AAV patients are more prone to future disease relapses

than AZA-treated patients

_{. Although immunosuppressive therapies have significantly}

improved the prognosis of the disease, treatment is often associated with significant

7

toxicity, including an enhanced risk of infection, myelosuppression, malignancy, and

cardiovascular disease. Therefore, less toxic and more specific treatment is needed for AAV.

Previously, B cell-depleting therapy using rituximab has been shown to be as effective

as cyclophosphamide for induction of remission in patients with newly diagnosed AAV,

despite not targeting (the autoAb-producing) plasma cells

_{. Given that the initial}

response to rituximab is seen within 72 hours of treatment, it is not likely to be due to

reduced Ab production alone

_{. It is well known that}

_{B cell-derived cytokines affect the}

differentiation of naive into effector Th cells

_.

_{Thus, the beneficial effect of rituximab}

may result from its indirect impact on Th cell responses. Therefore, it is important to

investigate the Ab-independent roles of B cells in AAV to reveal potential anomalies in B

cell functioning and their effect on Th cells.

Unravelling the role of cellular immunity (involving both B- and T cells) in the

pathogenesis of AAV could aid in the discovery of potential biomarkers to predict

relapses and the identification of more specific targets for therapeutic intervention. One

type of AAV, i.e. GPA, is particularly a relapsing disease in which ±60% of the patients

experience one or multiple relapses during the course of their disease

_{. Hence, it is}

essential to identify GPA patients at risk for relapse (already) during disease remission

in order to prevent these relapses and there is still a pressing need for novel and more

reliable biomarkers that accurately predict disease relapses in GPA patients. Therefore,

the aims of this thesis were to:

1. Study the functional role of Bregs and effector B cells in the pathogenesis of GPA

2. Assess the effect of immunosuppressive therapy on B cell functioning in GPA

3. Examine the B cell repertoire as biomarker for future GPA disease relapses

In Chapter 3, we determined the frequency of circulating Bregs (i.e. phenotypically

characterized as CD19

_CD24

_CD38

_{cells). The frequencies of circulating Bregs were}

significantly decreased in GPA patients receiving immunosuppressive therapy at the

time of measurement, whereas these frequencies were not different in untreated GPA

patients compared to healthy controls (HCs). Indeed, immunosuppressive treatment

is well known to induce lymphopenia in the circulation

_{. Hence, it is possible that}

the discrepancy in the literature with respect to Breg frequencies in remission GPA

patients (discussed in Chapter 2) exists because patients were not stratified according

to immunosuppressive therapy at the time of sampling

_{. Also, in other autoimmune}

diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)

numerical Breg deficiencies have been demonstrated, although in both studies

untreated and treated patients were included

_.

Functional studies on Bregs have shown that these cells can inhibit Th cell responses

_.

Together with the observations that in several autoimmune diseases frequencies and/

or suppressive function of Bregs are reduced

_{, this prompted us to study whether}

reductions in Breg frequencies in GPA patients had an impact on the distribution of

circulating eff ector Th cells in Chapter 3. To this end, we assessed the frequency

of circulating Bregs in relation to the distribution of both circulating Th

17 cells

(phenotypically characterized as CC-chemokine receptor (CCR) 6

_{CXC-chemokine}

receptor (CXCR) 3

_CCR4

_CCR7

_CD45RO

_CD4

_CD3

_{cells) and Th}

EM

1 cells (phenotypically

characterized as CCR6

_CXCR3

_CCD4

_CCR7

_CD45RO

_CD4

_CD3

_{cells) in GPA patients.}

As mentioned above, Breg frequencies were decreased in GPA patients treated with

immunosuppressive drugs, whereas these frequencies were not altered in untreated

GPA patients compared to HCs. The Th

17 cell frequencies were increased in both

treated and untreated GPA patients compared to HCs, whereas the Th

1 cell frequency

was decreased albeit in treated patients only. Importantly, we observed an inverse

correlation between Th

17 cell and CD24

_CD38

_{Breg frequencies in the circulation}

of untreated GPA patients whereas a positive correlation was found between these

Bregs and Th

1 cells. No correlations between these cell subsets were found in treated

patients. Although associative, these observations suggest that Bregs are important

regulators of Th

cells in untreated GPA patients. To investigate whether Bregs were

indeed capable of suppressing Th

cell responses, we sorted and co-cultured CD4

_Th

cells with either CD24

_CD38

_{Breg-depleted B cells or with total B cells of GPA patients}

in remission in the presence of CpG-deoxynucleotides (CpG) and Staphylococcal

enterotoxin B. As explained in Figure 1, we showed that depletion of CD24

_CD38

Bregs resulted in an increased IL-17

_{Th cell frequency, whereas no changes in IFNγ}

Th cell frequencies were detected in GPA patients in remission. These fi ndings are in

line with a study in tuberculosis patients, although Bregs in this study were defi ned as

CD19

_CD1d

_CD5

_.

Circulation

in vitro

Figure 1. Disturbed Th_EM17- and Th_EM1 cell frequency distribution within CD4+ _{Th cells and}

the in vitro eff ect of CD24hi_CD38hi _{Bregs on the Th cell response in GPA patients. Left: In the}

circulation of untreated GPA patients increased Th_EM17 cell frequencies are present, whereas the Th_EM1 cell frequencies are decreased compared to HCs. Right: In vitro co-cultures of total B cells or CD24hi_CD38hi _{Breg-depleted B cells with CD4}+ _{Th cells of GPA patients showed that Bregs reduced}

7

In support of our findings, studies assessing the B cell repertoire after B cell depletion

with rituximab in RA and SLE patients revealed that this treatment reduced the Th17

cell response and restored the Th cell balance

_{. In these patients, B cells with the}

CD24

_CD38

_{phenotype were the first B cell subset that emerged from the bone}

marrow after B cell depletion with rituximab and became the dominant circulating

B cell subset

_{. Indeed, it has been speculated that the effectiveness of rituximab}

in AAV can in part be ascribed to the expansion of Bregs and subsequent inhibition

of Th17 cell responses

_{. Alternatively, a recent study showed that in vitro CD8}

_{T cell}

pro-inflammatory cytokine response was decreased in AAV patients treated with

rituximab

_{. Moreover, a co-culture of B cells and naive CD8}

_{T cells of AAV patients}

resulted in increased pro-inflammatory cytokine production by these T cells compared

to HCs

_{. Thus, rituximab treatment might normalize the CD8}

_{T cell response and during}

B cell repopulation an enrichment in circulating Bregs may have a therapeutic effect by

inhibiting the pathogenic Th17 cells. However, as discussed before, the identification

and function of Bregs is, compared to Tregs, less well defined and we are only beginning

to understand the functions of these cells. Studies such as ours (Chapter 3) are required

to unravel the role of Bregs in autoimmune disease pathogenesis. Possibly, Bregs could

ultimately be used for in vitro expansion and autologous transfer to inhibit autoimmune

disease development. However, before we can even think of such therapeutic

applications, extensive functional studies are required first to really understand Breg

biology and behavior in health and disease.

Besides enhancing the suppressive function of Bregs, inhibiting activated effector

B cells might be a promising treatment option for GPA patients. Circulating B cells of

active GPA patients have been shown to be in an increased state of activation compared

to HCs as evidenced by increased CD38 expression

_{. Moreover, it has been recently}

demonstrated that activated B cells from patients with autoimmune disease (RA and

Sjogren’s syndrome (SS)) show increased phosphorylation levels of signaling molecules

downstream of the B cell receptor (BCR) complex that could potentially serve as

therapeutic targets

_{. One of these important molecules in B cells is Bruton’s tyrosine}

kinase (BTK), which is a pivotal signaling molecule that directly links BCR signaling to

B cell effector function. Therapeutic BTK blockade is already widely used to inhibit B

cell activation in chronic lymphocytic leukemia patients

_{and has recently been shown}

to ameliorate kidney disease in a lupus nephritis mouse model

_{. Thus, BTK might be}

a promising drug candidate for modulating B cell activation and effector function in

AAV. In Chapter 4, we examined the BTK protein levels and its phosphorylation status

in B cell subsets of GPA patients with active disease and in remission and HCs. We

showed that BTK protein levels were significantly increased in transitional and naïve

B cells of active GPA patients compared to remission patients and HCs (Figure 2). In

addition, increased BCR sensitivity to in vitro stimulation with anti-IgM, reflected by

increased BTK phosphorylation and signaling, and increased phospholipase C (PLC) γ2

phosphorylation, was detected in active GPA patients. Together, these results indicate

that in GPA patients newly emerging B cells are in a heightened state of activation,

which is in line with previous work in other autoimmune diseases i.e. RA and SS

_.

We next assessed the impact of a BTK blocker on B cell cytokine production, plasma

cell formation and, IgG and ANCA production in vitro. The BTK blocker used in our

study is a small molecule that binds to the phosphorylation site of the BTK protein,

thereby preventing phosphorylation and subsequent activation of the BCR signaling

pathway

_{. Upon in vitro BTK blockade, we found that the frequencies of IFNγ}

_{, IL-6}

and IL-10

_{B cells were decreased in peripheral blood mononuclear cell (PBMC) samples}

of active and remission GPA patients and HCs, whereas the TNFα

_{B cell frequency was}

only decreased in remission GPA patients. Interestingly, plasma cell formation was only

inhibited by the BTK blocker in remission patients, whereas no difference was found in

active GPA patients and HCs. Importantly, plasma cell formation was increased in active

GPA patients with and without addition of the BTK blocker to the culture compared to

remission patients. Lastly, total IgG was decreased in remission patients and HCs in the

presence of BTK blockade and seemed to decrease in active patients. Interestingly,

PR3-ANCA levels tended to be lower in samples cultured in the presence of the BTK blocker

although this did not reach statistical significance, possibly due to low sample size.

Breg- and Treg-derived IL-10 is classically known as an anti-inflammatory cytokine

capable of suppressing the activation of other immune cells

_{. In Chapter 4 we found}

that in vitro IL-10

_{Breg frequencies were decreased when a BTK inhibitor was added}

to PBMC cultures. At first glance, such an effect seems undesirable given the

well-known anti-inflammatory properties of IL-10. However, IL-10 is also well-known to exert

non-inhibitory functions on various immune cells including B cells (reviewed in

_{). For}

example, IL-10 has been shown to inhibit B cell apoptosis

_{, enhance B cell proliferation}

and differentiation into plasma cells

_{, and promote immunoglobulin class switching}

_.

Therefore, in the context of B cell-mediated autoimmune diseases, IL-10 could also be

regarded as a pro-inflammatory cytokine. This contention is supported by GWAS studies

showing cytosine-adenine repeat polymorphisms in the IL-10 gene of GPA patients

_,

multiple myeloma patients

_{and SLE patients}

_{. Functionally, these polymorphisms}

were associated with increased in vitro IL-10 production by lipopolysaccharide

stimulated PBMCs of multiple myeloma patients

_{. Moreover, polymorphisms in the}

IL-10 gene have been reported to correlate with increased autoAb production in SLE

patients

_{. Although the functional consequences of IL-10 polymorphisms in GPA}

remain to be investigated, a recent meta-analysis demonstrated that polymorphisms

in the IL-10 gene are associated with susceptibility for development of vasculitis, in

particular Behçets disease and GPA

_{. Collectively, these studies clearly indicate the}

7

P

No BTK Inhibition

BTK levels

BTK

P

BTK Inhibition

Figure 2. BTK protein and phosphorylation levels in B cell subsets of active and remission GPA patients and HCs and the eff ect of BTK blockade on B cell functions. Top: BTK protein and phosphorylation levels were increased in newly emerging B cells (transitional and naïve B cells) of active GPA patients. Bottom: In vitro BTK inhibition decreased (auto)Ab secretion, as well as IL-10 and pro-infl ammatory cytokine (i.e. IFNγ and IL-6) production by B cells.

important role of IL-10 in the pathogenesis of autoimmune diseases. Moreover, we

speculate that while IL-10 may be essential for maintaining peripheral tolerance to

self-Ags and thus prevent autoimmunity, once tolerance is lost it could in fact promote

autoimmune disease by stimulating the diff erentiation of B cells into autoAb-producing

plasma cells.

Based on our in vitro findings the effectiveness of BTK blockade for active GPA patients

can be questioned as, for example, plasma cell formation in active GPA patients was

not inhibited by the BTK blocker. One possible explanation why BTK blockade did not

inhibit plasma cell formation is that B cells of active patients are already in an elevated

state of activation and do not require BCR stimulation. Additionally, experimental

evidence exists indicating that BTK inhibition not only suppresses B cell activation but

also cell migration. The latter was demonstrated in B cell lines where BTK- and

PLCγ2-deficient B cell lines were found to be unable to migrate in vitro. Moreover, treatment of

primary B cells with BTK or PLCγ2 blockers inhibited B cell migration in vitro indicating

that phosphorylation of these proteins is essential in this process

_{. Thus, blocking}

BTK phosphorylation may be an attractive novel therapeutic strategy for AAV to not

only decrease effector B cell functioning but also effector B cell (e.g. memory B cell)

migration to sites of inflammation.

The current therapy options for remission maintenance for AAV include rituximab, AZA

or MMF combined with glucocorticoids. Interestingly, one open label clinical research

trial showed that MMF-treated AAV patients were more prone for disease relapses than

AZA-treated AAV patients

_{. Interestingly, the opposite is seen in SLE patients, where}

MMF is more effective in the maintenance of remission

_{. The effect of MMF and}

AZA on the immune system, and particularly on B cells, remains poorly understood.

We hypothesized that the increased relapse rate in MMF-treated AAV patients may be

due to a disbalance in Breg and effector B cell frequencies induced by MMF treatment.

Therefore, we investigated in Chapter 5 whether the distribution of Bregs and effector

B cells (identified based on their cytokine production) are differentially affected by

AZA and MMF in AAV patients. This study consisted of two parts. In part I, PBMCs of

untreated remission GPA patients and HCs were stimulated with CpG combined with

mycophenolic acid (MpA) or 6-mercaptopurine (6-MP) (i.e. the active compounds of

MMF and AZA, respectively) for three days. In part II, B cell functioning was determined

in GPA patients that were on maintenance therapy consisting of either AZA or MMF at

the time of blood sampling. To do so, we cultured PBMCs of these GPA patients and

stimulated the cells with CpG for three days. We showed that B cell proliferation was

decreased by MpA and 6-MP compared to CpG only, whereas no difference was found

in B cell proliferation in samples of actively treated GPA patients. Interestingly, in vitro

stimulation of PBMCs from GPA patients in the presence of MpA showed a decreased

IL-6

_{B cell frequency compared to 6-MP-treated GPA samples (Figure 3). Importantly,}

the IL-10

_{B cell frequency was decreased by MpA in HC samples only whereas 6-MP did}

not affect the cytokine-positive B cell frequencies at all. Although these results seem to

indicate that the cytokine profile of B cells in MpA-treated samples is shifted towards a

less pro-inflammatory state, these results need to be validated in larger patient cohorts.

7

Additionally, these immunosuppressive drugs are broad inhibitors of immune cell

activation not only aff ecting B cells but other immune cells as well.

B cell IL-10 regulatory effector TNF⍺

+ Azathioprine

/6-mercaptopurine

+ Mycophenolate

Mofetil

/Mycophenolic Acid

CpG

Figure 3. B cell cytokine production in PBMCs of remission GPA patients and HCs in vitro upon exposure to 6-MP or MpA and in CpG-stimulated PBMCs of GPA patients receiving AZA or MMF treatment. Top: The eff ect of CpG stimulation alone on B cell cytokine production. Middle: The eff ect of in vitro AZA or 6-MP on B cell cytokine production compared to MMF/MpA treatment. Bottom: The eff ect of in vitro MMF or MpA on B cell cytokine production compared to AZA/6-MP treatment.

After GPA patients have achieved remission, the most important question for the

treating clinician is how to prevent a relapse most eff ectively with the least amount of

toxicity. To accomplish this, clinical and/or serological disease related factors need to

be identifi ed that reliably predict the risk for disease relapse in each patient. To date,

the most extensively investigated disease-related factor in GPA to predict relapse is the

serum PR3-ANCA titer and changes herein over time. However, results of these studies

are inconsistent and the value of serial PR3-ANCA measurements in clinical practice has

been a matter of debate. A persistently positive ANCA titer during disease remission has

been reported to be associated with future disease relapses

_{, whereas others found}

that rather a rise in serum PR3-ANCA titer over time preceded a relapse

_{. In contrast,}

others did not find an association between serum PR3-ANCA levels and ensuing

disease relapses

_{. Indeed, a meta-analysis by Tomasson et al.}

_{showed that changes}

in serum PR3-ANCA titer over time are only weak predictors of disease relapses and

are ineffective in predicting disease relapses for all AAV patients. However, more recent

research suggests that serial PR3-ANCA measurements may be helpful in identifying

relapses in patients presenting with renal involvement and alveolar hemorrhage

_.

Additionally, PR3-ANCA levels should also be determined using novel methods (e.g. by

Phadia analyzer) to investigate whether such methods are more sensitive in detecting

rises in PR3-ANCA levels which may improve relapse prediction based on changes in

ANCA levels.

It has also been proposed that the prevalence of particular B cell subsets in the circulation

of AAV patients in remission may be an alternative disease related indicator for relapse

risk. The introduction of rituximab treatment particularly allowed for a unique way to

investigate whether specific distribution profiles of repopulated peripheral B cell subsets

associate with relapses

_{. Repopulation of circulating memory B cells at six months post}

last rituximab infusion was found to be associated with increased relapse risk, whereas

patients with naïve B cell repopulation experienced less relapses

_{. Interestingly,}

another study showed that increased proportions of circulating CD5

_{Bregs upon B cell}

repopulation in AAV patients were related to prolonged remission whereas patients

with lower CD5

_{Breg frequencies showed a shorter time to relapse}

_{. However, not all}

GPA patients are treated with rituximab. Thus, in Chapter 6 we examined whether B cell

subset frequencies could predict future disease relapses in GPA patients in remission

not treated with rituximab. We showed that an increased frequency of circulating

plasmablasts was associated with decreased relapse-free survival. Circulating

plasmablast frequencies showed a trend towards a decrease in the last blood sample

collected 1-6 months prior to the relapse, which might indicate plasmablast migration

to sites of inflammation in GPA. Therefore, we stained plasmablasts in kidney biopsies

and urine of active AAV patients with renal involvement. Together, these results indeed

suggest that plasmablasts migrate from the circulation to sites of inflammation and that

monitoring circulating plasmablast frequencies might be a useful indicator for future

disease activity.

Plasmablasts are a result of the germinal center reaction and are migrating to plasma cell

niches mainly in the bone marrow to become plasma cells. In contrast to plasma cells,

plasmablasts are capable of producing only low amounts of antibodies. Plasmablasts

7

are, like plasma cells, typically present in low numbers in the circulation

_{. Importantly,}

in other autoimmune diseases increased plasmablast frequencies were found to

correlate with autoAb levels and disease activity

_{. The data presented in}

Chapter 6 suggest that in patients with upcoming relapses, B cells are already activated

and instructed to diff erentiate towards plasmablasts. The decrease in plasmablasts in

the last sample before relapse might highlight diff erentiation into plasma cells and

migration of these cells to sites of infl ammation. Although plasmablasts were related to

decreased relapse free survival when present in higher frequencies, we did not detect

a correlation between plasmablast frequencies and ANCA titers. Nonetheless, others

have shown that both memory B cells and plasmablasts are the predominant B cell

subsets of GPA patients to react with PR3

_{. Recently, PR3-ANCA-positive B cells have}

been detected in infl amed tissues of GPA patients which is in line with our view that

autoreactive B cells migrate from the circulation to sites of infl ammation

_{. However, it}

is currently not known whether plasmablasts at sites of infl ammation can secrete

PR3-ANCA.

+

Future disease relapse Remission

effector regulatory

Plasmablasts

Urine Peripheral_Blood

Active Disease

Kidney

Figure 4. Plasmablasts as indicators for future disease relapses. During remission (top), future relapsing GPA patients demonstrated with increased plasmablast frequencies. During active disease (bottom), plasmablast frequencies were increased in the urine of GPA patients with renal involvement, whereas this frequency was lower in peripheral blood. Additionally, plasmablasts infi ltrated the infl amed kidney in these patients.

Although plasmablast frequencies might provide a novel marker to identify patients

at risk for relapse, a predictive B cell marker at an earlier stage of the disease is highly

preferred. Plasmablasts are already at a “late” differentiation stage and are in the process

of becoming Ab-producing plasma cells. Ideally, in AAV, but also in other autoimmune

diseases, the formation of pathogenic autoAb-producing cells is prevented. This would

be possible if patients at risk for relapse are identified earlier during disease remission

but this requires a marker that indicates increased activation of B cells at earlier

differentiation stages. BTK protein and its phosphorylation levels in newly emerging

B cells might provide such a B cell-specific early marker. In Chapter 4 we showed

that BTK levels were increased in transitional and naïve B cells of active patients only.

Importantly, these increased BTK levels correlated with B cell activation. Although in

our study BTK levels were not increased in remission GPA patients, it is likely that B cells

become increasingly activated in patients that are about to relapse. Currently, data on

BTK protein and phosphorylation levels in newly emerging transitional and naive B cells

of remission patients with approaching relapse is lacking. This is however of interest

because if newly emerging B cells in remission patients with future relapses indeed

show increased BTK levels, it would indicate increased B cell activation and might

identify patients at risk for relapse. The possibility of measuring B cell BTK levels as an

indicator for future relapse should be tested in a larger cohort with multiple fixed time

points to analyze changes in BTK levels over time.

Future Perspectives

The work presented in this thesis contributes to our knowledge on the role of B cells

in the pathogenesis of GPA and explored their potential as target for novel treatment

strategies and predictors of relapses. We have studied the distribution of B cell subsets

in GPA patients and found that an increased frequency of circulating plasmablasts was

associated with decreased relapse-free survival. Moreover, plasmablast frequencies

were found to be decreased in blood samples collected 1-6 months prior to relapse

of the disease and these cells could be detected in kidney biopsies and urine of GPA

patients with active renal disease. To this end, analyses of B cell subset distribution and

monitoring the frequency of plasmablasts in blood and urine could be informative as an

indicator of disease status and possibly aid in the recognition of an upcoming relapse in

AAV patients. Future studies in larger patient cohorts, including a cohort of

rituximab-treated GPA patients, are however necessary to substantiate whether plasmablast

frequencies are indeed predictive of relapses in GPA and should determine whether

these include plasmablasts that produce ANCA. Such studies will open up new avenues

for future use of these cells as markers for (upcoming) disease activity or as targets for

novel therapeutic strategies.

We also investigated the association between Bregs and the expanded Th17 cell

response in GPA patients. We observed an inverse correlation between circulating Bregs

7

and Th

17 cells in GPA patients, and in vitro Breg depletion resulted in an increased Th17

cell frequency. Thus, a reduction of circulating Bregs in GPA patients may contribute

to increased numbers of Th17 cells which release IL-17, a pro-inflammatory cytokine

implicated in cell migration and granuloma formation in GPA patients. Future studies

are required to identify the signals that induce Breg expansion, as this may provide clues

to develop novel strategies to control Th17 cell responses in GPA patients and perhaps

autoimmune diseases in general. Moreover, it is essential that consensus is reached on

Breg phenotype(s), e.g. based on (novel) surface or functional markers or combinations

thereof, to truly discover the regulatory potential of these cells. Only then the potential

of therapeutic Breg transfer upon ex vivo expansion can be investigated as a possible

treatment strategy to restore immune balance.

In addition to the disturbed B cell subset distribution, we demonstrated alterations in

the BCR signaling pathway in newly emerging transitional and naive B cells of active

GPA patients. These B cells showed increased BTK levels whereas blocking BTK activity

inhibited B cell cytokine and IgG production, and plasma cell formation in vitro. Hence,

BTK might be a novel therapeutic target to dampen B cell activation in GPA patients

and future preclinical and clinical trials should establish whether BTK is a potential

novel treatment option for these patients. Our findings on intracellular BCR signaling

molecules such as BTK may also hold promise for the discovery of novel biomarkers

for (upcoming) disease activity. To investigate the potential of BTK as a biomarker, the

dynamics of BTK expression in B cells should be investigated in larger (longitudinal)

studies of future-relapsing and non-relapsing AAV patients.

Classically, the therapeutic strategy for GPA consists of remission induction and

maintenance therapy using immunosuppressive medication. However, B cell depletion

therapy by rituximab is increasingly applied for remission induction and maintenance in

GPA patients. Although rituximab is an efficacious therapeutic strategy, it is not specific

for the autoreactive B cells since it depletes all B cells including Bregs. The suggested

Breg expansion or BTK blockade might provide an additional therapeutic strategy

for AAV, and this could be more specific by inhibiting Th17 cell activation and B cell

activation, respectively.

However, the most ideal therapy for autoAb mediated autoimmune diseases would

be one that specifically depletes autoreactive B cells only. Interestingly, Ellebrecht et

al. tested such a novel therapy to deplete autoreactive B cells in pemphigus vulgaris

(PV)

_{. PV is a severe autoimmune disease that involves blistering of the skin and}

oral mucosa and is characterized by autoAbs directed against keratinocyte adhesion

proteins (e.g. Dsg3). The authors engineered T cells with a chimeric autoAg receptor

(CAAR) and showed that these CAAR T cells specifically eliminated autoreactive

Dsg3-specific B cells in vitro and in a PV mouse model

_{. This elegant study provides proof of}

principle that this form of cellular immunotherapy might be a future approach to treat

autoimmune diseases. The fact that this arising therapy specifically targets autoreactive

B cells makes it a very promising research field that could ultimately lead to an effective

and durable treatment of AAV and other autoimmune diseases.

7

References

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