University of Groningen Modulation of T and B cell function in Granulomatosis with polyangiitis Lintermans, Lucas Leonard

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University of Groningen

Modulation of T and B cell function in Granulomatosis with polyangiitis

Lintermans, Lucas Leonard

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Lintermans, L. L. (2019). Modulation of T and B cell function in Granulomatosis with polyangiitis: Targeting Kv1.3 potassium channels. Rijksuniversiteit Groningen.

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527482-L-bw-Lintermans 527482-L-bw-Lintermans 527482-L-bw-Lintermans 527482-L-bw-Lintermans Processed on: 2-1-2019 Processed on: 2-1-2019 Processed on: 2-1-2019

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Granulomatosis with polyangiitis

Targeting Kv1.3 potassium channels

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Processed on: 2-1-2019 PDF page: 2PDF page: 2PDF page: 2PDF page: 2 Colofon

Layout: Douwe Oppewal Printing: Ipskamp Printing

ISBN: 978-94-034-1345-7 printed version 978-94-034-1344-0 e-book

Cover image: Stichodactyla helianthus by Florent Charpin (reefguide.org)

Copyright © 2018 by L.L. Lintermans. All rights reserved. Any unauthorized reprint or use of this material is prohibited. No part of this thesis may be reproduced, stored or transmitted in any form or by any means, without permission of the author or, when appropriate, of the publishers of the publications.

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Granulomatosis with polyangiitis

Targeting Kv1.3 potassium channels

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de

UHFWRUPDJQL¿FXVSURIGU(6WHUNHQ en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op PDDQGDJIHEUXDULRPXXU

door

Lucas Leonard Lintermans

JHERUHQRSPDDUW te Villa Maria, Oeganda

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4

Promotores

Prof. dr. P. Heeringa Prof. dr. C.A. Stegeman

Copromotores

Dr. W.H. Abdulahad Dr. A. Rutgers

Beoordelingscommissie

Prof. dr. A.L.W. Huckriede Prof. dr. S.P. Berger Prof. dr. B. Wilde

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5 Yannick van Sleen

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9

CHAPTER 1 General introduction and outline of the thesis

CHAPTER 2 T cells in vascular inﬂammatory diseases

Front. Immunol. 5, 504 (2014)

CHAPTER 3 Chemokine receptor co-expression reveals aberrantly

distributed T

_H

eﬀector memory cells in GPA patients

Arthritis Research & Therapy (2017) 19:136

CHAPTER 4 Kv1.3 blockade by ShK186 modulates CD4

+

_eﬀector

memory T-cell activity of patients with granulomatosis

with polyangiitis in vitro.

Submitted

CHAPTER 5 Kv1.3 channel blockade modulates the eﬀector function

of B cells in granulomatosis with polyangiitis

Front Immunol. 8:1205 (2017)

CHAPTER 6 Circulating

CD24

hi

_CD38

hi

_{regulatory B-cells correlate}

inversely with the frequency of TH

_EM

17-cells in

granulomatosis with polyangiitis patients

Accepted for publication; Rheumatology

CHAPTER 7 Summary, general discussion, and future perspectives

CHAPTER 8 Nederlandse

samenvatting

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11

GENERAL INTRODUCTION AND

OUTLINE OF THE THESIS

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12

ANCA-associated vasculitis

ANCA-associated vasculitides (AAV) are a group of autoimmune diseases characterized by a chronic, and often systemic, inﬂammation of medium- to small-sized blood vessels 1_{. AAV}

encompasses three clinically deﬁned disorders: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA) 1_{. In the}

majority of patients, the disease is hallmarked by the presence of anti-neutrophil cytoplasmic antibodies (ANCA). These autoantibodies are considered to play an important role in the pathogenesis of the diseases 2_{. In AAV, ANCA are mainly directed against proteinase-3 (PR3)}

and myeloperoxidase (MPO) 3_{.PR3 and MPO are enzymes present in cytoplasmic granules of}

neutrophils and become accessible for circulating ANCA on the surface of neutrophils after pre-activation (priming) of these cells. Generally, PR3-ANCA are present in the majority of patients with GPA, whereas in MPA patients MPO-ANCA is more prevalent 4_{. Patients with PR3-ANCA}

and MPO-ANCA are also characterized by diﬀerences in their clinical presentation. PR3-ANCA is strongly associated with granulomatous inﬂammation of the upper and lower respiratory tract and a more systemic presentation of the disease frequently involving the kidney whereas patients with MPO-ANCA often present with a renal limited form of vasculitis 5_.

Below, I brieﬂy introduce the cellular players that fulﬁll central roles in the pathogenesis of AAV and discuss how selective targeting of these pathogenic immune cells may hold therapeutic promise for patients with AAV, focusing mainly on GPA patients.

AAV pathogenesis

The etiopathogenesis of AAV is not completely understood. However, multiple cellular players have been proposed to be involved including i) neutrophils, expressing the ANCA target antigens, ii) B cells, being responsible for the production of ANCA, and iii) T cells, mediating the (auto)-inﬂammatory response in disease development 6_.

In AAV pathogenesis, it has been suggested that pro-inflammatory factors e.g. released due to an infection, trigger the disease. In particular, Staphylococcus aureus (S. aureus) has been shown to be an important risk factor for the occurrence of relapses in AAV and anti-bacterial treatment is beneficial in reducing the relapse rate in these patients 7, 8_{. Pro-inflammatory cytokines and}

chemokines that are released as a result of local or systemic infection cause priming of neutrophils, upregulation of endothelial adhesion molecules, and an expansion of circulating effector T cells. Neutrophil priming results in translocation of the ANCA antigens (i.e. PR3 and MPO) from their lysosomal compartments to the cell surface. Engagement of the ANCA with either PR3 or MPO on the cell surface and interaction of the Fc part of the antibody with Fc receptors activates neutrophils. This causes increased neutrophil adherence to the endothelium and transmigration through the vessel wall. ANCA-mediated neutrophil activation also triggers the production of reactive oxygen species (ROS) and induces neutrophil degranulation of proteolytic enzymes causing vessel wall damage. Meanwhile, the injury to the vessel wall in combination with the pro-inflammatory triggers elicit an adaptive inflammatory immune response recruiting T cells that further contribute to the development of vasculitis. Additional disbalances in the T cell

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13 compartment result in further release of pro-inﬂammatory cytokines promoting neutrophil priming and persistent activation of T cells that sustain the vascular inﬂammatory response in AAV.

T cell involvement in AAV

Besides ANCA mediated neutrophil responses, the pro-inﬂammatory environment that is created will also attract T cells from the adaptive immune system. The involvement of CD4+_{T helper}

(T_H) cells in the pathogenesis of AAV, in particular GPA, is supported by several observations. First, abundant T cell inﬁltrates can be detected in inﬂammatory lesions found in AAV and CD4+

T cells are a prominent component of the granuloma frequently observed in GPA 9_{. Second,}

soluble T cell activation markers are elevated in serum and plasma of AAV patients compared to controls and are associated with disease activity 10, 11_{. Third, ANCA antigen speciﬁc T cells have}

been detected in the circulation of AAV patients 12, 13_{. Fourth, the IgG subclass distribution of}

ANCA with a predominance of IgG1 and IgG4 subclasses implies isotype switching indicating a T cell dependent immune response 14_{. Collectively, these observations make it highly likely that T}

cell mediated inﬂammatory responses contribute importantly to AAV pathogenesis.

The CD4+_T_{cell population can be separated into four distinct subsets based on the surface}

expression of the phosphatase CD45RO and the lymph node homing chemokine receptor CCR7

15_{. Naïve T cells receiving a relatively weak T cell receptor (TCR) signal and antigen presenting}

cell-derived co-stimulation will proliferate and differentiate into long lived central memory T (T_CM) cells, whereas strong TCR stimulation or prolonged repeated stimulations favors the differentiation into effector memory T (T_EM) cells. Naïve T cells and T_CM cells express CCR7 and efficiently home to the lymph nodes and exert limited effector functions upon antigen exposure. T_EM cells lack the expression of CCR7 but express other chemokine receptors that facilitate migration to non-lymphoid sites of inflammation. These cells are poised for a rapid response to repeated antigen exposure by the production of effector cytokines. Therefore, it is plausible that CD4+_T

EM cells

may directly contribute to tissue injury and disease progression in GPA 15, 16_.

In GPA, a persistent expansion of CD4+_T

EM cells has been observed in the peripheral blood

of GPA patients in remission, but not in GPA patients with active disease 17_{. A follow up study}

revealed the presence of CD4+_T

EM cell in the urine of GPA patients, indicating that CD4 +_T

EM cells

migrate from the circulation to inﬂammatory lesion during active episodes of the disease 18_.

Moreover, the phenotypes of T cells found locally at the inﬂammatory sites in lung and kidney tissues mainly resemble those of memory T cells 9, 19_.

The CD4+_T

EM cell population consists of diﬀerent lineage-committed TH cell phenotypes

that can be distinguished according to surface makers and secreted signature cytokines. These phenotypes include T_H1 cells; characterized by CXCR3 and IFN-γ, T_H2 cells; characterized by CRTh2 and IL-4, T_H17 cells; characterized by CCR6 and IL-17, and, regulatory T (T_REG) cells; characterized by CD25 and the transcription factor FoxP3 20_{. In AAV, the T}

H cell polarization deviates from the

healthy situation depending on disease state (i.e. active disease vs remission) and/or disease category (i.e. localized vs generalized). For instance, a T_H1–type response is predominant in GPA

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patients with localized disease, whereas a T_H2-response is associated with more generalized disease 21-24_{. Furthermore, increased T}

H17-type responses reﬂected by elevated levels of IL-17 and

the presence of auto-antigen speciﬁc T_H17 cells are observed in GPA patients 25, 26_{. Together, these}

T_H responses contribute to the pro-inﬂammatory eﬀector response involved in the pathogenesis of GPA.

In addition, CD4+_T

EM cells also display cytotoxic features similar to natural killer (NK) cells 27_.

They have been shown to mimic features of NK cells by their surface expression of the natural killer group 2 member D (NKG2D). NKG2D can mediate cytoxic responses and tissue damage through speciﬁc interaction with its ligand MICA expressed on target cells 28, 29_{. Interestingly, it has}

been reported that NKGD2 is prefentaily expressed on circulating CD4+_T

EM cells and both NKG2D

and MICA are expressed in the granulomatous lesion in GPA patients 30_{. It is likely that killing}

mechanisms via NKG2D-MICA interaction contribute to vessel injury and disease progression in AAV-patients.

The observed abnormalities of the expanded CD4+_T

EM cell compartment in GPA patients are

in part attributed to deregulated expression of cytokines but may also be inﬂuenced by aberrant functioning of T_REG cells. Under normal physiological conditions T_REG cells have the capacity to suppress the activation, proliferation and eﬀector fucntions of CD4+_T

H cells. However, in AAV

several studies have reported an impaired functionality of the T_REG cells demonstrating that T_REG from AAV patients are not able to suppress the proliferation of CD4+_T

EM cells

31-33_{. Thus, the}

dysfunctional T_REG cells may cause expansion of the CD4+_T

EM population and disbalances in

eﬀector cells. To date, the underlying mechanisms responsible for the functional impairment of T_REG cells in AAV patients remains unclear.

Taken together, the persistent expansion of the CD4+_T

EM cells in combination with the lack

of inhibitory mechanisms by T_REG cells may promote T_EM cell eﬀector functions and migration to inﬂamed tissues in AAV. Therefore, CD4+_T

EM cells constitute a potentially interesting cellular

target for pharmacological intervention in GPA patients. B cell involvement in AAV

In AAV pathogenesis B cells are considered crucial because these cells are the precursors for plasma cells that produce the ANCA (like the PR3-ANCA IgG in GPA patients). However, accumulating evidence indicates that beside their antibody-producing role, B cells also exert multiple other functions that inﬂuence immune responses. B cells are eﬀective antigen presenting cells (APCs) and can regulate T cell responses by providing co-stimulatory signals and secretion of cytokines

34, 35_{. Depending on the cytokines secreted, B cells can be divided into eﬀector cells producing}

pro-inflammatory cytokines or regulatory B (B_REG) cells producing anti-inflammatory cytokines. Effector B cells can stimulate T_H1 and T_H17 cell responses by the secretion of IFN-γ, IL-6, and, TNF-α

36, 37_{. These eﬀector cytokines (i.e. TNF-α) are mainly produced by B cells from the memory B cell}

compartment, including unswitched memory B cells (CD27+_IgD+_{) and class-switched memory B}

cells (CD27+_IgD-₎38_{. In GPA patients it has been found that both memory B cell phenotypes are}

decreased in the peripheral blood irrespective of the disease state 39_{. Currently the reason for}

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15 this decrease remains unclear. However, B cells have been detected in the granulomatous lesions of AAV patients 40_{. Therefore, it cannot be excluded that the memory B cells migrate from the}

circulation to sites of inﬂammation and exert their eﬀector functions locally similar as the CD4+

T_EMcells.

In contrast to their pro-inflammatory effector functions, B cells can also present anti-inflammatory regulatory functions via secretion of IL-10 and TGF-β that inhibit T_H cell responses and modulate the number of T_REG cells 41, 42_{. Currently, the phenotypic identification of B}

REG cells

remains controversial and relies on the detection of IL-10 production. However, it has been suggested that B_REG cells can be identiﬁed based on the high expression of surface CD24 in combination with either CD38 or CD27 43_{. Interestingly, studies have shown that alterations}

in numbers and/or function of CD24hi_CD38hi_B

REGS are associated with progression of several

autoimmune diseases and these cells are able to inhibit CD4+_T

H responses

44, 45_{. Therefore, similar}

to CD4+_T

EM cells, it could be beneficial to selectively target the pro-inflammatory effector B cells

within the memory B cell compartment without impairing the regulatory function of B cells. Current therapy

The current treatment recommendations in the management of AAV are based on the severity of the disease and the organs involved. High dose glucocorticoids in combination with cyclophosphamide (CYC) is generally used as treatment for induction of remission in AAV patients 46, 47_{. Alternatives to CYC such as methotrexate (MTX) or mycophenolate mofetil (MMF)}

are, compared to CYC, inferior for induction of remission in patients with either non-severe disease or patients that do not tolerate CYC well 48, 49_{. More recently, B cell depletion by rituximab}

(RTX, anti-CD20) treatment has been shown to be equally eﬃcacious as CYC for induction of remission in AAV 50, 51_{. Subsequent to the initial therapy aimed to induce remission, patients}

receive maintenance therapy to prevent disease relapses. The maintenance treatment regimen consists of azathioprine (AZA) or MMF often in combination with low-dose glucocorticoids 52, 53_.

MTX is another option for maintenance treatment and has been shown to be similarly eﬀective in sustaining remission compared to AZA but tends to be associated with more severe adverse events in AAV patients 54_{. Interestingly, recent data indicate that RTX is superior to AZA in}

maintaining CYC induced remission 55_{. However, optimal dosing regimens, long-term safety and}

eﬃcacy, as well as cost eﬀectiveness have still to be addressed.

Overall, these current treatment strategies in the management of AAV have changed AAV from fatal diseases to chronic (relapsing) diseases. However, many patients still experience relapses during the course of their disease 56, 57_{. PR3-ANCA patients especially are at risk for disease}

relapse, which in 30-50% of the patients occurs within 5 years after diagnosis 58_{. Renewed disease}

activity exposes patients to more immunosuppressive therapy and accumulating organ damage. Therefore, new treatment options are needed to avoid drug related toxicity and prevent the accumulation of organ damage due to the chronic course of the (frequently) relapsing disease. Preferably, such new treatments should speciﬁcally target pathogenic cellular players involved in the pathophysiology of AAV.

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Ion channels on Lymphocytes

As described above, neutrophils, T cells and, B cells are closely connected in the pathogenesis of AAV. In particular, the effector T and B cells position themselves as interesting therapeutic targets because of their pro-inflammatory properties. Ion channels comprise a network that perform vital functions in the cellular homeostasis, activation and differentiation of T and B lymphocytes59_.

Of particular interest are potassium (K+_{) channels that serve to regulate the membrane potential}

and calcium signaling in lymphocytes. Human T lymphocytes express two types of K+_channels,

namely the voltage-gate Kv1.3 potassium channel and the calcium-activated KCa3.1 potassium channel. Moreover, the expression of Kv1.3 and KCa3.1 channels on T lymphocytes depends on the state of activation and diﬀerentiation of a given T lymphocyte subset 60_.

T cells at each diﬀerentiation state have either a quiescent or activated state when encountered by an antigen. Patch clamp analysis revealed that quiescent T naïve, T_CM, and T_EM cells express about 200 – 300 Kv1.3 channels and 5 – 35 KCa3.1 channels per cell (table 1) 61_{. The}

expression-pattern of these channels changes upon T cell activation, leading to altered channel phenotypes in the diﬀerent T cell subsets. Activated T naïve and T_CM cells upregulate KCa3.1 channels to 500 channels per cell, whereas T_EM cells increase Kv1.3 expression to 1500 channels per cell with little change in KCa3.1 expression levels 61_{. The switch of potassium channel phenotype signiﬁcantly}

aﬀects the responsiveness of these cells to Kv1.3 or KCa3.1 blockers. Therefore, T_EM cells are highly sensitive to Kv1.3 channels blockers, while T naïve and T_CM cells are more sensitive to KCa3.1 channel blockers.

One of the earliest events in T cell activation is the increase in intracellular calcium concentrations 62, 63_{. The Kv1.3 channels play a critical role in this process}64_{. Antigen presentation}

to the T cell receptor leads to rapid release of calcium from endoplasmic reticulum (ER) stores. Depletion of the ER Ca2+_{stores causes Ca}2+_{release-activated calcium (CRAC) channels to open}

in the membrane ensuring extracellular calcium to enter the cell. The inﬂux of Ca2+_raises

the intracellular calcium concentration that subsequently culminates in cell activation and proliferation. The large influx of calcium through the CRAC channels induces cell depolarization, which, if left unchecked, induces a reduction in calcium influx. However, the driving force for calcium entry is restored by membrane hyperpolarization induced by the efflux of potassium through the Kv1.3 and KCa3.1 channels. The tight interplay between calcium influx through CRAC channels and potassium efflux through Kv1.3 and KCa3.1 channels underlies the oscillating changes in calcium concentrations necessary for T cell activation.

Similar to T cells, human B cells undergo a comparable diﬀerentiation process. Naïve B cells (IgD+_CD27-_{) diﬀerentiate into unswitched memory B cells (IgD}+_CD27+_{), and upon}

repeated stimulation these cells diﬀerentiate further into class-switched memory B cells (IgD

-CD27+_{) lacking IgD but expressing either IgG, IgA, or IgE on their cell surface. The expression}

of Kv1.3 and KCa3.1 channels on B cells is identical to the channel expression patterns during diﬀerentiation and activation as it does for T cells 65_{. Naïve and unswitched memory B cells, like}

their T cell counterparts (T naïve and T_CM cells), up-regulate KCa3.1 channels upon activation,

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17 whereas class-switched memory B cells, like T_EM cells, up-regulate Kv1.3 channels upon activation

65_{. Interestingly, quiescent class-switched memory B cells express much higher Kv1.3 levels}

compared to quiescent T_EM cells (table 1).

Like T cells, the pharmacological sensitivity of B cells to potassium channel blockers parallels their potassium channel expression pattern. KCa3.1 speciﬁc blockers inhibit the proliferation and activation of naïve and unswitched memory B cells, whereas Kv1.3 blockers suppress the proliferation and activation of class-switched memory B cells 65_.

Table 1 | Number of Kv1.3 and channels per cell in T and B cell subsets

Lymphocyte Potassium Channel Naïve T cells (CD45RO-_CCR7+₎ TCM cells (CD45RO+_CCR7+₎ TEM cells (CD45RO+_CCR7-₎ Quiescent Active Quiescent Active Quiescent Active

T cell Kv1.3 300 300 300 300 300 1500 Naïve B cells (IgD+_CD27-₎ Unswitched memory B cells (IgD+_CD27+₎ Class-switched memory B cells (IgD-_CD27+₎

Quiescent Active Quiescent Active Quiescent Active

B cell Kv1.3 100 90 255 180 2430 3270

T_EM cells are main players in mediating the pathophysiological processes of various autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, Type 1 diabetes mellitus, as well as in GPA 61, 66, 67_{. As described above, these T}

EM cells express high numbers of Kv1.3 channels upon

activation, which lend themselves to selective targeting by Kv1.3 channel blockers 61, 66_{. Targeting}

these T_EM cells without aﬀecting the T naïve and T_CM cells represents a promising and more speciﬁc way for treating autoimmune diseases avoiding generalized immunosuppression. Furthermore, the Kv1.3 channels expressed as homotetramers, have a functionally restricted tissue distribution for lymphocytes, and therefore represent attractive therapeutic targets in T_EM cell mediated autoimmune disorders.

Kv1.3 inhibitors are found in many venoms including that of sea anemones. In 1995, a potent potassium channel blocker was extracted from the Caribbean sea anemone Stichodactyla

helianthus and termed Stichodactyla helianthus K+_{channel toxin (ShK)}68_{. Soon after the discovery}

of the native peptide, the peptide was successfully synthesized, and its three-dimensional structure was determined 69_{. Further extensive studies of its structure, selectivity, biological}

activity in conjunction with the generation of analogs with increased selectivity and stability, resulted in a synthetic form of ShK that blocks the Kv1.3 channels in T_EM cells with picomolar aﬃnity 70_{. Subsequently, several studies demonstrated that activation of disease-associated}

(autoreactive) T_EM cells can be inhibited by a ShK-mediated Kv1.3 blockade. Selective blockade of the Kv1.3 channels has proven eﬃcacious in preventing and/or treating animal models of delayed type hypersensitivity, type 1 diabetes, rheumatoid arthritis and multiple sclerosis without inducing generalized immunosuppression 66, 71-73_.

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Aim and outline of this thesis

Advances in the treatment of GPA have led to increased patient survival. However, the prolonged exposure of patients to generalized immunosuppressive therapy carries a heavy burden of adverse events including opportunistic infections and drug related toxic effects. To minimize or circumvent these therapy related adverse effects, tapering or discontinuation of treatment is required. Consequently, GPA patients suffer from frequent disease relapses where each relapse is associated with the risk of cumulative organ damage. This emphasizes the need for improved treatment strategies that are more specific and less toxic for GPA patients. Such improved therapeutic options should preferably be directed to the key cellular players in GPA pathogenesis.

The main aim of this thesis was to investigate the effect of the highly specific Kv1.3 channel inhibitor ShK-186 on the effector functions of CD4+_T

EM cells and B cells to provide

proof of principle for Kv1.3 blockade as a potential novel treatment strategy for GPA with high specificity towards these pathogenic cellular players. The effector functions of T and B cells were determined in GPA patients and it was investigated whether specific blockade of Kv1.3 channels was effective in reducing the pro-inflammatory functions of these cells. In addition, we characterized the phenotype of circulating CD4+_T

EM subsets in GPA patients in relation with

the clinical presentation of the disease in these patients. Finally, a potential interplay between regulatory B cells and the expanded T_H17 population in GPA patients was investigated.

In chapter 2, we reviewed the literature regarding the role of T cells in systemic autoimmune and chronic inflammation. The current knowledge regarding the behavior of T cells in these two distinct inflammatory conditions was discussed to illustrate the characteristics of T cell features in AAV and atherosclerosis. Particular attention was given to the different T cell phenotypes, the role of effector memory T cell responses and the modulation of the effector T cell responses.

In chapter 3 we investigated the distribution of diﬀerentiated T cell phenotypes based on the co-expression of chemokine receptors. We delineated diﬀerences in the distribution of CD4+

T_EM phenotypes and analyzed whether these cells associated with the heterogeneous clinical presentation of the disease.

Chapter 4 assessed the eﬀect of the Kv1.3 channel blocker (ShK-186) on the pro-inﬂammatory properties of CD4+_T

EM cells from GPA patients compared to CD4 +_T

EM cells from healthy individuals

in vitro.

Besides T cells, Kv1.3 channels are expressed on all B cells among which class-switched memory B cells in particular express high levels of Kv1.3 channels. Therefore, in chapter 5, we characterized the distribution of circulating B cell subsets in GPA patients and studied the eﬀect of ShK-186 on B cell cytokine production, proliferation and (PR3-ANCA) IgG production.

Accumulating evidence indicates that B cells modulate T cell responses. In particular, a subset of B cells characterized by high expression of CD24 and CD38, termed regulatory B cells (B_REG), has been reported to exert regulatory functions modulating the distribution of CD4+_T

H subsets.

In chapter 6 we hypothesized that numerical alterations in CD24hi_CD38hi_B

REG cells may explain

the expansion of the T_H17 population in GPA patients. To test this hypothesis, we assessed the

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19 frequency of circulating CD24hi_CD38hi_B

REG cells and TH17-cells in GPA patients and investigated

the functional impact of these B_REGcells on the expanded frequency of the T_H17-cells in vitro. Finally, chapter 7 summarizes and discusses the main ﬁndings and future perspectives of the research presented in this thesis in the context of the current literature.

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T CELLS IN VASCULAR

INFLAMMATORY DISEASES

Lucas L. Lintermans1_{, Coen A. Stegeman}2_{, Peter Heeringa}3_{, Wayel H. Abdulahad}1 1 _{Department of Rheumatology and Clinical Immunology,}2 _{Department of Nephrology,} 3 _{Department of Pathology and Medical Biology, University of Groningen, University Medical}

Center Groningen, Groningen, Netherlands

Front. Immunol. 5, 504 (2014)

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ABSTRACT

Inflammation of the human vasculature is a manifestation of many different diseases ranging from systemic autoimmune diseases to chronic inflammatory diseases, in which multiple types of immune cells are involved. For both autoimmune diseases and chronic inflammatory diseases several observations support a key role for T lymphocytes in these disease pathologies, but the underlying mechanisms are poorly understood. Previous studies in several autoimmune diseases have demonstrated a significant role for a specific subset of CD4+_{T cells termed effector memory}

T cells. This expanded population of effector memory T cells may contribute to tissue injury and disease progression. These cells exert multiple pro-inflammatory functions through the release of effector cytokines. Many of these cytokines have been detected in the inflammatory lesions and participate in the vasculitic reaction, contributing to recruitment of macrophages, neutrophils, dendritic cells, NK cells, B cells and T cells. In addition, functional impairment of regulatory T cells paralyzes anti-inflammatory effects in vasculitic disorders. Interestingly, activation of effector memory T cells is uniquely dependent on the voltage-gated potassium Kv1.3 channel providing an anchor for specific drug targeting. In this review, we focus on the CD4+_{T cells in the}

context of vascular inflammation and describe the evidence supporting the role of different T cell subsets in vascular inflammation. Selective targeting of pathogenic effector memory T cells might enable a more tailored therapeutic approach that avoids unwanted adverse side effects of generalized immunosuppression by modulating the effector functions of T cell responses to inhibit the development of vascular inflammation.

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1. INTRODUCTION

Vasculitides comprises a group of rare diseases, characterized by inflammation of the blood vessel walls. The clinical manifestations are dependent upon the localization, the type of vessel involved as well as the nature of the inflammatory process. Vasculitis constitutes, in most cases, as a primary autoimmune disorder, but can also be secondary to other conditions. The underlying conditions to secondary vasculitis are infectious diseases, connective tissue disorders, or hypersensitivity disorders. In general, primary vasculitides are systemic diseases with variable clinical manifestations making it difficult to classify. According to the latest Chapel Hill Consensus Conference, primary systemic vasculitides can be divided into seven main entities of which three are most common; large vessel vasculitis, medium vessel vasculitis and, small vessel vasculitis (1). The group of large vessel vasculitides (LVV) affects the aorta and its major branches. The two major variants of LVV are giant cell arteritis (GCA) and Takayasu’s arteritis (TA). Medium vessel vasculitis (MVV) is vasculitis that predominantly affects medium arteries defined as the main visceral arteries and their branches. The two major categories are polyarteritis nodosa (PAN) and Kawasaki disease (KD). Small vessel vasculitis (SVV) is divided into anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and immune complex SVV (2). AAV is characterized by necrotizing vasculitis with few or no immune deposits that predominantly affects small vessels which lead to systemic organ damage. AAV are associated with the presence of circulating ANCA that are directed against proteinase-3 (PR3) or myeloperoxidase (MPO), proteins in the cytoplasmic granules of neutrophils. This group of systemic vasculitis includes granulomatosis with polyangiitis (GPA), primarily associated with antibodies to PR3-ANCA and microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), both principally associated with antibodies to MPO-ANCA.

Besides autoimmune disorders related to vascular inflammation, a more common chronic vascular inflammatory disease is atherosclerosis. Clinical evidence indicates that patients suffering from large and medium sized vessel vasculitis show accelerated atherosclerosis (3). In small vessel vasculitis this relation is less well defined. However, many patients with small vessel vasculitis carry several risk factors (e.g. impaired renal function, persistent proteinuria and increased level of C-reactive protein) that contribute to the acceleration of the atherosclerotic process (3,4). Enhanced oxidation processes, persistently activated T cells and reduced numbers of regulatory T cells are among the many pathophysiological factors that play a role in the acceleration of atherogenesis (5). Both vasculitis and atherosclerosis, although in nature different forms of chronic conditions, reveal similarities in T cell repertoire that occur within the process of vascular inflammation.

This review provides an overview of the role of adaptive immune mechanisms in vascular inﬂammation focusing on the T lymphocytes in particular. The main emphasis will be on the role of eﬀector memory T (T_EM) cells in vasculitis (i.e. AAV and atherosclerosis) and the potential therapeutic interventions for modulating the activity of these cells.

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2. T LYMPHOCYTES: KEY PARTICIPANTS IN VASCULAR

INFLAMMATION

T cells are recruited to the vessel wall in conjunction with macrophages, but in lesser quantity. In the blood vessel wall or tissues, T cell responses are initiated by signals generated via the association of TCR complexes with specific peptide-MHC protein complexes on the surface of antigen presenting cells (APCs) and through signals provided by costimulators expressed on APCs. The responses to antigen and costimulators include synthesis of pro-inflammatory mediators (e.g. IFN-γ) cellular proliferation, differentiation into effector and memory cells and performance of effector functions. These initial events further amplify the inflammatory response, aggravating disease progression. Different T cell subsets exist that can influence vascular inflammation in various ways. In the last decade, substantial progress has been made in the characterization of T cell mediated responses in vascular inflammation.

2.1 T cell involvement in AAV and atherosclerosis

In ANCA-associated vasculitis (AAV) it has been postulated that ANCA in vivo bind to surface expressed auto-antigens (PR3 or MPO) on primed neutrophils which subsequently activates the neutrophils (6). These activated neutrophils enhance neutrophil degranulation and the release of cytotoxic products that promote endothelial cells damage leading to vascular inflammation and injury (6). This initial inflammatory response mediated by the innate immune system creates a pro-inflammatory (micro)environment to attract cells from the adaptive immune system. In the case of autoimmune mediated vascular pathologies, like AAV, loss of self-tolerance and continuous antigen presentation also contributes to the involvement of the adaptive immune system. The contribution of T cell mediated immune responses in vascular inflammation is most likely because infiltrating T cells are detected in inflammatory lesions observed in the microvascular bed of kidney, lung and in nasal biopsies from AAV patients (7-11). In accordance with these findings, soluble T cell activation markers (soluble interleukin-2-receptor (sIL-2R) and soluble CD30) are elevated in plasma or serum and have been shown to be associated with disease activity in AAV (12-15). Also, ANCA antigen specific T cells have been detected in AAV (16,17). Moreover, the IgG subclass distribution of ANCA, predominantly consisting of IgG1 and IgG4 implies isotype switching of ANCA for which T cells are required (18). Importantly, Ruth

et al. demonstrated a pivotal role of T cells in the expression of crescentic glomerulonephritis

(19). They induced experimental anti-MPO-associated crescentic glomerulonephritis by immunizing C57BL/6 mice with human MPO followed by subsequent challenge with anti-glomerular basement membrane (anti-GBM) antibodies. Mice depleted of T cells at the time of administration of anti-GBM antibodies developed significantly less glomerular crescent formation and displayed less cell influx in glomeruli compared with control mice. Interestingly, specific T cell depleting therapies with anti-CD52 antibodies (Alemtuzumab) or anti-thymocyte globulin can induce remission in refractory AAV patients (20,21).

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29 Atherosclerosis is considered a chronic inﬂammatory disease, characterized by a slowly progressing passive lipid accumulation in large and medium-sized blood vessels that ultimately leads to the formation of plaques. Both innate and adaptive immunity are involved in this process. Ait-Oufella

et al. recently reviewed the role of the adaptive immune response in atherosclerosis and discussed

the role of dendritic cells (DCs) in the control of T cell involvement in atherosclerosis (5). Classically, DCs accumulate in the atherosclerotic plaque through direct chemokine mediated recruitment. DCs take up (atherosclerotic-specific) antigens such as ApoB100 and LDL and become activated and mature. Subsequently, DCs migrate to draining lymph nodes, where they can present antigens to naïve T cells. After activation these T cells develop into effector cells, clonally expand and enter the bloodstream. When effector T cells are recruited into atherosclerotic plaques they are reactivated by antigens presented by local macrophages and DCs, boosting the immune response. In human atherosclerotic lesions, the ratio of macrophages to T cell has been reported to be approximately 10:1, thus T cells are not as abundant as macrophages. However, because T cells are activated in the lesions resulting in the production of pro-atherogenic mediators, they can importantly contribute to lesion growth and disease aggravation. The first evidence of T cell involvement in atherosclerosis came with the demonstration that MHC class II positive cells and T cell cytokines (e.g. IFN-γ) are expressed in human atherosclerotic plaques (22). Later, the presence of T cells was observed in atherosclerotic plaques in humans (23,24) and mice (25,26). These observations only demonstrated the association of T cell with atherosclerosis but did not revealed the role of T cells in atherogenesis. However, Zhou et al. demonstrated a specific role of T cells in atherogenesis using an animal model of atherosclerosis. They showed that transfer of CD4+_{T cells into ApoE}-/-_{mice crossed with immunodeficient mice (scid/scid mice) fully reversed}

the atheroprotection provided by T and B cell deﬁciency (27).

Taken together, these observations indicate that T cell mediated immunity is an important contributor to the pathogenesis of vascular diseases such as AAV and atherosclerosis. In line with this, different T cell populations have been identified in vascular inflammation as will be discussed below. Figure 1 presents a proposed mechanism of the T cell mediated vascular inflammatory process.

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Aberrant T helper (T_H) cell polarization has been described in patients with vascular diseases. The involvement of diﬀerent T_H cells subsets in the pathogenesis of vascular disease has been suggested to depend on disease activity/stage and whether the disease is localized or systemic. In AAV, analysis of patients sera for soluble markers associated with either T_H1 cells (IFN-γ, sCD26) or T_H2 cells (IL-4, IL-5, IL-10, IL-13, sCD23 and sCD30) revealed a shift towards a T_H2-type response in patients with active generalized disease, whereas a T_H1-type response is predominant in patients with localized disease (28,29). Consistent with these observations, analysis of nasal granulomatous lesions from AAV patients demonstrated a relative increase of cells expressing T_H1-associated markers such as IFN-γ and CD26 during localized disease, whereas the T_H2-associated marker IL-4 was found in generalized AAV (11). In addition, Lamprecht et al. compared chemokine receptors on

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Figure 1 | Proposed pathophysiological mechanism of T cell mediated vascular inﬂ ammation.

Vascular infl ammation is initiated by a pro-infl ammatory trigger such as an infection. Release of pro-infl ammatory cytokines causes priming of neutrophils, upregulation of adhesion molecules on endothelial cells and an expansion of circulation eff ector T cells. Activation of primed neutrophils enhances vessel wall adherence and the transmigration capacity of the neutrophils. Production of reactive oxygen species and degranulation of fully activated primed neutrophils causes damage to vascular endothelial cells. This acute injury together with pro-infl ammatory triggers elicits an innate infl ammatory response that recruits T lymphocytes, which replace the neutrophils and either resolves or mediate the development of vasculitis. In this pro-infl ammatory environment the innate immune system with antigen-presenting cells (APCs) and T cells start to mediate the infl ammatory response. Distinct cytokine patterns in combination with a defect in regulatory T (TREG) cell function

or frequency results in expansion of eﬀ ector memory T (T_EM) cells. The dysbalance in the homeostasis of T_REG cells and T_EM cells, results in additional releases of pro-inﬂ ammatory cytokines promoting neutrophil priming and persistent activation of TEM

cells. Expanded circulating T_EM cells upregulate their killer immunoglobulin-like receptor (NKG2D) and interact with their ligand major histocompatibility complex class-I chain-related molecule A (MICA) on vascular endothelial cells. This event results in the migration of T_EM cells into target tissues, drive granuloma formation leading to tissues destruction in a perforin-dependent and granzyme-dependent way, ending up in vasculitis. The T cell driven vascular infl ammatory response is a multistep process and has diff erent therapeutic possibilities. For this purpose, selective T_EM cell modulation might be benefi cial to regulate the TEM cell activity, proliferation and migration. Other therapeutic options are, modulation of T cell activation by interfering

with costimulatory molecules, depletion of T cells, inhibition of T cell migration or neutralizing secreted pro-inﬂ ammatory cytokines. (This ﬁ gure was created using Visi ScienceSlides®_Software).

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31 peripheral blood-derived T cells. The inducible inﬂammatory T_H1-type chemokine receptor CCR5 was more prominent in the granulomatous lesions of AAV patients (30).

Similar to AAV, a study on cytokine expression in advanced human atherosclerotic plaques confirmed the dominance of pro-inflammatory T_H1 cytokines (IFN-γ, TNF-α and IL-2) (31). Genetic deficiency in IFN-γ or its receptor in ApoE-/-_{mice reduced atherosclerotic lesion formation}

and enhanced plaque stability (32), whereas exogenously administered IFN-γ enhanced atherosclerosis in ApoE-/-_{mice (33). Intriguingly, it seems that the protective eﬀect of IFN-γ}

deﬁciency is restricted to male ApoE-/-_{mice (34). In addition, several studies revealed that}

intervention in IL-12 or IL-18 gene, or receptor function was found to reduce plaque development in mouse models of atherosclerosis (35-37). Furthermore, administration of these cytokines accelerated disease progression (38,39). Collectively, these data point towards a pro-inflammatory T_H1 response in atherosclerosis. However, the role of T_H2 immune responses in atherosclerosis is controversial. IL-4, the signature cytokine of the T_H2 lineage, is not frequently observed in human atherosclerotic plaques (31). Moreover, experimental studies examining the involvement of T_H2 cells are contradictory, some showing pro-atherosclerotic effects (36,40), whereas others show no or athero-protective effects (41,42).

Overall, the balance between T_H1 cells and T_H2 cells plays a key role in the development of vascular inﬂammation. Interestingly, in the last decade T_H17 cells have emerged as a new CD4+_{T cell subset characterized by secretion of IL-17A and other cytokines including IL-17F, IL-21}

and IL-22. These cells are considered another major pathogenic eﬀector subset involved in the development of inﬂammatory and autoimmune diseases (43).

IL-17 has been reported to promote the release of the pro-inflammatory cytokines IL-1β and TNF-α from macrophages (44), which are essential for priming and activation of neutrophils. Furthermore, this pro-inflammatory milieu induces CXC chemokine release (45) and up-regulation of endothelial adhesion molecules (46) responsible for the recruitment of neutrophils to the site of inflammation (47). These pro-inflammatory events suggest that IL-17 may directly contribute to the acute vascular inflammatory response in AAV. Convincing experimental evidence that support this notion comes from several studies. Hoshino et al. demonstrated that neutrophils produce IL-17A and IL-23 in response to MPO-ANCA creating local conditions to promote T_H 17-mediated autoimmunity (48). In addition, Gan et al. showed that immunization of C57BL/6 mice with murine MPO resulted in MPO-specific dermal delayed type hypersensitivity and systemic IL-17A production (49). Upon injection of low dose anti-GBM antibodies these mice developed glomerulonephritis. In contrast, IL-17A deficient mice were nearly completely protected from disease induction due to reduced neutrophil recruitment and MPO deposition (49). Consistent with this finding, Odobasic et al. demonstrated that IL-17A contributes to early glomerular injury, but it paradoxically, attenuates the severity of fully established crescentic disease by limiting the T_H1 responses (50). They used a mouse model of crescentic anti-GBM glomerulonephritis assessing the renal injury and immune responses in IL-17A-/-_{and in wild-type (WT) mice.}

Crescentic glomerulonephritis was enhanced in IL-17A-/-_{mice, with increased glomerular T cell}

accumulation and augmented T_H1 responses (50). In contrast, mice lacking IL-12(p35), the key T_H