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Vivo T Cells In+Effector and Regulatory CD4

GITR Triggering Induces Expansion of Both

NolteBoon, Jörg Hamann, Rene A. W. van Lier and Martijn A.Gisbergen, Felix M. Wensveen, Robert M. Hoek, Louis Ronald W. van Olffen, Nathalie Koning, Klaas P. J. M. van

http://www.jimmunol.org/content/182/12/7490doi: 10.4049/jimmunol.0802751

2009; 182:7490-7500; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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GITR Triggering Induces Expansion of Both Effector andRegulatory CD4� T Cells In Vivo1

Ronald W. van Olffen,* Nathalie Koning,*† Klaas P. J. M. van Gisbergen,* Felix M. Wensveen,*Robert M. Hoek,2* Louis Boon,‡ Jorg Hamann,* Rene A. W. van Lier,* and Martijn A. Nolte3*

Glucocorticoid-induced TNF receptor family-related protein (GITR) is expressed on activated and regulatory T cells, but its role onthese functionally opposing cell types is not fully understood. Here we describe that transgenic expression of GITR’s unique ligand(GITRL) induces a prominent increase of both effector and regulatory CD4� T cells, but not CD8� T cells. Regulatory T cells fromGITRL transgenic mice are phenotypically activated and retain their suppressive capacity. The accumulation of effector and regulatoryT cells is not due to enhanced differentiation of naive T cells, but is a direct result of increased proliferation. Functional consequencesof increased numbers of both regulatory and effector T cells were tested in an autoimmune model and show that GITR stimulation isprotective, as it significantly delays disease induction. These data indicate that GITR regulates the balance between regulatory andeffector CD4� T cells by enhancing proliferation of both populations in parallel. The Journal of Immunology, 2009, 182: 7490–7500.

M embers of the TNF receptor superfamily are able todirectly and indirectly affect the course of an immuneresponse (1). The glucocorticoid-induced TNF recep-

tor family-related protein (GITR)4 is a member of this family andhas been implicated in regulating both innate and adaptive immuneresponses (2, 3). Regulatory T cells are well known for their ex-pression of GITR, although this receptor is also expressed on ac-tivated nonregulatory T cells, as well as B cells, monocytes andmacrophages, dendritic cells, and mast cells (3–7). Its ligand(GITRL) can be found on a variety of cells, including dendriticcells, macrophages, and B cells (7–9). GITRL is transiently up-regulated on these APCs upon stimulation via the transcriptionfactor NF-1 (8), and it most likely exerts its main function duringinflammatory responses. Indeed, agonistic Abs and (cells express-ing) recombinant GITRL enhance T cell proliferation in vitro uponTCR triggering (2, 5, 6, 8, 10), which suggests that GITR acts asa costimulatory factor for T cells. GITR stimulation on regulatoryT cells in coculture with effector T cells has been suggested toneutralize the suppressive capacity of regulatory T cells (5, 6, 8).However, it was later shown that GITR ligation on regulatory Tcells does not directly affect their suppressive capacity, but thatGITR stimulation on nonregulatory T cells allows them to escape

suppression by regulatory T cells (11). GITR is not essential forregulatory T cell function, as regulatory T cells from GITR�/�

mice display a normal capacity to suppress T cell proliferation invitro (2, 12). This leaves unanswered what the function of GITR ison regulatory T cells.

Studies using GITR�/� mice showed that the absence of GITRwas protective in several disease models, which was attributed toan impaired effector function of T cells (13–15). Correspondingly,studies that have directly addressed the function of GITR on Tcells in vivo by deliberate stimulation of the receptor with agonis-tic Abs concluded that GITR has a proinflammatory role within theimmune system through its costimulatory effects on T cells (6, 7,16, 17). However, these Abs have their limitations when studyingthe impact of GITR stimulation in complex disease models, inparticular since anti-GITR Abs have been reported to cause deple-tion of regulatory T cells (7). Moreover, recent studies on the crys-tal structure of GITRL have revealed that this ligand can exist inmultiple oligomerization states that depend on binding to the re-ceptor (18, 19), and it therefore remains to be addressed whethercross-linking GITR with agonistic Abs exerts the same down-stream effects as signaling induced by membrane-bound GITRL.Thus, to properly address the consequence of direct GITR stimulationon T cell function in vivo, we generated transgenic (TG) mice inwhich GITRL is constitutively expressed on B cells. Our findingsdemonstrate that GITR stimulation in vivo very effectively increasesthe absolute number of both effector and regulatory CD4� T cellsthrough enhanced proliferation of both cell types. In agreement withincreased regulatory T cell numbers, GITRL TG mice showed amarked delay in disease onset upon induction of experimental auto-immune encephalomyelitis (EAE), an experimental model for multi-ple sclerosis. We propose that GITR plays an important role in theregulation of both regulatory and effector CD4� T cell numbers invivo by enhancing their turnover.

Materials and MethodsGeneration of GITRL TG mice

cDNA encoding murine GITRL was obtained via PCR on total spleniccDNA and cloned into the pGEM-T plasmid (Promega). This construct wasdigested with NotI and XhoI to obtain a 600-bp fragment containing themGITRL cDNA, which was cloned into the NotI-XhoI site of a CD19-pC3plasmid (kindly provided by Patrick Derksen, Academic Medical Center,

*Department of Experimental Immunology, Academic Medical Center, University ofAmsterdam, Amsterdam, The Netherlands; †Netherlands Institute for Neuroscience,an Institute of the Royal Netherlands Academy for Arts and Sciences, Amsterdam,The Netherlands; and ‡Bioceros, Utrecht, The Netherlands

Received for publication August 21, 2008. Accepted for publication April 6, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by a VICI Grant (to R.A.W.v.L.) and a VIDI Grant (toM.A.N.) from The Netherlands Organization of Scientific Research.2 Current address: Department of Neuromedical Genetics, Netherlands Institute forNeuroscience, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands.3 Address correspondence and reprint requests to Dr. Martijn A. Nolte, Department ofExperimental Immunology, Academic Medical Center, Meibergdreef 9, 1105AZ Am-sterdam, The Netherlands. E-mail address: m.a.nolte@amc.nl4 Abbreviations used in this paper: GITR, Glucocorticoid-induced tumor necrosisfactor receptor family-related protein; GITRL, GITR ligand; EAE, experimental au-toimmune encephalomyelitis; TG, transgenic; WT, wild type.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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The Netherlands), resulting in the GITRL expression construct under con-trol of the human CD19 promoter. This construct was linearized via AatIIdigestion (see Fig. 1A) and microinjected into pronuclei of C57BL/6 fer-tilized oocytes and implanted into pseudopregnant female C57BL/6 mice.Transgenic founders were identified by PCR analysis of tail or ear DNA,using the following PCR primers: pC3s1 (5�-GCAGTGACTCTCTTAAGGTAGCC-3�) and mGITRL4a (5�-CTTGAGTGAAGTATAGATCAGTGTA-3�). Three GITRL TG founder lines (RW14, RW18, RW20) werepropagated by mating with wild-type (WT) C57BL/6 mice, and offspringwere tested for the presence of the transgene by PCR analysis of tail or earDNA with the same primers.

Mice

GITRL TG mice were maintained on a C57BL/6 background and bred inthe animal department of the Academic Medical Center (Amsterdam, TheNetherlands) under specific pathogen-free conditions. Mice were used at6–24 wk of age, age- and sex-matched within experiments, and were han-dled in accordance with institutional and national guidelines. All experi-ments have been reviewed and approved by the Academic Medical CenterAnimal Ethics Committee. For measurement of in vivo T cell proliferation,mice were injected i.p. with 1 mg of BrdU (Sigma-Aldrich) and sacrificedfor analysis 16 h later.

Cell staining and flow cytometry

Single-cell suspensions were obtained by mincing the specified organsthrough 40-�m cell strainers (BD Biosciences). Erythrocytes were lysedwith an ammonium chloride solution and cells were subsequently countedusing an automated cell counter (Casy; Scharfe System). Cells (5 � 105 to5 � 106) were collected in staining buffer (PBS with 0.5% BSA; Sigma-Aldrich) and stained for 30 min at 4°C with Abs in the presence ofanti-CD16-CD32 (clone 2.4G2). The following fluorescently or biotin-labeled mAbs (and clone names) were obtained from BD Pharmingen:anti-B220 (RA3-6B2), anti-CD3� (145-2C11), anti-CD4 (L3T4), anti-CD8 (Ly-2), anti-CD62L (clone MEL-14), and anti-CD69 (H1.2F3); orfrom eBioscience: anti-GITRL (ebioYGL386), anti-FoxP3 (NRRF-30),anti-GITR (DTA-1), anti-CD44 (IM7), anti-CTLA4 (4C10-4B9), anti-PD1(RMP1-30), anti-CD134 (OX86), anti-CD27 (LG.7F9), anti-CD103(M290), and anti-CD45.1 (104 or A20). PE-conjugated anti-CD25 wasobtained from Miltenyi Biotec. For the detection of biotinylated Abs,streptavidin-PE (Caltag Laboratories), streptavidin-allophycocyanin (BDPharmingen), or streptavidin-conjugated PerCP-Cy5.5 (BD Pharmingen)was used. Intracellular stainings for FoxP3 and/or BrdU were performedusing Foxp3 fixation/permeabilization concentrate and diluent (eBioscience),according to the manufacturer’s protocol. For BrdU staining, FITC-conju-gated anti-BrdU/DNase (BD Pharmingen) was added during the FoxP3staining step and stained for 25 min at room temperature. Data were col-lected on a FACSCalibur or FACSCanto (BD Biosciences) and were an-alyzed with using FlowJo software (Tree Star).

Intracellular cytokine staining

To determine direct ex vivo cytokine production, splenocytes were platedat 1 � 106 cells/well in a 96-well round-bottom plate and stimulated with1 ng/ml PMA and 1 �M ionomycin. After 2 h of incubation at 37°C, 1�g/ml of the protein-secretion inhibitor brefeldin A was added (Sigma-Aldrich) and cells were cultured for another 4 h. Afterward, cells werewashed and stained for CD3 and CD4 or CD8, followed by fixation andpermeabilization (BD Biosciences). Cells were then incubated for 30min with fluorescently labeled Abs against IFN-�, IL-2, IL-10, or IL-17(eBioscience), thoroughly washed, and analyzed by flow cytometry.

T cell proliferation assay

To analyze the effect of GITR engagement on the proliferative capacity ofWT responder cells, T cells were enriched from spleens of WT mice bynegative selection using CD19� beads (Miltenyi Biotec). T cell-enrichedsplenocytes were labeled with 0.25 �M CFSE in PBS at 37°C for 10 minand stimulated with 100 ng/ml anti-CD3 (clone 145-2C11) for 3 days in thepresence of irradiated (10 Gy) WT or GITRL TG B cells with or without200 U/ml IL-2. B cells were isolated by positive selection using CD19�

beads (Miltenyi Biotec). For the analysis of the expression of CD25 andCD69, non-CFSE-labeled enriched WT T cells were used, stimulated sim-ilarly, and analyzed after 1 day. To determine the effects of GITR ligationon IL-2 production in vitro, T cell-enriched splenocytes were stimulated asdescribed above, in the presence of 10 �g/ml blocking anti-CD25 Ab(clone PC61) to prevent IL-2 consumption. Culture supernatant was har-vested after 1 day of stimulation and frozen at �20°C. The IL-2 ELISA

(BD Biosciences) was performed according to instructions from themanufacturer.

Regulatory T cell assay

Splenic CD4�CD25� (responder cells) and CD4�CD25high (regulatorycells) T cells were isolated by cell sorting using a FACSAria (BD Bio-sciences) and purity of sorted populations was consistently �96%. Re-sponder cells were mixed with regulatory T cells at different ratios in 96-well tissue culture plates. The cells were stimulated with 10 �g/ml solubleanti-CD3 (clone 145-2C11) plus irradiated (10 Gy) WT splenocytes(APCs) at 37°C for 72 h. Hereafter, cells were pulsed for 16 h with 1 �Ciof 3H-TdR ([methyl-3H]thymidine; Amersham Pharmacia)/well, and incor-poration of 3H-TdR was determined using a beta plate scintillation counter(Wallac; 1450 Microbeta Plus liquid scintillation counter). Data are pre-sented as percentage proliferation compared with maximum responder cellproliferation of triplicate assays.

Adoptive transfer of naive T cells into WT and GITRL TG mice

For adoptive transfers, naive (CD25�) CD4� T cells were purified fromspleens and peripheral lymph nodes of Ly5.1 mice by negative selectionusing the CD4�CD25� regulatory T cell isolation kit (Miltenyi Biotec).Purified CD4�CD25� cells (purity �90%) were labeled with 0.25 �MCFSE in PBS at 37°C for 10 min and injected after washing (with orwithout 1 � 106 in 200 �l of PBS) i.v. into WT and GITRL TG recipientmice. Distribution and phenotype of transferred cells was analyzed 3 dayslater by flow cytometry.

EAE induction

EAE was induced by s.c. immunization of mice in the hind flanks using 50�g of MOG35–55 peptide in CFA containing 1 mg/ml heat-inactivated My-cobacterium tuberculosis (Difco) on day 0. Mice also received 200 ng ofpertussis toxin (Sigma-Aldrich) i.v. on days 0 and 2. Disease severity wasassessed according to the following scale: 0, no disease; 1, flaccid tail; 2,loss of hind leg spreading reflex; 3, hind limb weakness; 4, unilateral hindlimb paralysis; 5, bilateral hind limb paralysis; 6, abdominal paralysis; 7,moribund; 8, dead. All mice were sacrificed 14 days following EAE in-duction, after which brain and spinal cord was frozen in Tissue-Tek(Sakura Finetek) at �80°C for immunohistochemical analysis.

Immunohistochemistry

Cryostat sections (8 �m) of spinal cord and brain of 4 WT and 4 TG micewere fixed in acetone, containing 1% H2O2 for 10 min. Then, sections wereincubated with monoclonal rat anti-mouse Abs to CD68 (a kind gift fromSiamon Gordon, Oxford, U.K., clone FE-11), CD4, and FoxP3 (eBioscience),diluted in PBS with 8% BSA, 10% normal mouse serum, and 0.05% NaN3

for 1 h at 4°C. After washing in PBS, the sections were incubated withanti-rat HRP diluted in PBS, 8% BSA, 10% normal mouse serum, 0.05%NaN3, and 350 mM NaCl. Staining was visualized with diaminobenzidine(Sigma-Aldrich) applied for 10 min. Sections were counterstained withheamatoxylin for 30 s, dehydrated, and mounted in Entallan (Merck). Asnegative controls, primary Abs were either left out or substituted with anisotype control Ab. No immunoreactivity was seen for all negativecontrols.

For the visualization of cellular infiltrates in organs obtained from WTand GITRL TG mice, cryostat sections (7 �m) of the thyroid, kidney, liver,stomach, and small and large intestine were stained by Diff-Quick (DadeBehring) according to the manufacturer’s instructions and analyzed bylight microscopy.

Statistical analysis

Statistical analysis of the data was performed using the unpaired Student’st test or Wilcoxon rank-sum test where mentioned. For the EAE experi-ments, the effect on mean clinical score was assessed by calculating thearea under the curve using the trapezoidal rule, followed by the Wilcoxonrank-sum test. Differences in cumulative incidence were analyzed on a perday basis using a �2 test of a contingency table.

ResultsGeneration of B cell-specific GITRL TG mice

To study the function of GITR on T cells in vivo, we generated Bcell-specific GITRL TG mice by expressing GITRL cDNA undercontrol of the human CD19 promoter (Fig. 1A). Through micro-injection of fertilized oocytes, we acquired three founder lines(RW14, RW18, and RW20), which were identified by genomic

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PCR analysis (Fig. 1B). GITRL TG mice were fertile, born atexpected Mendelian frequencies and appeared as healthy as theirlittermate controls. Flow cytometry showed that GITRL was in-deed significantly expressed on B cells in all founder lines, withhighest expression on the RW18 line (Fig. 1, C and D). As receptorshedding upon ligand stimulation is a hallmark of various TNFreceptor superfamily members (20–24), we determined the expres-sion of GITR on T cells. T cells from GITRL TG mice showeddecreased GITR expression compared with WT mice, which cor-related with the level of GITRL expression (Fig. 1, E and F). Thisindicates that GITR is indeed functionally engaged by its ligand inthese mice. The data shown below are obtained from experimentswith the GITRL TG RW18 line, although a similar, but less pro-nounced phenotype was also found in the other founder lines (datanot shown).

GITRL TG mice have more CD4� effector memory-like andregulatory-type T cells

To establish the effects of GITR triggering in vivo, we analyzed theprimary and secondary lymphoid organs of GITRL TG mice. T celldifferentiation in the thymus of these mice was comparable to WTlittermates (data not shown), as was cellularity of bone marrow,thymus, and peripheral and mesenteric lymph nodes (Fig. 2A).However, spleens of GITRL TG mice contained significantly moreleukocytes than WT mice (Fig. 2A). This increase was due to el-evated numbers of CD4� T cells (Fig. 2, B and C), whereas num-bers of B cells and CD8� T cells were not significantly altered(Fig. 2, B and C). Analysis of FoxP3 expression indicated that asubstantial part of this increase in CD4� T cells could be attributedto an enlarged regulatory T cell compartment, as up to three timesmore CD4�FoxP3� regulatory T cells were present in the spleensof GITRL TG mice (Fig. 2, D and E). Phenotypic analysis ofFoxP3�CD4� T cells indicated that the nonregulatory fraction wasalso affected in GITRL TG mice, as significantly more CD4� Tcells with an effector memory-like (CD44�CD62L�) and centralmemory-like (CD44�CD62L�) phenotype were identified (Fig. 2,D and E). This increase of CD4� T cells with either a regulatoryor a memory-like phenotype in GITRL TG mice apparently did notdevelop at the cost of the naive CD4� population, since absolute

numbers of naive CD4� T cells were comparable with WT litter-mates (Fig. 2E). No differences were found for CD8� T cells withrespect to their naive, effector memory-like and central memoryphenotype (Fig. 2, F and G). Corroborating the specific increase inCD4� effector T cells in GITRL TG mice, splenocyte stimulationwith PMA-ionomycin showed increased production of the effectorcytokines IL-2 (Fig. 2H) and IFN-� (Fig. 2, I and J) by CD4�, butnot CD8� T cells. Consistent with the increase in regulatory T cellnumbers, we observed a trend toward more IL-10-producing CD4T cells, but this difference was not significant (Fig. 2, K and L).These data thus indicate that GITR triggering in vivo enhanced thenumber of both regulatory and effector CD4� T cells.

Distribution and activation status of regulatory T cells inGITRL TG mice

To determine whether the strong increase of regulatory T cells inGITRL TG mice was restricted to the spleen, we analyzed thepresence of these cells in bone marrow, thymus, peripheral andmesenteric lymph nodes, and liver in these mice. We found thatGITRL TG mice have a systemic increase in regulatory T cellnumbers, as all analyzed compartments, except for the bone mar-row, showed a significantly higher fraction of FoxP3�CD4� cellscompared with WT mice (Fig. 3A).

Next, we analyzed the activation status of splenic regulatory andnonregulatory T cells. Apart from the described changes in CD44and CD62L expression (see Fig. 2, D and E), nonregulatory CD4�

T cells in GITRL TG mice were comparable with their WT coun-terparts on the basis of several other costimulatory and activationmolecules (Fig. 3B). On the other hand, we found that CD4�

FoxP3� regulatory T cells from GITRL TG mice consistently ex-pressed lower levels of CD25, CD62L, and CTLA4 compared withtheir counterparts in WT mice (Fig. 3, B and C). Additionally, theexpression of PD1 was increased in GITRL TG mice, while a largefraction of regulatory T cells from GITRL TG mice expressed theadhesion molecule CD103 (�E integrin) on their surface (Fig. 3, Band C). The expression of OX40, CD27, and CD69 was compa-rable to WT mice (Fig. 3, B and C). As FoxP3� regulatory T cellscan be divided in two functionally distinct subsets, namely naive(CD62L�CD103�) and effector regulatory T cells (CD62L�

FIGURE 1. Generation of B cell-specific GITRL TG mice. A, Sche-matic representation of the hCD19-mGITRL DNA construct. The humanCD19 promoter (hatched bar) is fol-lowed by a chimeric intron (whitebar), mGITRL cDNA (dotted bar),and a poly(A) tail (black bar). B, PCRanalysis of genomic tail DNA fromWT or GITRL TG mice (founder linesRW14, RW18, and RW20). C, Repre-sentative staining for GITRL onsplenic B220� cells from WT, RW14,RW18, and RW20 mice and (D) theexpression of GITRL on splenicB220� B cells as the average geomet-ric mean fluorescence intensity(GeoMFI) � SD for three mice pergroup. E, Representative staining forGITR expression on splenic CD3�

cells from WT, RW14, RW18, andRW20 mice and (F) the expression ofGITR on CD3� T cells as the averagegeoMFI � SD. ��, p � 0.005.

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CD103�) (25), we conclude that constitutive GITR stimulation notonly leads to more regulatory T cells, but specifically stimulatesthe formation of regulatory T cells with an effector phenotype.

GITR engagement in vivo does not affect the suppressivecapacity of regulatory T cells

To determine whether the altered phenotype of regulatory T cellsin GITRL TG mice mirrored a change in their function, we per-formed in vitro proliferation assays, in which WT responder Tcells (CD4�CD25�) were stimulated with anti-CD3/CD28 in thepresence of increasing numbers of regulatory T cells (CD4�

CD25�) from WT or GITRL TG mice. From these experiments itcan be concluded that regulatory T cells from GITRL TG mice arefully capable of suppressing responder T cell proliferation andwere equally anergic as regulatory T cells from WT mice (Fig.4A). This conclusion challenges previous reports, which have sug-gested that GITR stimulation on regulatory T cells is sufficient toabrogate their suppressive capacity (5, 6).

We also analyzed the susceptibility of GITRL TG- vs WT-de-rived responder T cells to the suppressive capacity of WT regula-tory T cells, as it has been reported that GITR stimulation allowsT cells to escape suppression by regulatory T cells (11). Theseexperiments revealed that responder T cells from GITRL TG micecould still be adequately suppressed by regulatory T cells (Fig.4B), thereby indicating that chronic GITR stimulation in vivo isnot sufficient to induce an enduring state of insensitivity to regu-latory T cell activity. Instead, it rather suggests that this previously

described capacity of GITR is only effective when given togetherwith TCR stimulation (11).

To establish whether regulatory T cells in GITRL TG mice areindeed functionally active in vivo, we examined several organs of12-mo-old GITRL TG mice for cellular infiltrates as a sign oforgan inflammation and autoimmunity. Loss of function of regu-latory T cells has been associated with increased numbers of au-toreactive T cells, which can induce several forms of autoimmu-nity, including thyroiditis, glomerulonephritis, gastritis, andinflammatory bowel disease (26–28). However, despite the strongincrease of effector CD4 T cells observed in lymphoid organs ofGITRL TG mice (Fig. 2), we did not find any sign of inflammationor cellular infiltration in either the thyroid gland, kidney, liver,stomach, or intestines of these mice (Fig. 4C). This indicates thatGITR stimulation does not impair the function of regulatory Tcells in vivo, and considering the increase of effector T cells, thissuggests that the regulatory T cells in GITRL TG mice are rathercompetent in preserving the homeostasis within this more activeimmune system.

GITR costimulation enhances CD4� T cell proliferation andIL-2 production

To investigate whether the increase in effector and regulatory typeT cells could be explained by an increased survival potential me-diated through GITR signaling, we examined the expression pro-file of �40 pro- and antiapoptotic proteins using an advanced PCRapproach called multiplex ligation-dependent amplification (29).

FIGURE 2. GITRL TG mice have more effector and regulatory type CD4� T cells. A, Absolute number of cells in spleen, bone marrow (BM), thymus,peripheral (pLN), and mesenteric (mLN) lymph nodes in 4- to 8-wk-old WT (open bar) and GITRL TG (filled bar) mice. Percentage and absolute numberof (B) T and B cells or (C) CD4� and CD8� T cells in the spleen of WT and GITRL TG mice. D and E, Percentage and absolute number of splenicregulatory (FoxP3�) and nonregulatory (FoxP3�) CD4� T cells in WT and GITRL TG mice. Nonregulatory CD4� T cells were subdivided in naive(CD44�CD62L�), effector memory (EM) (CD44�CD62L�) and central memory (CM) (CD44�CD62L�) cells. F and G, Percentage and absolute numberof naive, EM, and CM cells of splenic CD8� T cells. Production of (H) IL-2, (I and J) IFN-�, and (K and L) IL-10 by CD4� or CD8� T cells in WT andGITRL TG mice after stimulation with PMA-ionomycin. �, p � 0.05; ��, p � 0.005. Data represent the average value � SD of three to five mice and arerepresentative of two to four independent experiments.

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However, this comprehensive analysis did not reveal any signifi-cant differences in the apoptotic gene expression profile of naive,effector, and regulatory CD4 T cells isolated from GITRL TG micecompared with WT mice (data not shown).

Next, we set out to determine the effects of GITR triggering onT cell proliferation. CFSE-labeled T cells from WT mice werestimulated with suboptimal anti-CD3 in a 1:1 ratio with WT or

GITRL TG irradiated B cells for a period of 3 days. We found thatincreased GITRL availability enhanced CD4� T cell proliferation,but did not affect CD8� T cell proliferation (Fig. 5A). The en-hanced proliferation of CD4� T cells via GITR engagement wasno longer apparent when extra IL-2 was added to these cultures,indicating that GITR engagement affects the early proliferative ca-pacity of CD4� T cells. We found that increased GITR ligation

FIGURE 3. Systemic increase ofregulatory T cells via GITR signaling.A, The percentage of FoxP3� cellswithin the CD4� T cell compartmentin spleen, bone marrow (BM), thy-mus, peripheral (pLN) and mesenteric(mLN) lymph nodes, and liver in WTand GITRL TG mice. B, Phenotypicanalysis of splenic FoxP3� (filledgraph) and FoxP3� (open graph)CD4� T cells for surface expressionof CD25, CTLA4, PD1, OX40,CD27, CD62L, CD69, and CD103 inWT and GITRL TG mice. C, Averagegeometric mean fluorescence inten-sity (GeoMFI � SD) or percentagepositive cells (% � SD) of these mol-ecules on FoxP3� CD4� T cells. �,p � 0.05; ��, p � 0.005. Data arerepresentative of two independent ex-periments with each at least threemice per group.

FIGURE 4. Regulatory T cellfunction of WT and GITRL TG mice.A, Ability of purified WT and GITRLTG regulatory (CD4�CD25�) T cellsto suppress WT responder (CD4�

CD25�) T cells. B, Ability of WTregulatory T cells to suppress prolif-eration of WT and GITRL TG re-sponder T cells. Cells were cultured atdifferent ratios for 4 days with 10�g/ml soluble anti-CD3 mAb in thepresence of irradiated WT spleno-cytes as APCs; for the final 16 h[3H]thymidine was added and incor-poration was measured. Data are de-picted as the percentage proliferationcompared with responder T cellsalone (average of triplicate wells �SD) and are representative of two in-dependent experiments. C, Wright-Giemsa staining of cryosections fromthyroid gland, kidney, liver, stomach,and small and large intestine of 12-mo-old WT and GITRL TG mice.Data are representative of two miceper group.

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raised the percentage of CD4� T cells entering cell division (Fig. 5B),as well as the number of divisions that these cells underwent (Fig.5C). Since the addition of IL-2 enhanced T cell proliferation to asimilar extent as the addition of GITRL TG B cells, we questionedwhether GITR stimulation induced IL-2 production. Indeed, when

WT T cells were stimulated with anti-CD3, the addition of GITRLTG B cells induced almost 3-fold more IL-2 than WT B cells (Fig.5D). Moreover, GITR ligation increased the expression of CD25 andCD69 on CD4� T cells, confirming the enhanced IL-2 production andincreased activation induced by GITR stimulation (Fig. 5, E and F).

FIGURE 5. GITR costimulation invitro enhances CD4� T cell proliferationand IL-2 production. A, Anti-CD3-in-duced proliferation of CFSE-labeled WTT cells cultured for 3 days in the presenceof WT (filled graph) or GITRL TG (opengraph) B cells with or without IL-2. Trip-licate wells analyzed for (B) the averageprecursor frequency and (C) the averagedivision index (i.e., the number of divi-sions that the dividing population under-went). D, IL-2 concentration in superna-tants (average of triplicate wells � SD)after stimulating WT T cells as in A for24 h in the presence of a blocking Abagainst CD25 to prevent IL-2 consump-tion. Expression of CD25 (E) and CD69(F) on CD4� T cells stimulated as in Aafter 24 h. G, Naive WT CD4�CD25� Tcells from Ly5.1 mice were CFSE-la-beled and injected i.v. in WT (filledgraph) or GITRL TG (open graph) mice.Donor cells, gated on CD45.1�CD4� Tcells, were analyzed 3 days after transferfor expression of CFSE, CD103, andCD62L expression. A representativestaining from three mice per group isshown. �, p � 0.05; ��, p � 0.005.

FIGURE 6. GITR triggering en-hances proliferation of effector andregulatory T cells in vivo. A, Repre-sentative intracellular staining forFoxP3 and Ki-67 on splenic CD4� Tcells from WT and GITRL TG miceand (B) the percentage Ki-67� cellsof FoxP3� and FoxP3�CD4� T cells(average � SD). C, Representativeintracellular staining for BrdU andFoxP3 on splenic CD4� T cells fromWT and GITRL TG mice 16 h afteri.p. injection of 1 mg BrdU. D, Thepercentage BrdU� cells of FoxP3�

and FoxP3� CD4� T cells (aver-age � SD). Characterization of pro-liferating FoxP3� and FoxP3�CD4�

T cells based on CD62L (E) orCD103 (F) expression. The percent-age of BrdU� cells in each fraction isdepicted for WT (open bar) andGITRL TG (filled bar) mice (aver-age � SD). Data are representativefor two independent experiments witheach at least three mice per group. �,p � 0.05; ��, p � 0.005.

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To determine whether constitutive GITR triggering alone wassufficient to induce activation and/or proliferation of naive T cellsin vivo, we isolated naive nonregulatory CD4�CD25� T cellsfrom Ly5.1� WT donor mice, labeled them with CFSE, and trans-ferred them into WT or GITRL TG (Ly5.2�) recipients. Threedays after transfer, naive T cells transferred to both WT andGITRL TG mice showed no CFSE dilution and did not alter theirexpression levels of CD62L or CD103 (Fig. 5G). Thus, despite thefact that GITRL TG mice contained more T cells with an effectormemory-like phenotype, these data imply that stimulation throughGITR alone is not sufficient to induce activation or proliferation ofnaive T cells. However, when TCR triggering is provided, GITR

stimulation enhanced the production of IL-2 and increased prolif-eration of CD4� T cells in vitro.

GITR engagement in vivo increases the proliferation of effectorand regulatory CD4� T cells

As GITR engagement could directly and specifically enhanceCD4� T cell proliferation, we investigated the proliferative capac-ity of CD4� T cells in WT and GITRL TG mice in vivo. Asmeasured by Ki-67 expression, GITRL TG mice had more non-regulatory T cells (CD4�FoxP3�) in cell cycle in the spleen thandid WT mice (24 � 2.2% in GITRL TG mice vs 14 � 1.2% in WTmice) (Fig. 6, A and B). The fraction of regulatory T cells

FIGURE 7. Delayed EAE induc-tion though GITR ligation. A, The cu-mulative incidence and (B) the aver-age clinical score following EAEinduction in WT (�) and GITRL TGmice (f). Experiment depicted con-tains 10 animals per group and is rep-resentative of two independent exper-iments. Significant differences (p �0.05) of the area under the curve weredetermined by Wilcoxon rank sumtest (highlighted area). The absolutenumbers of (C) CD4� T cells and (D)IFN-�- and IL-17-producing CD4� Tcells on day 5 following EAE induc-tion in the draining (inguinal and lum-bar) and nondraining (axillary andbrachial) lymph nodes from WT andGITRL TG mice are shown. E, Per-centage regulatory (FoxP3�) and (F)nonregulatory effector (FoxP3�

CD62L�) T cells within the CD4�

compartment was determined in pe-ripheral blood following immuniza-tion (day 0) and pertussis toxin injec-tion (days 0 and 2) of WT (�) andGITRL TG (f) mice. Data representthe average � SD of four mice. As-terisks denote significant differencesbetween WT and GITRL TG mice ona particular day (�, p � 0.05; ��, p �0.005); black dots denote significantdifferences between consecutive daysfor either group (●, p � 0.05; ●●,p � 0.005. Cellular infiltrates in brainand spinal cord were analyzed formice with a clinical score of 5 and inwhich disease onset occurred 4–5days before experimental endpoint.G, Size of cellular infiltrates in thespinal cord of WT and GITRL TGmice. Representative immunohisto-chemical staining and quantificationof the spinal cord of WT and GITRLTG mice shows the presence of (H)CD68� macrophages, (I) CD4� Tcells, and (J) FoxP3� regulatory Tcells on a heamatoxylin backgroundstaining (�20 magnification). Arrowindicates a single FoxP3� cell. Forquantification purposes, at least sixinfiltrates were analyzed per stainingper mouse.

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(CD4�FoxP3�) that stained positive for Ki-67 was not signifi-cantly different between WT and GITRL TG mice, but the fractionof Ki-67� cells within the regulatory T cell compartment is alreadyhigh (�30%) in WT mice (Fig. 6, A and B).

To directly assess T cell proliferation in vivo, WT and GITRLTG mice were injected i.p. with BrdU and incorporation of thiscompound was analyzed 16 h later (Fig. 6, C and D). Within thenonregulatory CD4� T cell compartment, we found that GITRLTG mice contained more BrdU� cells than did WT mice and thisincrease was most pronounced in the CD62L� effector fraction(Fig. 6C–E). A GITR-mediated increase in proliferation was evenmore profound for regulatory T cells, as the percentage ofCD4�FoxP3� T cells that had incorporated BrdU had more thandoubled compared with WT littermates (Fig. 6, C and D). In thiscase, it was the CD62L�CD103� fraction of regulatory T cellsthat showed the most profound increase in BrdU incorporation(Fig. 6, E and F). No effect of GITRL overexpression on BrdUincorporation was found in CD8� T cells (data not shown). Over-all, these data indicate that GITR affects the numbers of regulatoryT cells as well as the memory/effector pool of nonregulatory CD4�

T cells in vivo by regulating their proliferation.

Enhanced GITR ligation delays EAE

To examine the significance of the expansion of both regulatoryand effector CD4� T cells on a complex immune response in vivo,GITRL TG mice were subjected to EAE, an experimental modelfor multiple sclerosis. We selected this model, as it induces auto-immunity by selective depletion and loss of function of regulatoryT cells through treatment of immunized mice with pertussis toxin,which probably facilitates autoreactive T cells to develop andcause nerve damage in the CNS (30, 31). It is therefore conceiv-able that GITRL TG mice are protected from disease (i.e., limbparalysis) because they have more regulatory T cells, but it couldalso be that they display enhanced susceptibility to EAE comparedwith WT mice, because of their increased effector T cell formation.We found that regulatory mechanisms dominated the EAE re-sponse in GITRL TG mice, as they had a significant delay in dis-ease onset compared with WT mice, which correlated with a lowerclinical score (Fig. 7, A and B). Although disease was delayed inGITRL TG mice, it was not inhibited, as the cumulative incidenceand clinical score were similar between both groups at the exper-imental endpoint (Fig. 7, A and B), suggesting that autoreactivecells did develop in these mice.

To obtain more insight in the mechanism of this difference indisease progression, we examined the CD4� T cell response indraining lymph nodes from WT and GITRL TG mice early afterimmunization. After 5 days, the impact of the immunization wasreadily visible in both mouse strains, as the draining (inguinal andlumbar), but not nondraining (axillary and brachial), lymph nodeshad considerably increased in size (data not shown). In contrast towhat might be expected from the delay in disease progression, wefound that the development of effector CD4� T cells was not per-turbed in GITRL TG mice, as they contained even more effectorCD4� T cells than did WT mice (Fig. 7C). Absolute numbers ofregulatory CD4� T cells were also increased in GITRL TG mice(Fig. 7C). Restimulation of lymph node cells with PMA-ionomy-cin corroborated an increase in effector T cells, as GITRL TG micecontained more IFN-�-producing cells, whereas IL-17 productionwas not affected (Fig. 7D). Thus, GITR stimulation does not neg-atively affect the formation of autoreactive T cells following EAEinduction, but, if anything, rather enhances it.

To further investigate the relative contribution of regulatory andeffector CD4� T cells in disease development in these mice, weexamined the levels of both cell types in circulation following

EAE induction. During the first 9 days, the fraction of regulatoryCD4� T cells decreased in both groups of mice, but GITRL TGmice continuously displayed more regulatory T cells in circulationthan did WT mice (Fig. 7E). Both GITRL TG and WT miceshowed a disease-related increase of effector type CD4� T cells inperipheral blood, but this occurred later in GITRL TG than in WTmice, which is interesting, as it correlates with the observed delayin disease development (Fig. 7F). At 14 days after immunization,when both mouse strains had a comparable clinical score, therewere no significant differences in effector or regulatory CD4� Tcells in the blood of GITRL mice compared with WT mice.

Finally, to investigate whether the observed differences in EAEsignified merely a difference in time or also in quality of the im-mune response, we analyzed the cellular infiltrates in the CNS ofmice with a similar clinical score. The infiltrated areas were com-parable in size between GITRL TG and WT mice (Fig. 7G). More-over, these mice showed a comparable influx of macrophages (Fig.7H) and CD4� T cells (Fig. 7I) in the CNS, and the influx ofFoxP3� cells was very low in both groups (Fig. 7J). Therefore,these data suggest that GITR engagement does not change thequality of this autoimmune response, but rather delays disease de-velopment. This is not due to a decrease in the formation of dis-ease-related effector type CD4� T cells, but might be related to analtered recirculation of these cells.

DiscussionSince its discovery in 1997, GITR has been the focus of manystudies that address its biological function in cellular immunology(2,4–6,8). These studies have indicated that GITR has costimula-tory effects during T cell activation, but it is still not fully under-stood at what level GITR triggering affects both T cell activationand regulatory T cell function and how this influences immuneresponses in vivo. Here we describe that in vivo GITR stimulationthrough its natural ligand increased absolute numbers of both ef-fector and regulatory type CD4� T cells. Detailed analysis re-vealed that this accumulation was a direct consequence of en-hanced proliferation of both cell types in GITRL TG mice. Theincrease of effector and regulatory CD4� T cells was not at theexpense of the naive CD4� T cell pool. Together with the findingthat transferred naive WT T cells do not get activated in GITRLTG mice (Fig. 5G), this indicates that enhanced GITR triggering isnot sufficient to activate naive T cells, but that this process is stillfully dependent on TCR activation. When TCR triggering is pro-vided, GITR stimulation does enhance the expansion of newlyactivated CD4� T cells, as can be concluded from the in vitrostimulation (Fig. 5A–C) and EAE immunization experiments (Fig.7C). However, the BrdU incorporation experiments indicate thatconstitutive GITR triggering also enhances proliferation of effectorand regulatory CD4� T cells during the steady-state situation (Fig.6E). To what extent the TCR is also required for this increasedlevel of homeostatic proliferation in GITRL TG mice is not yetclear, as this expansion might also be driven by increased avail-ability of cytokines like IL-2. Transfer experiments with TCR-transgenic T cells could shed further light on this issue.

GITR ligation in vivo does not affect the anergic state of regu-latory T cells in vitro, nor does it influence the suppressive func-tion of regulatory T cells (Fig. 4A). The fact that GITRL TG micedo not develop any sign of organ inflammation or autoimmunity(Fig. 4C), despite the expansion of effector T cells, supports thisnotion and indicates that regulatory T cells are fully functional invivo in these mice and maintain homeostasis. The hypothesis thatGITR regulates the size, but not the function, of the regulatory Tcell pool is supported by the observation that GITR�/� mice have

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normally functioning regulatory T cells, but fewer absolute num-bers (2, 11). In vitro studies have shown that agonistic anti-GITRAbs can induce proliferation of regulatory T cells in vitro in anIL-2-dependent manner, also without affecting their suppressiveactivity (5, 11). We found that GITRL expression on B cells in-creased IL-2 production by CD4� T cells in vivo (Fig. 2H) and invitro (Fig. 5A), which is most likely a direct effect, as GITR cross-linking with Abs can induce IL-2 production through TRAF-5(TNFR-associated factor-5)-mediated NF-�B activation (32).These results have two important implications for our understand-ing of the biological function of GITR on T cells. First, sinceregulatory T cells depend on exogenous IL-2 for their proliferation(33), these findings indicate that GITR drives proliferation of bothregulatory and effector T cells through the induction of IL-2 fromthe latter. Second, it explains why GITR stimulation on nonregu-latory T cells allows them to escape suppression by regulatory Tcells (11), since it was recently shown that regulatory CD4� Tcells exert their suppressive function through consumption of IL-2produced by activated T cells, leading to apoptosis of the latter(34). Since GITR triggering increases the production of IL-2, non-regulatory T cells can thereby escape from or delay cytokine dep-rivation-induced apoptosis. These implications fit in a previouslypostulated model for GITR function (7), in which it was also sug-gested that when GITRL expression decreases at the end of animmune response, this would render effector T cells susceptible tosuppression by an expanded, activated regulatory T cell pool.Transgenic GITRL expression does not allow us to test this hy-pothesis in our system, but it is worth following up on this idea, asit implies that GITR is indirectly involved in termination of a Tcell response.

Detailed analysis revealed that GITR ligation in vivo modifiedthe expression of several key proteins expressed by regulatory Tcells (Fig. 3). We found that the IL-2 receptor is down-regulatedon regulatory T cells of GITRL TG mice, which is most likely adirect consequence of increased IL-2 consumption driving en-hanced proliferation (33). This is in agreement with recent findingsthat homeostatically proliferating regulatory CD4� T cells in vivoexpress lower levels of the IL-2 receptor than do nonproliferatingcells (35). Furthermore, GITRL TG mice contained more regula-tory T cells with an activated phenotype, expressing low levels ofCD62L and high levels of CD103 (Fig. 3) (25). This is interesting,because we found that BrdU predominantly incorporated in theCD62L� and CD103� population of regulatory T cells in GITRLTG mice (Fig. 6). This would thus indicate that GITR ligationinduces proliferation of CD62L�CD103� regulatory T cells andthat during this proliferation they become activated and accumu-late as CD62L�CD103� regulatory T cells. This would be inagreement with an earlier study, which described that regulatory Tcells with a high turnover down-modulate CD62L after several celldivisions (36). Since CD62L is required for HEV-dependent lym-phocyte entry into lymph nodes and CD103 is an integrin neces-sary for the homing and retention of cells at inflammatory sites,these data suggest that regulatory T cells in GITRL TG mice aremore prone to enter (inflamed) peripheral tissues than secondarylymphoid organs compared with their WT counterparts. Indeed,we found that liver and bone marrow of GITRL TG mice accu-mulate more CD62L�CD103� regulatory T cells than do WTmice (data not shown), but since the supply of regulatory T cellsis also increased in these mice, it requires more specific migrationexperiments to adequately address this issue.

An intriguing finding from our analysis of GITRL TG mice isthat the functional consequences of GITR engagement were re-stricted to CD4� T cells, as no effects on the proliferation or ef-fector cell formation of CD8� T cells could be detected, neither in

vitro nor in vivo (Figs. 2 and 5A–C and data not shown). This is incontrast with other studies in which a role for GITR on CD8� Tcell responses was demonstrated, using agonistic GITR Abs orGITR�/� mice (17, 37, 38). We found that both CD4� and CD8�

T cells in GITRL TG mice had down-modulated surface expres-sion of GITR compared with WT mice (Fig. 1 and data notshown), which indicates that GITR was functionally engaged by itsligand on both cell types. In WT mice, GITR expression is higheron CD4� nonregulatory T cells than on CD8� T cells (2) (and datanot shown), which could be the reason why GITRL expression hasa stronger effect on CD4� T cells than on CD8� T cells. Thismight also relate to the finding that the costimulatory effect ofGITR cross-linking with an anti-GITR Ab is apparent at a loweranti-CD3 concentration in CD4� T cells than in CD8� T cells(11). Moreover, GITR up-regulation following T cell activation isdependent on CD28 engagement in CD4� cells but not CD8� Tcells (11, 38, 39). Thus, although GITR functions on both CD4�

and CD8� T cells, it is differently regulated in these subsets. In ourhands, deliberate triggering of GITR on CD8� T cells in vivo byits natural ligand clearly does not translate into functional conse-quences, or at least not as strong as the effects found on CD4� Tcells.

The synchronized expansion of regulatory and effector CD4� Tcells that is induced upon GITR stimulation might seem contra-dictory for protective immunity, as these cell types obviously haveopposite functions. However, recent in vivo studies have shownthat regulatory T cells expand with similar kinetics as effectorCD4� T cells upon HSV-2 infection (40) or immunization withFreund’s complete adjuvants (41), so that their ratio remains rel-atively constant. Coincident expansion of regulatory and effector Tcells could be a direct consequence of responsiveness of regulatoryT cells to IL-2 produced by effector T cells (42), and our datasuggest that GITR could play a role in this process. It is most likelythat the simultaneous increase of regulatory and effector T cells isthe reason why GITRL overexpression induces a mild phenotypecompared with transgenic overexpression of other members of theTNF superfamily, such as CD70, OX40L, 4-1BBL, and LIGHT,which leads to severe immunopathology induced by effector Tcells (20, 43–47). The clinical consequence of an immune re-sponse might even depend on this ratio of effector vs regulatory Tcells, as the experimental induction of both adjuvant arthritis andtype 1 diabetes correlates with an increase of this ratio (48–50).The same might apply for the EAE model, as depletion of regu-latory T cells resulted in enhanced disease progression and severity(51). We found that the delay in disease induction observed inGITRL TG mice was not due to a inhibition in the formation ofeffector CD4� T cells in the draining lymph nodes (Fig. 7C), butrather correlated with a delay in the increase of effector CD4� Tcells in circulation (Fig. 7, E and F). As no differences were ob-served in final disease severity nor cellular infiltrates in the brainparenchyma, these observations suggest that GITR stimulation en-hances both formation of effector and regulatory CD4� T cells inlymph nodes and might delay autoimmunity by regulating emigra-tion of effector CD4� T cells from the lymph nodes. WhetherGITR triggering has a direct effect on the egress of activated Tcells from lymph nodes or that this is an indirect effect mediated byregulatory T cells awaits further investigation.

In conclusion, we have shown that GITR serves as a costimu-latory molecule in that it induces proliferation of regulatory as wellas effector CD4� T cells in vivo. We suggest that up-regulation ofGITRL on APCs during the initiation of an immune response,through the increase of proinflammatory stimuli, enhances IL-2production and thereby the proliferation of cognate CD4� T cells,

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which also makes them less susceptible to suppression by regula-tory T cells. At the same time, GITRL expression during this earlyphase induces the expansion of regulatory T cells, aided by thepresence of exogenous IL-2 from proliferating nonregulatory Tcells. These regulatory T cells might be important to re-establishthe status quo of the immune system at later stages of the response.

AcknowledgmentsWe thank Alex de Bruin, Sten Libregts, and Dr. Koen van der Sluijs fortechnical assistance, as well as the staff of the animal facility of the Aca-demic Medical Center for excellent animal care. We appreciate the dedi-cated assistance from Dr. Marian van Roon and coworkers in generatingthe GITRL TG mice. We thank Hella Aberson and Dr. Eric Eldering for thedevelopment of the murine apoptosis multiplex ligation-dependent ampli-fication probe set and Dr. Janneke Samsom for providing us with reagentsfor the IL-2 ELISA. Finally, we thank Drs. Janneke Samsom, EstherNolte-’t Hoen, Eric Eldering, and Kris Reedquist for critical reading of themanuscript and helpful discussions.

DisclosuresThe authors have no financial conflicts of interest.

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