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Pediatric immune thrombocytopenia: Catching platelets

Laarhoven, A.G.

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Citation for published version (APA):Laarhoven, A. G. (2015). Pediatric immune thrombocytopenia: Catching platelets.

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Download date: 07 Aug 2020

General

Discussion

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This thesis encompasses research performed on the topic of pediatric immune

thrombocytopenia (ITP). To investigate the pathogenesis of ITP, we used

samples collected as part of the TIKI study (Treatment with or without IVIg in

Kids with acute ITP), in which children with newly diagnosed ITP are enrolled

and subsequently randomized into those receiving intravenous immunoglobulin

(IVIg) or not, to study whether IVIg will prevent development of chronic ITP.

This primary outcome of the TIKI study will be evaluated after inclusion of a

cohort of 200 cases. At the time of our studies, 138 children were enrolled. In this

thesis we focused on the putative role of regulatory T cells (Treg) in ITP, which

among other things, play an important role in protecting us from autoimmune

diseases. Furthermore, we studied Fc gamma receptor (FcγR) polymorfisms in

the pathogenesis of ITP in children and the role of FcγR profiles on the

immediate response of the platelet count upon treatment with IVIg. Finally, the

effects of platelet autoantibodies in functional platelet responses and their

correlation with bleeding tendency in children with chronic ITP were

investigated.

Treg in pediatric ITP

Immune thrombocytopenia is an autoimmune disease of heterogeneous origin,

of which the pathogenesis is not completely understood. To complicate matters

further, there are indications that the underlying pathology in ITP differs, not

only between newly diagnosed and chronic ITP, but also between disease onset

at childhood or adult age. To date, the majority of research addresses ITP

pathology in adult chronic ITP. In many cases, only those who fail to respond to

first line treatment are investigated, because patients and blood samples are

more easily available from this group. In the past decade, research on ITP has

increasingly focused on the role of effector T cells (Teff) and concomitantly, on

Treg. Autoreactive T cells, responding to several epitopes of glycoprotein IIIa

(GPIIIa), were identified in ITP patients.1;2 Curiously, T cells with similar

specificity can apparently be found in all healthy individuals, as was shown by

Filion et al. who tested 25 randomly assigned healthy blood bank donors who

were negative for anti-GPIIIaIIb antibodies. However, these healthy donor cells

were in an anergic state, indicating loss of tolerance in ITP patients.1;3 FOXP3-

positive Tregs are pivotal cells in maintaining peripheral tolerance, which makes

them a promising target in autoimmunity research.4 In addition, Ephrem et al.

demonstrated that development of induced experimental autoimmune

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encephalomyelitis (EAE) in mice could be prevented by IVIg treatment. The

preventive effect was associated with increased Treg numbers with enhanced

suppressive function. In addition, after depletion of Tregs with anti-CD25

monoclonal antibodies 10 days before EAE induction, IVIg therapy failed

altogether,5 indicating that IVIg might function via Tregs. It has to be noted

though that activated Teff may also have been depleted by this method, as they

carry CD25. In addition, Nishimoto et al. observed that syngeneic nude mice

injected with Treg depleted T cells spontaneously developed an ITP in 36% of

the cases, whereas all remained healthy when Treg were maintained. Moreover,

the mice that developed ITP also developed antiplatelet antibodies.6 Finally,

although the literature is contradicting, many studies have been performed

describing decreased Treg numbers and/or function in adult chronic ITP.7-16

Taken together, we hypothesized that also in children Treg might play a crucial

role in disease development, and are imperative to IVIg to exerts its beneficial

effect. We assumed that Treg numbers and function would be decreased in

newly diagnosed pediatric ITP patients, and would be restored or even

enhanced following IVIg therapy. However, in chapter 2 we show that Treg

number and suppressive function is similar to healthy controls, both during

diagnosis and upon recovery. In a cross-over model in which we co-cultured

PBMCs obtained at diagnosis together with Tregs from time point recovery and

vice versa, we demonstrated that Teff at diagnosis are more active. Remarkably,

Treg obtained at recovery failed to suppress these Teff altogether. Treg obtained

at diagnosis, on the contrary, suppressed these Teff just fine. This could point at

a compensatory mechanism in which Treg activity is enhanced at the onset of

the disease in response to the inflammatory environment. This phenomenon has

been described in mice.17 Similar findings of adequate Treg function and

enhanced Teff activity have been reported in juvenile idiopathic arthritis (JIA),

systemic lupus erythematosus (SLE) diabetes mellitus type 1 (DMT1) and other

autoimmune diseases.18-21 All in all, studies proposing increased resistance to

suppression of Teff rather than malfunctioning Treg in autoimmune diseases are

accumulating. In our studies we even observed Treg obtained at diagnosis

which seemed to have enhanced suppressive capacity and thus were well

adapted to the situation at hand. This, however, was only a trend and sample

size was small.

Our research is subject to some limitations, especially the small sample size,

limited sample volume of only 5-10 ml of peripheral blood, and the artificial

nature of our in vitro assays, which can never be fully representative of what

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occurs in vivo. In addition, we investigated Treg in general, since we were

unable to test antiplatelet antigen specific Treg specifically, and used peripheral

blood instead of splenic cells. Zhang et al. described that platelet antigen-

specific-Teff are only adequately suppressed by Treg sharing that specificity,

and that it only occurs in the presence of APCs primed for this antigen.22 Also,

Aubin et al. demonstrated in mice that the positive effect of IVIg in

downregulating antigen specific T cell responses, is established by direct

interaction with FcγR bearing APCs instead of T cells.23 These are remarkable

findings, worth further exploration, especially the necessary intercession of

primed APCs. Furthermore, we focused on newly diagnosed or persistent ITP in

children, who recovered spontaneously, thus our findings might be specific for

this cohort. We chose to address this specific group of patients because we

wished to study an ITP patient cohort without possible influences of

immunomodulating therapy. Moreover, we aimed to study the most common

natural course of disease of ITP in children, of which 75% does recover

spontaneously. In addition, we performed a pilot experiment in which we

assessed Treg suppressive capacity in 3 children with chronic ITP, showing

strong suppressive function equal to the healthy control. Our findings convinced

us to reject our initial hypothesis, since no clues were found pointing at

decreased Treg numbers or malfunction underlying disease pathogenesis in

newly diagnosed pediatric ITP. Preceding this line of thought, strengthened by

the observations of Zhang and Aubin,22;23 we no longer expect IVIg to function

primarily via Treg up-regulation. Therefore, we shifted our focus from Treg in

ITP to FcγR bearing phagocytic cells.

FcγR polymorfisms in ITP

Other important players in ITP pathogenesis are the FcγR bearing cells, who are

responsible for the destruction of opsonised platelets, particularly in the

spleen.24-25 While FcγRI is a high affinity receptor, which can even bind

monomeric antibodies, and as such is often occupied in vivo due to the high IgG

levels in serum of 3.31-11.64 g/L in a 1 year old increasing to 7.23 - 16.85 g/L at

adult age,26 its role in phagocytosis in vivo is not completely clear.27 Low-affinity

FcγRs on the contrary, consisting of the activating FcγRIIIa, FcγRIIIb, FcγRIIa,

FcγRIIc and the inhibiting FcγRIIb, are known to bind cells or pathogens

opsonised with IgG, and are capable to mediate antibody-dependent cell-

mediated cytotoxicity (ADCC) and phagocytosis. On macrophages, DCs and in

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rare cases B cells, the balance of activating and inhibiting FcγRs might determine

how and if the opsonized targets are processed and presented. On B cells the

inhibiting FcγRIIb can downregulate the cells’ activation and proliferation, and

as such function as a negative feedback for antibody production.28;29 Besides

playing a role in current ITP disease models, the relevance of FcγRs is

underlined by the success rate of IVIg treatment and splenectomy.30

Recently, research has focused on SNPs and CNVs of the genes encoding the

family of FcγR, namely the FCGR. Some of this genetic variation is known to

alter FcγR affinity and/or function and level of expression. To date, many FcγR

polymorfisms have been associated with a variety of autoimmune diseases, such

as SLE,31-33 antibody positive RA,34 ITP35-41 and others.42-44 Though an association

with FCGR CNVs has never been described in ITP, many studies identified

SNPs with an increased prevalence in ITP. Initial studies in this field have been

performed in heterogeneous and small groups of patients, resulting in

conflicting data.35;36;38-41;45;46 However, most of these studies report enhanced

FCGR3A*158V allele frequency in ITP, which has an increased affinity for IgG1,

IgG3 and IgG4.35;41;47 Recently, an increased incidence of the so-called

FCGR2C*ORF has been described, which results in FcγRIIC expression on

monocytes, B cells28 and NK cells, in contrast to the regular stop that turns

FCGR2C into a pseudogene35. To date, it is known that the FCGR2C*ORF allele

has to be further specified in order to distinguish between a ‘classical’ C-ORF

and a ‘non classical’ NC-ORF’, as the latter does not lead to FcγRIIC expression

due to an alternative splice site mutation.48 In chapter 3 we studied FCGR allele

distribution in 138 children with newly diagnosed ITP. Because FcγR

distribution is known to vary among ethnic background, we restricted our study

to individuals from Caucasian origin. Furthermore, all FCGR2 and FCGR3 alleles

are clustered in a region of 1q22, within a distance of 0.5 MB, therefore

haplotypes comprising linked combinations of the various FCGR2 and FCGR3

alleles may be present. We confirmed the increased allele frequency of

FCGR3A*V158 in newly diagnosed pediatric ITP (41.6% vs 32.8% in healthy

controls, P=.02). However, by correcting for the high linkage disequilibrium

(LD) between FCGR3A*V158 and FCGR2C*C-ORF (0,96),49 we showed that the

increased allele frequency of FCGR3A*V158 is a concomitant finding to the

enhanced prevalence of genotype FCGR2C*C-ORF (33.3% vs 19.6% in healthy

controls, P=.005) in combination with FcγRIIC promoter haplotype 2B.2 (34.8%

vs 19.6% in healthy controls, P=.002). Although the observation that enhanced

prevalence of FCGR3A*V158 in pediatric ITP is a confounder to the increased

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incidence of FCGR2C*C-ORF, it might still contribute actively to disease

development. FCGR3*158V results in a higher affinity for IgG and might

therefore augment binding and internalization of opsonized platelets.

Expression of FcγRIIC might culminate in: first, an altered balance in activating

and inhibiting FcγRs, resulting in enhanced phagocytic activity; second, it might

alter the inhibitory signaling of FcγRIIB expressed on B cells, being the only

activating FcγR present on these cells, leading to autoantibody production28 and

third it may play a role in the onset of pediatric ITP following a viral infection,

as FcγRIIC is present on NK cells where it amplifies ADCC activity, especially

under inflammatory conditions.35;48;50;51 No functional effects, indicating a certain

dis- or advantage of the FCGR2C 2B.2 promoter polymorphism, have been

described.33

In addition, we observed that the FCGR2B promoter haplotype 2B.4, which is

associated with a 1.5 fold increase in FcγRIIB expression and functional

activity,33;52 is more prevalent in our patient cohort, a finding which is highly

counterintuitive. Mice which were FcγRIIB deficient developed a SLE like

symptoms.53 In addition, mice were prone to autoimmune diseases when their

FcγRIIB promoter was subject to polymorphisms resulting in lower FcγRIIB

expression levels.54;55 When FcγRIIB expression was restored to normal levels

autoimmunity was prevented.56 On the contrary, Su et al. proved that in human

SLE patients FcγRIIB promoter haplotype 2B.4 was overrepresented compared

to healthy individuals, like in our ITP patients.57 We could not explain

mechanistically how augmented inhibitory function would predispose to

development of ITP, unless the increased expression functionally enhances the

immune response, perhaps through mechanisms that are not related to B cell

activation directly. Evidence for enhanced antigen presentation of intact

antigens through IgG and FcγRIIB has been provided on mouse dendritic cells.58

Another possibility would be that FcγRIIB expression levels are not solely

dependent on the promoter haplotype. Indeed, Wu et al. described lower

FcγRIIB expression on splenic macrophages of refractory adult ITP patients,59

which does not add up with the increased incidence of the 2B.4 promotor

haplotype in ITP. Furthermore, Su et al. demonstrated that there are indeed

subtleties in expression levels of FcγRIIB per cell subset. By showing a down-

regulation of FcγRIIB expression on memory- and plasma B cells in SLE

patients, compared to their naïve B cells, which is contrary to expression

patterns in healthy controls.29;33;52 Together, these data show that genotype

cannot be adopted in a linear fashion to expression levels on cells per se.

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Next to investigating the FcγR profile in patients versus controls, we were

intrigued by the possibility that FcγR isotypes might predict a patients’ response

to IVIg, especially since IVIg among others, is thought to function via FcγR by

either blocking FcγR or adjusting the balance of activating and inhibiting FcγR.60

We formulated our hypothesis based on some murine models, demonstrating

that IVIg function depends on FcγRIIB, and upregulates the inhibitory FcγR.61-62

During our research it became known that the conclusions drawn from the

murine models were probably due to different mice strains rather than FcγRIIB

dependent.63 We hypothesized that patients with the FcγRIIB promoter

haplotype 2B.4, and the common homozygous FcγRIIB*I232 genotype, would be

more responsive to IVIg therapy. This would be not only because of its

augmented expression levels, but also because FcγRIIB*I232 dampens activating

receptors more efficiently than FcγRIIB*T232.64 Indeed, in the acute pediatric ITP

patients the homozygous FcγRIIB*I232 genotype, in combination with promoter

haplotype 2B.4, predicted a good response to IVIg in 93% of the cases, and was

also associated with a quick spontaneous recovery in 44% of the patients in the

observatory group. Moreover, we showed in all three individuals with a

homozygous FcγRIIB*T232 genotype in our series that they failed to respond

altogether. These data add up to the previous finding of Bruin et al. that

pediatric patients with a heterozygous FcγRIIB*I/T232 genotype are more prone

for developing chronic ITP compared to patients with a homozygous

FcγRIIB*I232 genotype.65 Unfortunately, none of the participants in that study

carried the homozygous FcγRIIB*T232 variant. Although our finding is

intruiging, in particular because FcγRIIB*T232 is associated with impaired

FcγRIIB function,31;39;65;66 it has to be considered carefully. Homozygous

FcγRIIB*T232 is very rare in Caucasians.49 Moreover, we could not ensure

whether the observed outcome was IVIg specific, since no patients were carrier

of homozygous FcγRIIB*T232 in our observation group.

In contrast, two children with chronic ITP who carried the homozygous

FcγRIIB*T232 genotype did respond to IVIg therapy. This could point to a

different mechanism in acute and chronic ITP.65 Another explanation would be

the small group size as well as the varying time intervals after which the platelet

count has been determined in this study. Homozygous FcγRIIB*T232 might be

associated with an impaired short term response to IVIg. Whereas, after a few

weeks, the more long term response is probably determined by other factors.

Thus, no consensus on the effect of IVIg treatment in patients carrying the

homozygous FcγRIIB*T232 could be reached.

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Still, our studies do illustrate that SNP and CNV analysis might yield insights in

both disease pathogenesis and working mechanisms of immunomodulating

therapies, and as such enable personalized medicine in the future. FcγR

polymorphisms on APCs are in particular of interest in ITP pathogenesis, since

the subtle differences in binding and processing of opsonized platelets might

determine how they are presented, and thus whether autoreactive T cells are

activated and e.g. a TH1 response is favored. The APCs might be the crucial cells

in determining onset of ITP or disease outcome. Nevertheless, genotyping

studies are prone to a severe limitation, since they only provide a rough estimate

of responsiveness to a certain stimulus. In vivo, FcγR expression levels are very

flexible depending on the immune status of an individual. To that end,

functional data would be very helpful. However, research in this area in

challenged by great similarities among FcγR which complicate staining in flow

cytometry and functional experiments, like phagocytic or ADCC assays.

Ideally, APCs from patients of which the FcγR profile is known should be

tested. By testing these cells in phagocytic or ADCC assays, or co-culturing these

cells together with opsonised platelets and antigen specific T cells, to assess T

cell activation and proliferation, might lead to a deeper understanding of the

presumed pivotal role of APCs in ITP development.

Platelet defects in chronic pediatric ITP

In addition to pondering fundamental research questions addressing the

development of, and pathogenesis underlying pediatric ITP, we wondered why

the degree of thrombocytopenia does not necessarily predict bleeding. In

chapter 5 we hypothesized that not only platelet numbers but also platelet

function could be affected in ITP patients, resulting in the variety of bleeding

phenotypes observed. Since the golden standard in platelet function tests, the

light transmission aggregometry test, is not reliable with platelet counts below

50*109/L, we developed two new functional assays suitable for individuals with

low platelet counts.67-69 The first assay, the micro platelet aggregation test,

assesses platelet function directly, by measuring patients’ platelets capability to

form aggregates with control platelets in response to an agonist of choice.

Because autoantibodies associated with ITP are mainly directed at GPIIbIIIa,

and to a lesser extent to GPIbIX, we thought that activation pathways operating

via these glycoproteins were the most likely to be affected. Therefore, we chose

PMA and Ristocetin as agonists respectively.70-72 The second assay, the platelet

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reactivity assay, determines to which extent platelets are activated in response to

an agonists by measuring the speed and quantity of P-selectin expression, to

assess platelet degranulation, and GPIIbIIIa opening, to demonstrate the

conformational stage and ability to bind fibrinogen thereby indicating a pro-

aggregation state. Both tests complement each other, whereas the first provides

a clear cut functional outcome it does not distinguish between possible factors

influencing the final platelet aggregation, other than the pathway of the agonist

of choice. The second assay though gives detailed information on receptor

pathways and specific manifestations of platelet activation, however the overall

picture of platelet function is lacking.

We tested our hypothesis in a well-defined cohort of chronic pediatric ITP

patients, who were characterized as either a mild or a severe bleeding

phenotype based on the modified scale of Buchanan.73;74 Patients were part of the

CINKID study as described in chapter 4. We observed decreased platelet

aggregation in response to PMA activation in patients with a severe bleeding

phenotype. In addition, the reactivity assay showed most predominantly a

lower expression level of P-selectin in response to ADP as well as the need of

higher concentrations of ADP to open GPIIbIIIa in patients with a severe

bleeding phenotype. Results augmented each other. Obviously, since

autoantibodies against GPIIbIIIa are most common in ITP patients, it is tempting

to suggest the involvement of autoantibodies blocking proper platelet function

to explain our results. Probably a similar mechanism takes place as is known to

cause acquired Glanzmanns’ disease, in which autoantibodies interact with

platelet function rather than opsonizing them leading to destruction by splenic

macrophages.70;75 Nevertheless, we failed to prove such a mechanism, since we

were unable to perform direct autoantibody testing in our patients’ samples.

Indirect MAIPA and PIFT were performed instead, however, they have such a

low sensitivity and so few were considered positive that no conclusions could be

drawn.76-78 It is of interest to explore what causes the observed platelet defect

found in ITP patients with a severe bleeding phenotype. To that extent more

sensible tests to detect autoantibodies are needed, in which a small amount of

blood is sufficient. An alternative though, would be to test adult ITP patients,

from whom a larger amount of blood can be drawn for testing, to enable direct

autoantibody detection tests even in individuals with low platelet counts.

If indeed platelet function is crucial in developing severe bleeding tendency in

patients with a form of thrombocytopenia, our assays are of great value. They

GENERAL DISCUSSION

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might serve as a tool to predict bleeding tendency and thereby lead to

personalized medicine, in which medical treatment or platelet transfusions are

reserved for those at risk of severe bleeding, and vice versa, adverse effects

caused by treatment are withheld from patients who do not need therapy per se.

However, several limitations have to be overcome before they can be used as

such. First, platelet function has to be assessed in a variety of thrombocytopenic

patients, to determine whether platelet malfunction is indeed a requisite for

severe bleeding. Second, in healthy individuals platelet parameters are known to

be stable over time.79 This might not apply for ITP patients, or patients suffering

from other autoimmune diseases, in which the course of disease is often

unpredictable. Many autoimmune diseases, such as SLE, M.Crohn, JIA and ITP

express a pattern characterized by flares of disease activity alternated by periods

of mild disease activity or even remission. This makes it less likely that a single

measurement will represent a persons’ innate platelet reactivity, although it is

possible that measurements performed during disease activity are

representative for following flares within an individual. Therefore, longitudinal

studies have to be performed. Moreover, it would be of great value to start

testing in newly diagnosed patients and follow the natural course of disease. In

addition, one has to realize that both assays are performed in vitro, implying that

in vivo factors that might influence platelet function as well, such as endothelial

function and plasma factors, are not taken into account.

In conclusion, the studies described in this thesis encompassed various

approaches in pediatric ITP research. A diagnostic track was pursued by the

development of laboratory assays to determine platelet function in individuals

with low platelet counts. Hereby showing that some chronic pediatric ITP

patients have functional platelet defects associated with severe bleeding

tendency irrespective of platelet counts.

Furthermore, by focusing on fundamental research to elucidate the pathogenesis

of newly diagnosed pediatric ITP we showed that Treg function well and are

present in normal numbers, contrary to the majority of the previous findings in

chronic adult ITP patients. Due to this finding we rejected the hypothesis that

the effect of IVIg is based for an important part on restoring normal Treg

number and function. And likewise, the idea of Treg therapy in pediatric ITP

patients. Therefore, we shifted our attention to a possible crucial step prior to T

cell activation, namely FcγR. FcγR play an important role in humoral and

adaptive immune responses and as such might be involved in both clearance of

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Ig-opsonised platelets, as well as presenting them via APCs thereby inducing T

and B cell activation. Indeed, we demonstrated that several FcγR

polymorphisms, which are known to affect presence, expression level and

function of the FcγR, are associated with newly diagnosed ITP, and even

possible, yet contradicting, affect the outcome of IVIg function. These findings

are interesting, both to obtain a deeper understanding of the mechanism of

disease and to predict disease outcome or even effect of therapy. However, there

is still a long way to go, in which expression levels and function of FcγR per cell

subset have to be determined and where possible related to genotype. It would

be even more interesting to set up experiments with patients’ FcγR-bearing cells,

expose them to opsonized platelets and read out autoantigen-specific T cell

responses. Yet, even then it will be in vitro experiments, which might not be

representative for what occurs in vivo. Thus, although promising, personalized

medicine in pediatric ITP still seems one bridge too far.

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