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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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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
GENERAL DISCUSSION
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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
GENERAL DISCUSSION
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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.
GENERAL DISCUSSION
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Reference List
1. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive T cells to platelet GPIIb-IIIa in
immune thrombocytopenic purpura. Role in production of anti-platelet
autoantibody. J.Clin.Invest 1998;102:1393-1402.
2. Sukati H, Watson HG, Urbaniak SJ, Barker RN. Mapping helper T-cell epitopes
on platelet membrane glycoprotein IIIa in chronic autoimmune
thrombocytopenic purpura. Blood 2007;109:4528-4538.
3. Filion MC, Proulx C, Bradley AJ et al. Presence in peripheral blood of healthy
individuals of autoreactive T cells to a membrane antigen present on bone
marrow-derived cells. Blood 1996;88:2144-2150.
4. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune
tolerance. Cell 2008;133:775-787.
5. Ephrem A, Chamat S, Miquel C et al. Expansion of CD4+CD25+ regulatory T
cells by intravenous immunoglobulin: a critical factor in controlling
experimental autoimmune encephalomyelitis. Blood 2008;111:715-722.
6. Nishimoto T, Satoh T, Takeuchi T, Ikeda Y, Kuwana M. Critical role of
CD4(+)CD25(+) regulatory T cells in preventing murine autoantibody-mediated
thrombocytopenia. Exp.Hematol. 2012;40:279-289.
7. Aboul-Fotoh L, bdel Raheem MM, El-Deen MA, Osman AM. Role of
CD4+CD25+ T cells in children with idiopathic thrombocytopenic purpura.
J.Pediatr.Hematol.Oncol. 2011;33:81-85.
8. Audia S, Samson M, Guy J et al. Immunologic effects of rituximab on the human
spleen in immune thrombocytopenia. Blood 2011;118:4394-4400.
9. Bao W, Bussel JB, Heck S et al. Improved regulatory T-cell activity in patients
with chronic immune thrombocytopenia treated with thrombopoietic agents.
Blood 2010;116:4639-4645.
10. Li J, Wang Z, Hu S, Zhao X, Cao L. Correction of abnormal T cell subsets by
high-dose dexamethasone in patients with chronic idiopathic thrombocytopenic
purpura. Immunol.Lett. 2013; 154:42-8
11. Ling Y, Cao X, Yu Z, Ruan C. Circulating dendritic cells subsets and
CD4+Foxp3+ regulatory T cells in adult patients with chronic ITP before and
after treatment with high-dose dexamethasome. Eur.J.Haematol. 2007;79:310-
316.
CHAPTER 6
162
12. Liu B, Zhao H, Poon MC et al. Abnormality of CD4(+)CD25(+) regulatory T cells
in idiopathic thrombocytopenic purpura. Eur.J.Haematol. 2007;78:139-143.
13. Sakakura M, Wada H, Tawara I et al. Reduced Cd4+Cd25+ T cells in patients
with idiopathic thrombocytopenic purpura. Thromb.Res. 2007;120:187-193.
14. Stasi R, Cooper N, Del PG et al. Analysis of regulatory T-cell changes in patients
with idiopathic thrombocytopenic purpura receiving B cell-depleting therapy
with rituximab. Blood 2008;112:1147-1150.
15. Yu J, Heck S, Patel V et al. Defective circulating CD25 regulatory T cells in
patients with chronic immune thrombocytopenic purpura. Blood 2008;112:1325-
1328.
16. Zahran AM, Elsayh KI. CD4+CD25+High FoxP3+ Regulatory T Cells, B
Lymphocytes, and T Lymphocytes in Patients With Acute ITP in Assiut Children
Hospital. Clin.Appl.Thromb.Hemost. 2014;20:61-7
17. Koch MA, Tucker-Heard G, Perdue NR et al. The transcription factor T-bet
controls regulatory T cell homeostasis and function during type 1 inflammation.
Nat.Immunol. 2009;10:595-602.
18. Schneider A, Rieck M, Sanda S et al. The effector T cells of diabetic subjects are
resistant to regulation via CD4+ FOXP3+ regulatory T cells. J.Immunol.
2008;181:7350-7355.
19. Schneider A, Long SA, Cerosaletti K et al. In active relapsing-remitting multiple
sclerosis, effector T cell resistance to adaptive T(regs) involves IL-6-mediated
signaling. Sci.Transl.Med. 2013;5:170ra15.
20. Venigalla RK, Tretter T, Krienke S et al. Reduced CD4+,CD25- T cell sensitivity
to the suppressive function of CD4+,CD25high,CD127 -/low regulatory T cells in
patients with active systemic lupus erythematosus. Arthritis Rheum.
2008;58:2120-2130.
21. Wehrens EJ, Mijnheer G, Duurland CL et al. Functional human regulatory T cells
fail to control autoimmune inflammation due to PKB/c-akt hyperactivation in
effector cells. Blood 2011;118:3538-3548.
22. Zhang XL, Peng J, Sun JZ et al. De novo induction of platelet-specific
CD4(+)CD25(+) regulatory T cells from CD4(+)CD25(-) cells in patients with
idiopathic thrombocytopenic purpura. Blood 2009;113:2568-2577.
23. Aubin E, Lemieux R, Bazin R. Indirect inhibition of in vivo and in vitro T-cell
responses by intravenous immunoglobulins due to impaired antigen
presentation. Blood 2010;115:1727-1734.
GENERAL DISCUSSION
163
6 6
24. Kuwana M, Okazaki Y, Ikeda Y. Splenic macrophages maintain the anti-platelet
autoimmune response via uptake of opsonized platelets in patients with
immune thrombocytopenic purpura. J.Thromb.Haemost. 2009;7:322-329.
25. McMillan R. Antiplatelet antibodies in chronic immune thrombocytopenia and
their role in platelet destruction and defective platelet production.
Hematol.Oncol.Clin North Am. 2009;23:1163-1175.
26. Mc Auley D. Common Laboratoy (LAB) values [I]. 2012. Retrieved 24-10-2014
from: http://www.globalrph.com/labs i.htm
27. Poel CE van der, Spaapen RM, van de Winkel JG, Leusen JH. Functional
characteristics of the high affinity IgG receptor, FcgammaRI. J Immunol.
2011;186:2699-2704
28. Li X, Wu J, Ptacek T et al. Allelic-dependent expression of an activating Fc
receptor on B cells enhances humoral immune responses. Sci Transl Med.
2013;5:1-20
29. Su K, Yang H, Kimberly RP et al. Expression Profile of FcγRIIb on Leukocytes
and Its Dysregulation in Systemic Lupus Erythematosus. J Immunol.
2007;178:3272-3280
30. Rodeghiero F, Michel M, Gernsheimer T et al. Standardization of bleeding
assessment in immune thrombocytopenia: report from the International
Working Group. Blood 2013;121:2596-2606.
31. Kono H, Kyogoku C, Suzuki T et al. FcgammaRIIB Ile232Thr transmembrane
polymorphism associated with human systemic lupus erythematosus decreases
affinity to lipid rafts and attenuates inhibitory effects on B cell receptor
signaling. Hum.Mol.Genet. 2005;14:2881-2892.
32. Morris DL, Roberts AL, Witherden AS et al. Evidence for both copy number and
allelic (NA1/NA2) risk at the FCGR3B locus in systemic lupus erythematosus.
Eur J Hum Genet. 2010;18:1027-1031
33. Su K, Wu J, Edberg JC et al. A Promotor Haplotype of the Immunoreceptor
Tyrosine-Based Inhibitory Motif-Bearing FcgRIIb Alters Receptor Expression
and Associates with Autoimmunity. I. Regulatory FCGR2B Polymorphisms and
Their Association with Systemic Lupus Erythematosus. J.Immunol.
2004;172:7186-7191.
34. McKinney C, Broen JC, Vonk MC et al. Evidence that deletion at FCGR3B is a
risk factor for systemic sclerosis. Genes Immun. 2012;13:458-460
CHAPTER 6
164
35. Breunis WB, van ME, Bruin M et al. Copy number variation of the activating
FCGR2C gene predisposes to idiopathic thrombocytopenic purpura. Blood
2008;111:1029-1038.
36. Carcao MD, Blanchette VS, Wakefield CD et al. Fcgamma receptor IIa and IIIa
polymorphisms in childhood immune thrombocytopenic purpura.
Br.J.Haematol. 2003;120:135-141.
37. Cooper N, Stasi R, Cunningham-Rundles S et al. Platelet-associated antibodies,
cellular immunity and FCGR3a genotype influence the response to rituximab in
immune thrombocytopenia. Br J Haematol. 2012;158:539-547.
38. Eyada TK, Farawela HM, Khorshied MM et al. FcgammaRIIa and FcgammaRIIIa
genetic polymorphisms in a group of pediatric immune thrombocytopenic
purpura in Egypt. Blood Coagul.Fibrinolysis 2012;23:64-68.
39. Floto R.A., Clatworthy MR, Heilbronn KR et al. Loss of function of a lupus-
associated FcgRIIb polymorphism through exclusion from lipid rafts. Nat.Med.
2005;10:1056-1058.
40. Foster CB, Zhu S, Erichsen HC et al. Polymorphisms in inflammatory cytokines
and Fcgamma receptors in childhood chronic immune thrombocytopenic
purpura: a pilot study. Br.J.Haematol. 2001;113:596-599.
41. Koene HR, Kleijer M, Algra J et al. FcgRIIIa-185V/F Polymorphism Influences
the Binding og IgG by Natural Killer Cell FcgRIIIa, Independently of the
FcgRIIIa-48L/R/H Phenotype. Blood 1997;90:1109-1114.
42. Zhou XJ. Cheng FJ, Qi YY et al. FCGR2B and FCRLB gene polymorphisms
associated with IgA nephropathy. PLoS One. 2013;8:e61208
43. Zhou XJ. Lv JC, Yu L et al. FCGR2B gene polymorphism rather than FCGR2A,
FCGR3A and FCGR3B is associated with anti-GBM disease in Chinese. Nephrol
Dial Transplant. 2010;25:97-101.
44. McKinney C. Broen JC, Vonk MC et al. Evidence that deletion at FCGR3B is a
risk factor for systemic sclerosis. Genes Immun. 2012;13:458-460
45. Bruin M, Bierings M, Uiterwaal C et al. Platelet count, previous infection and
FCGR2B genotype predict development of chronic disease in newly diagnosed
idiopathic thrombocytopenia in childhood: results of a prospective study.
Br.J.Haematol. 2004;127:561-567.
46. Fujimoto TT, Inoue M, Shimomura T et al. Involvement of Fcg receptor
polymorphism in the therapeutic response of idiopathic thrombocytopenic
purpura. Br.J.Haematol. 2001;115:125-130.
GENERAL DISCUSSION
165
6 6
47. Wu J, Edberg JC, Redecha PB et al. A novel polymorphism of FcgammaRIIIa
(CD16) alters receptor function and predisposes to autoimmune disease.
J.Clin.Invest 1997;100:1059-1070.
48. Heijden J. van der, Breunis WB, Geissler J et al. Phenotypic variation in IgG
receptors by nonclassical FCGR2C alleles. J.Immunol. 2012;188:1318-1324.
49. Nagelkerke SQ, Breunis WB, Tacke CEA et al. Comprehensive genotyping of the
FCGR 2/3 locus reveals extensive ethnic variation, linkage disequilibrium and
novel chimeric genes. Manuscript in preparation
50. Metes D, Ernst LK, Chambers WH et al. Expression of functional CD32
molecules on human NK cells is determined by an allelic polymorphism of the
FcgammaRIIC gene. Blood. 1998;91:2369-80
51. Lejeune J, Piègu B, Gouilleux-Gruart V et al. FCGR2C genotyping by
pyrosequencing reveals linkage disequilibrium with FCGR3A V158F and
FCGR2A H131R polymorphisms in a Caucasian population. MAbs. 2012;4:784-
787
52. Su K, Li X, Edberg JC et al. A Promotor Haplotype of the Immunoreceptor
Tyrosine-Based Inhibitory Motif-Bearing FcgRIIb Alters Receptor Expression
and Associates with Autoimmunity. II. Differential Binding of GATA4 and Yin-
Yang1 Transcription Factors and Correlated Receptor Expression and Function.
J.Immunol. 2004;172:7192-7199.
53. Bolland S, Ravetch JV. Spontaneous autoimmune disease in Fc(gamma)RIIB-
deficient mice results from strain-specific epistasis. Immunity. 2000;13:277-85
54. Pritchard NR, Cutler AJ, Uribe S et al. Autoimmune-prone mice share a
promoter haplotype associated with reduced expression and function of the Fc
receptor FcgammaRII. Curr Biol. 2000;10:227-30.
55. Jiang Y, Hirose S, Abe M et al. Polymorphisms in IgG Fc receptor IIB regulatory
regions associated with autoimmune susceptibility. Immunogenetics.
2000;51:429-35
56. McGaha TL, Sorrentino B, Ravetch JV. Restoration of tolerance in lupus by
targeted inhibitory receptor expression. Science. 2005;307:590-3
57. Su K, Wu J, Edberg JC et al. A promoter haplotype of the immunoreceptor
tyrosine-based inhibitory motif-bearing FcgammaRIIb alters receptor expression
and associates with autoimmunity. I. Regulatory FCGR2B polymorphisms and
their association with systemic lupus erythematosus. J Immunol. 2004;172:7186-
91
CHAPTER 6
166
58. Bergtold A, Desai DD, Gayhane A et al. Cell surface recycling of internalized
antigen permits dendritic cell priming of B cells. Immunity. 2005;23:503-14
59. Wu Z, Zhou J, Prsoon P et al. Low expression of FCGRIIB in macrophages of
immune thrombocytopenia-affected individuals. Int.J.Hematol. 2012;96:588-593.
60. Durandy A, Kaveri SV, Kuijpers TW. Intravenous immunoglobulins-
understanding properties and mechanisms. Clin. Exp. Immunol. 2009;158:2-13
61. Crow AR, Song S, Freedman J et al. IVIg mediated amelioration of murine ITP
via FcgRIIB is independent of SHIP1, SHP-1 and Btk activity. Blood
2003;102:558-560.
62. Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatory Activity of IVIG
Mediated Through the Inhibitory Fc Receptor. Science 2001;291:484-486.
63. Leontyev D, Katsman Y, Branch DR. Mouse background and IVIG dosage are
critical in establishing the role of inhibitory Fcγ receptor for the amelioration of
experimental ITP. Blood. 2012;119:5261-4
64. Floto RA, Clatworthy MR, Heilbronn KR. Loss of function of a lupus-associated
FcγRIIb polymorphism through exclusion from lipid rafts. Nat.Med.
2005;11:1056-58
65. Bruin M, Bierings M, Uiterwaal C et al. Platelet count, previous infection and
FCGR2B genotype predict development of chronic disease in newly diagnosed
idiopathic thrombocytopenia in childhood: results of a prospective study. Br J
Heamatol. 2004;127:561-7
66. Li X, Wu J, Carter RH et al. A novel polymorphism in the Fcgamma receptor IIB
(CD32B) transmembrane region alters receptor signaling. Arthritis Rheum.
2003;48:3242-3252.
67. Rand ML, Leung R, Packham MA. Platelet function assays. Transfus Apher Sci.
2003;28:307-317.
68. Rodgers RP, Levin J. A critical reappraisal of the bleeding time. Semin Thromb
Hemost. 1990;16:1-20.
69. Picker SM. In-vitro assessment of platelet function. Transfus Apher Sci.
2011;44:305-319.
70. De Cuyper IM, Meinders M, van d, V et al. A novel flow cytometry-based
platelet aggregation assay. Blood 2013;121(10):e70-e80.
GENERAL DISCUSSION
167
6 6
71. Bonnefoy A, Yamamoto H, Thys C et al. Shielding the front-strand beta 3 of the
von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein
Ibalpha. Blood 2003;101(4):1375-1383.
72. Gadisseur A, Hermans C, Berneman Z et al. Laboratory diagnosis and molecular
classification of von Willebrand disease. Acta Haematol. 2009;121(2-3):71-84.
73. Buchanan GR, Adix L, Buchanan GR, Adix L. Grading of hemorrhage in
children with idiopathic thrombocytopenic purpura. J.Pediatr. 2002;141:683-688.
74. Bennett CM, Rogers ZR, Kinnamon DD et al. Prospective phase 1/2 study of
rituximab in childhood and adolescent chronic immune thrombocytopenic
purpura. Blood. 2006;107:2639-42
75. Porcelijn L, Huiskes E, Maatman R, de KA, de HM. Acquired Glanzmann's
thrombasthenia caused by glycoprotein IIb/IIIa autoantibodies of the
immunoglobulin G1 (IgG1), IgG2 or IgG4 subclass: a study in six cases. Vox
Sang. 2008;95(4):324-330.
76. Brighton TA, Evans S, Castaldi PA, Chesterman CN, Chong BH. Prospective
evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in
idiopathic thrombocytopenic purpura and other immune thrombocytopenias.
Blood 1996;88(1):194-201.
77. Hagenstrom H, Schlenke P, Hennig H, Kirchner H, Kluter H. Quantification of
platelet-associated IgG for differential diagnosis of patients with
thrombocytopenia. Thromb.Haemost. 2000;84(5):779-783.
78. Warner MN, Moore JC, Warkentin TE, Santos AV, Kelton JG. A prospective
study of protein-specific assays used to investigate idiopathic thrombocytopenic
purpura. Br.J.Haematol. 1999;104(3):442-447.
79. Siebers RW, Wakem PJ, Carter JM. Long-term intra-individual variation of
platelet parameters. Med.Lab Sci. 1989;46(1):77-78.