Review

The Biology of IgG Subclasses and Their Clinical

Relevance to Transplantation
Nicole M. Valenzuela, PhD1 and Stefan Schaub, MD, MSc2,3

Abstract: Immunoglobulin G (IgG) is the dominant immunoglobulin and can be divided into 4 distinct subclasses. The evolution
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of IgG subclass switches is regulated by interaction with T cells and follows a 1-way direction (IgG3 → IgG1 → IgG2 → IgG4).
Based on their structure, the 4 IgG subclasses can initiate different effector function such as complement activation, recruitment
of various cells by Fc receptors, and agonistic signaling. Using current assays for HLA antibody detection as a template and
replacing the generic reporter antibody with IgG subclass–specific reporter antibodies, it is possible to investigate the IgG
subclasses of HLA antibodies. There are 15 different IgG subclass compositions possible. Based on the capability to activate
the complement system and the class switch direction, 3 arbitrary patterns can be defined (ie, only complement-binding sub-
classes [IgG3 and/or IgG1], expansion to noncomplement-binding subclasses [IgG3 and/or IgG1 plus IgG2 and/or IgG4],
and switch to noncomplement-binding subclasses [IgG2 and/or IgG4]). The latter group accounts for less than 5%, whereas
the former 2 groups have a similar prevalence close to 50%. In the past 5 years, several studies correlated the IgG subclass
pattern with occurrence of antibody-mediated rejection and allograft outcomes. Because of differences of the used IgG sub-
class assay, the time point of analyses, and the definition of outcomes, a clear picture has not emerged yet. Future needs are
standardization of the assay, a more detailed knowledge of the initiated effector functions, and more well-designed clinical
studies also looking at changes of the IgG subclass pattern over time.

(Transplantation 2018;102: S7–S13)

BIOLOGY OF IMMUNOGLOBULIN G SUBCLASS
PRODUCTION
Immunoglobulin Genomic Organization
Antibodies are heterodimeric glycoproteins composed
of 2 light chains and 2 heavy chains. Both the light and
heavy chains have variable regions that form the contact
site for the antigen (paratope) and constant regions that
enable protein assembly and mediate effector functions.
Received 4 November 2016. Revision received 9 December 2016.
Accepted 13 December 2016.
1
UCLA Immunogenetics Center, Department of Pathology and Laboratory
Medicine, University of California, Los Angeles, CA.
2
Transplantation Immunology and Nephrology, Department of Biomedicine, University
Hospital Basel, Basel, Switzerland.
3
HLA-Diagnostic and Immunogenetics, Department of Laboratory Medicine,
University Hospital Basel, Basel, Switzerland.
The authors declare no funding or conflicts of interest.
S.S. and N.V. contributed equally to the outline, research and writing of
the manuscript.
Correspondence: Nicole Valenzuela, PhD, UCLA Immunogenetics Center, Department
of Pathology and Laboratory Medicine, University of California, Los Angeles, Room
1-520, 1000 Veteran Ave, Los Angeles, CA 90095. (npyburn@mednet.ucla.edu);
and Stefan Schaub, MD, MSc, Clinic for Transplantation Immunology and
Nephrology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland.
(stefan.schaub@usb.ch).
Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.
ISSN: 0041-1337/18/10201S-S7
DOI: 10.1097/TP.0000000000001816
The isotype and subclass are determined by the constant
region of the heavy chain encoded by discrete loci in the
immunoglobulin (Ig) complex. The human Ig system is di-
vided into 5 isotypes: IgM, IgD, IgA, IgE, and IgG. IgA and
IgG can be further subdivided into several subclasses. The
heavy chain constant region genes are ordered μ, δ, γ3, γ1,
α1, γ2, γ4, ε, and α2.
The Basics of Class Switching
The molecular mechanisms by which B cells class switch
have been extensively studied. IgM is the first subclass to be
produced, and only antigen-experienced B cells undergo class
switching to other isotypes. The nature of the antigen influ-
ences IgG subclass production, with thymus (T)–independent
polysaccharide or glycolipid antigens typically inducing IgM
and IgG2 and protein antigens canonically stimulating
IgG1 and IgG3. Production of most terminal IgG4 subclass
is usually seen only with chronic antigen stimulation, such as
in the setting of allergy or parasitic infection. T-dependent
protein antigens stimulate follicular B cells that typically re-
quire CD4+ T cell signaling to generate class-switched Igs
and for the establishment of memory B cells. Briefly, for-
mation of a synapse between T and B cells facilitates
CD40-CD40L (CD154) interactions that prime the B cell.
Cytokines signal the B cell to switch the isotype and subclass
of Ig and to secrete Ig (elegantly reviewed in a study by
Stavnezer et al1). A B cell that has switched to a downstream
isotype or subclass is incapable of switching back to a more
upstream isotype.

Transplantation ■ January 2018 ■ Volume 102 ■ Number 1S www.transplantjournal.com S7

Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.

S8 Transplantation ■ January 2018 ■ Volume 102 ■ Number 1S www.transplantjournal.com

B cells that express the more terminal subclasses IgG2 and

monocytes
and some
FcγRIIIB

neutrophils
IgG4 often retain remnants of upstream switch regions,2

+++

+++
NA2

-
suggesting that class switch recombination occurs in a step-
wise manner. Moreover, the variable regions of IgG2 and
IgG4 often exhibit more extensive somatic hypermutation
than IgG1 or IgG3 and may be of higher antigen affinity than

monocytes
and some
earlier subclasses.

FcγRIIIB

neutrophils
+++

+++
NA1
It is known that the T follicular–associated interleukin

-
(IL)-21 and TH2-associated cytokines such as IL-4,
IL-13, and IL-10 influence Ig isotype and subclass produc-
tion.3,4 But to date, no dedicated switch factor for any

FcγRIIIA-V158
given Ig subclass has been identified, and the signals

NK cells
governing specification of which isotype or subclass is

+++

+++
+

+
used are incompletely defined. Effort is needed to further
elucidate the signals controlling subclass specification, par-
ticularly in the setting of alloimmunization.

FcγRIIIA-F158

NK cells
+++
++
–

-
STRUCTURE AND EFFECTOR FUNCTIONS
OF IGG SUBCLASSES
Although the constant regions of the different gamma

dendritic cells
subclasses are more than 90% homologous, their effector

B cells, and
neutrophils,
functions and affinity for antigen can vary significantly. IgG1

FcγRIIB

Monocytes,
++
is by far the most abundant subclass in serum, and IgG3 has

+
-
the shortest half-life of only 7 days. The constant region of
IgG3 has the longest hinge region, yielding the most flexibility
and accessibility to effector molecules. The hinge regions of
IgG2 and IgG4 are much shorter, and these molecules exhibit

Monocytes and
FcγRIIA-R131

neutrophils
more rigidity. Variations in the species of N-linked glycan +++

+++
at the heavy chain constant region 2 (CH2) can dramatically

+
-
affect antibody effector function.5 Contact sites for engaging
complement C1q and Fc gamma receptors (FcγRs) overlap
in this CH2 domain, and this is where much of the diversity
between IgG subclasses is observed.
Monocytes and
FcγRIIA-H131

neutrophils
Complement
+++

+++
++

IgG3 and IgG1 exhibit the most efficient activation of the

Fc-receptor expression and IgG subclass–related effector functions

classical complement cascade. Under conditions of high an-

tibody titer or high antigen density, IgG2 is capable of acti-
vating complement as well, but IgG4 is generally incapable
Monocytes and

neutrophils

(Table 1). The complement C1 complex binds to the Fc region

activated
FcγRI
+++

+++
+++

of IgG via C1q-CH2 interactions. Key amino acid residues in

-

IgG that are required for C1q binding have been identified.7
Because host cells express a constellation of complement
regulatory proteins antagonizing the complement cascade,
only very high titers of antibody and/or the stronger
Complement

C1q binding subclasses may be able to trigger terminal

activation

++++
+++

complement activation leading to formation the membrane

+

attack complex and cell lysis. Complement components C3d

and C4d, commonly detected as part of the histological
diagnosis of antibody mediated rejection, are products of
cleavage by regulatory proteins and are remnants of
Abundance

different stages of complement activation in the vasculature.

++++
++
+
+

Interestingly, IgG4 molecules are prone to dissociation,

forming half molecules of 1 light chain and 1 heavy chain.
IgG4 may compete with other subclasses for antigen binding
Partially adapted from.6

and block complement activation.8 Given that IgG4 often

Cell expression
TABLE 1.

has the highest affinity for antigen but the lowest capacity
to elicit effector functions, it has been proposed that chronic
antigen exposure stimulates IgG4 as a mechanism of limiting
IgG1
IgG2
IgG3
IgG4

the humoral immune response.

Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.

© 2017 Wolters Kluwer
Fc Receptors
Antibodies enlist adaptive and innate immune cells to carry
out effector functions such as phagocytosis, degranulation and
antibody-dependent cell-mediated cytotoxicity. Receptors
for the Fc regions of IgA, IgG, IgE, and IgM are expressed
on a variety of lymphoid and myeloid cells, including B cells,
monocytes, natural killer (NK) cells, neutrophils, mast cells,
and occasionally, T cells. The human FcγR system is com-
posed of 3 major classes, FcγRI (CD64), FcγRII (CD32)
and FcγRIII (CD16). FcγRII and FcγRIII each have several
isoforms. Only the FcγRIIb exhibits inhibitory functions
via an ITIM; the other FcγRs are activating. The unique
FcγR molecules have discrete affinities for different repertoires
of IgG subclasses and patterns of expression6 (Table 1). In
humans, FcγRIIA, FcγRIIIA, and FcγRIIIB are polymorphic,
with allelic variants exhibiting differences in affinity for IgG
subclasses relevant in infectious disease, autoimmunity, and
response to therapeutic antibodies as well as transplant
outcomes.9 Association of a coding polymorphism in
FcγR IIA has also been associated with graft survival in
retransplant candidates (reviewed in study by Bournazos
et al10). We observed that in vitro recruitment of
monocytes by HLA antibody–activated endothelial cells
varies depending on the IgG subclass of HLA antibody
and the monocyte FcγR repertoire.11 In brief, IgG3 and
IgG1 bind well to monocyte FcγRI, neutrophil FcγRIIA,
and FcγRIIIB, and NK cell FcγRIIIA. IgG2 can only be
effectively recognized by 1 allele of FcγRIIA, whereas
IgG4 is only bound by FcγRI (see Table 1 for more detail).
Agonistic Signaling
In addition to the discussed Fc-mediated effector functions,
antibodies are capable of activating agonistic signaling on di-
merization of target antigens. Many groups have described
that the intracellular signaling cascades downstream of
HLA cross-linking by antibodies in antigen-presenting cells
and vascular cells. Of relevance to transplantation, antibod-
ies to HLA class I have been shown to trigger endothelial
and smooth muscle cell activation of proliferative and inflam-
matory pathways via calcium signaling, mammalian target of
rapamycin, and ERK12-14 that can also be detected in allo-
grafts during rejection.15 It is hypothesized that such agonistic
signaling promotes endothelial dysfunction and transplant
vasculopathy. In theory, all IgG subclasses should be capable
of cross-linking HLA molecules and triggering activation
of endothelial cells in the allograft vasculature, although
this remains to be determined experimentally.
MEASUREMENT OF HLA-SPECIFIC IGG
SUBCLASSES
An accurate assay to measure HLA-specific IgG subclasses
requires targets carrying stable amounts of HLA molecules
and IgG subclass–specific reporter antibodies with a high sen-
sitivity and specificity. The first studies investigating HLA-
specific IgG subclasses used cells as the HLA-molecule
source and various reporter antibodies with often not-well-
characterized specificities.16,17 The introduction of single
HLA-antigen beads (SA) revolutionized the ability to detect
HLA antibodies. Using this test, system with IgG subclass–
specific reporter antibodies has emerged as the preferred
assay to detect HLA-specific IgG subclasses. Based on the
current literature and our own experience (Hönger et al18),
Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.
Valenzuela and Schaub S9
we recommend using the following reporter antibodies be-
cause they have a very low cross-reactivity: IgG1, clone
HP6001; IgG2, clone HP6002; IgG3, clone HP6050; and
IgG4, clone HP6025.
The readout of the SA assay is mostly given as mean fluo-
rescence intensity (MFI), which reflects the number of bound
HLA antibodies to the beads. It is important to notice that
IgG subclass–specific SA assays produce different MFI values
compared with the generic SA assay using a polyspecific re-
porter antibody binding to all IgG subclasses. In a very clean
experimental system, we observed 60% lower MFI for IgG1,
76% lower MFI for IgG2, 37% lower MFI for IgG3, and
50% higher MFI for IgG4 compared with the generic SA
assay (Hönger et al18). Furthermore, the background signal
in the IgG subclass–specific assay is often substantially lower
than in the generic SA assay. Therefore, it seems reasonable
to use for each IgG subclass, and each serum as well as each
bead, a different MFI cutoff to assign a positive re-
sult.19,20 On the other hand, an arbitrary MFI cutoff for
all IgG subclass–specific assays might be easier to use and
allows for better comparison among different studies.21,22
Although not absolutely correct from a scientific point of
view, we favor a pragmatic approach to standardize the
IgG subclass–specific assay for clinical studies. We suggest
using the previously mentioned IgG subclass–specific re-
porter antibodies all at the same concentration and to ap-
ply 1 arbitrary MFI cutoff for all IgG subclass–specific
assays, which is 50% lower than the one used for the ge-
neric SA assay.
IGG SUBCLASS PATTERN OF HLA ANTIBODIES
Given the 4 IgG subclasses, 15 different combinations with
at least 1 detectable IgG subclass are possible. Based on the
complement-activating capability and the class switch
evolution of IgG, 3 “biological” groups can be defined:
(i) “complement-binding subclasses only” group (ie, IgG1
and/or IgG3), (ii) “expansion to noncomplement-binding
subclasses” group (ie, IgG1 and/or IgG3 and IgG2 and/or
IgG4), and (iii) “switch to noncomplement-binding sub-
classes” group (ie, IgG2 and/or IgG4).
Three studies provided details regarding IgG subclass pat-
terns, which are summarized in Figure 1.19,20,22 Despite using
different assay conditions and investigating patients at differ-
ent clinical stages, the IgG subclass patterns are remarkably
similar. IgG1 was the most frequently observed isolated
subclass (25%-30%), followed by IgG3 (5%), and IgG2
(2%) and IgG4 (2%). With respect to the “biological” groups,
the “complement-binding subclasses only” group accounted for
37% to 48% and the “expansion to noncomplement-binding
subclasses” group for 47% to 62% of all IgG subclass pattern.
The “switch to noncomplement-binding subclasses” group
was rarely observed (1%-5%). Importantly, a single, re-
stricted subclass response is also quite rare.
Lowe et al20 found that IgG subclasses induced by blood
transfusions were more often restricted to the “complement-
binding subclasses only” group, whereas pregnancies
and graft failure more often provoked an expansion to
noncomplement-binding subclasses. Interestingly, Arnold
et al23 reported that an expansion to noncomplement-binding
subclasses in patients with de novo donor-specific antibodies
to HLA (HLA-DSA) after renal transplantation was associated
with a higher frequency of ABMR. Altogether, these data
S10 Transplantation ■ January 2018 ■ Volume 102 ■ Number 1S
FIGURE 1. IgG subclass patterns of HLA antibodies observed in three different populations.18,19,21
suggest that an expansion to noncomplement-binding sub-
classes indicates an advanced immune response stimulated by
a longer and more intense antigen exposure. It is still unknown,
under which conditions a complete switch to noncomplement-
binding subclasses with loss of IgG1/3-producing plasma cells
will occur.
CLINICAL STUDIES INVESTIGATING THE UTILITY
OF IGG SUBCLASS ASSAYS
The presence of HLA-DSA pretransplant or posttransplant
is associated with an increased risk for ABMR and allograft
loss. However, individual patients with HLA-DSA demon-
strate a wide spectrum ranging from uneventful clinical
courses to severe ABMR with subsequent allograft loss.
Several studies investigated whether an IgG subclass analy-
sis of HLA-DSA helps to improve prediction of ABMR and
allograft loss.
Table 2 summarizes those 10 studies that analyzed all 4 IgG
subclasses. Because of the different settings (eg, pretransplant vs
posttransplant), different IgG subclass assay protocols and
cutoffs, and different definitions of outcomes, it is currently
impossible to draw any firm conclusions. It seems that the
pretransplant IgG subclass pattern is not very predictive for
ABMR and allograft loss. By contrast, the posttrans-
plant presence of high IgG3 levels or an expansion to
noncomplement-binding subclasses likely provide diagnostic
and prognostic value beyond the generic SA assay regarding
prediction of the ABMR phenotype and the clinical course of
ABMR. Whether this information is clinically helpful to
guide treatment strategies is still unknown.
There are several other studies that investigated only 1 sin-
gle IgG subclass regarding prediction of allograft failure.
Such a study design might introduce a bias because the effect
of other individual IgG subclasses and specific IgG subclass
mixtures cannot be fully investigated. We only highlight
2 studies in this context. Everly et al28 found that de novo
Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.
www.transplantjournal.com
posttransplant HLA-DSA being IgG3 and IgM positive are
associated with a high risk of renal allograft failure. O'Leary
et al29 observed a trend toward inferior survival of liver allo-
graft recipients with pretransplant IgG3 positive HLA-DSA.
A very interesting topic is the dynamic evolution of HLA-
DSA IgG subclasses from pretransplant to posttransplant.
So far, this has only been systematically investigated by
Khovanova et al25 using sera obtained within the first 30 days
posttransplant. They observed changes in the IgG subclass pro-
files, but the correlation with clinical outcomes was rather weak.
Another interesting question is the biological significance
of IgG2/IgG4 HLA-DSA when present together with IgG1/
IgG3 HLA-DSA. As previously mentioned, some clinical
studies suggest that an expansion to these noncomplement-
binding subclasses indicates an advanced immune response
enabled by ongoing T cell help. These IgG2/IgG4 antibodies
might induce complement activation in synergy with IgG1/
IgG3 antibodies if they target different epitopes on the same
HLA molecule.30 On the other hand, if the IgG2/IgG4 antibod-
ies are directed against the same epitope as the IgG1/IgG3 anti-
bodies, they could potentially block IgG1/IgG3-dependent
complement activation. We found in in vitro experiments
that this blocking effect starts when IgG2/IgG4 are present
in 2-fold higher concentrations than IgG1/IgG3 and it is al-
most complete at a 10-fold excess (Hönger et al18).
FUTURE NEEDS
The humoral immune response against HLA antigens is
very complex, because the target is highly polymorphic; the
process is dynamic and has different effector functions largely
depending on the IgG subclasses. Therefore, deciphering
the precise role of IgG subclasses requires progress in
several areas.
First, the IgG subclass assay needs to be standardized to
such an extent that comparison between studies is possible.
 TABLE 2.
Summary of studies investigating all 4 IgG subclasses simultaneously regarding associated clinical outcomes

IgG subclass
Analyzed samples Author, year detection Ab Assay (cutoff ) Organ (n) Population Clinical association
17
© 2017 Wolters Kluwer

Pretransplant Griffiths et al, 2004 IgG1—8C/6-39 Flow cross match with Kidney (20) Patients with pretransplant • Skewing toward IgG1 (only IgG1 pos.)
IgG2—HP6014 surrogate cells class I DSA compared with skewing away from IgG1
IgG3—HP6050 (=IgG2/3/4 dominant) associated with
IgG4—HP6025 lower graft survival
Pretransplant Hönger et al, 201119 IgG1—HP6001 Single antigen beads Kidney (74) Patients with pretransplant DSA • Subclass profile not associated with
IgG2—31-7-4 (individual for each development and severity of ABMR
IgG3—HP6050 subclass and bead) • Isolated IgG2/4 likely less ABMR
IgG4—HP6025
Pretransplant and Gao et al, 200416 Not reported Flow cross match Kidney (7) Not reported • 1/7 kidney allografts were lost due to
posttransplant Liver (6) hyperacute ABMR. This patient had
high pretransplant IgG3.
Pretransplant and Cicciarelli et al, 201324 IgG1—HP6001 Single antigen beads Kidney (3) Patients with pretransplant DSA • Case series (no statistical analysis)
posttransplant IgG2—HP6002 (likely MFI > 500) Heart (4)
IgG3—HP6047
IgG4—HP6023
Pretransplant and Khovanova et al, 201525 IgG1—4E3 Single antigen beads Kidney (80) Patients with pretransplant DSA • Pretransplant IgG1 presence and IgG4
posttransplant IgG2—31-7-4 (individual for levels associated with rejection
IgG3—HP6050 each subclass) within 30 days
IgG4—HP6025 • Pretransplant IgG1 levels and IgG4
presence associated with allograft failure
• Posttransplant presence or levels of
IgG1/IgG4 were associated with rejection
and graft failure
Pretransplant and Lefaucheur et al, 201622 IgG1—HP6001 Single antigen beads Kidney (186) Patient with pretransplant DSA • IgG3 associated with clinical ABMR and
posttransplant and Viglietti et al, 201626 IgG2—31-7-4 (mostly MFI > 500) (n = 110) and patients decreased death-censored graft survival
IgG3—HP6050 with posttransplant DSA • IgG4 associated with subclinical ABMR
IgG4—HP6025 (n = 186; de novo and
persisting pretransplant DSA)

Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.

Posttransplant at time Kannabhiran et al, 201227 IgG1—4E3 Single antigen beads Kidney (11) Patients with ABMR • No associations found (sample size
of rejection and IgG2—HP6002 (MFI > 1000) receiving bortezomib limitation discussed)
after treatment IgG3—HP6050
IgG4—HP6025
Posttransplant at time Kaneku et al, 201221 IgG1—4E3 Single antigen beads Liver (39) Patients with chronic rejection • Presence of multiple IgG subclasses is
of rejection IgG2—HP6002 (MFI > 500) (n = 39) and controls associated with chronic rejection
IgG3—HP6050 (n = 66) • IgG3 associated with graft loss
IgG4—HP6025
Valenzuela and Schaub

Posttransplant Arnold et al, 201423 IgG1—HP6001 Single antigen beads Kidney (94) Patients with posttransplant • Expansion from IgG1/3 alone to
IgG2—31-7-4 (likely MFI > 1000) de novo DSA IgG1/3 plus IgG2/4 had a higher
IgG3—HP6050 rate of ABMR but similar allograft survival
IgG4—HP6025
S11
S12 Transplantation ■ January 2018 ■ Volume 102 ■ Number 1S
Second, because patients typically mount a mixed subclass
alloimmune response, the individual contribution of each
subclass can be difficult to dissect in these studies. It is unclear
whether the predominance of IgG4 late in transplant and its
association with chronic rejection reflects the particular
pathogenic effect of this subclass or rather is indicative of
sustained alloimmune activation.
Third, the clinical relevance of each subclass is beginning
to be revealed, but the mechanisms of graft injury elicited
by different donor specific IgG subclasses are mostly un-
characterized. Certainly, complement activation is a key path-
ological mechanism of graft injury, but it is not absolutely
required for rejection in humans or in animal models.16,31
NK cells and macrophages also seem to play an important role
during ABMR.31-33 However, the contributions of FcγR-
mediated functions and differential capacity of IgG sub-
classes to elicit antibody-dependent cell-mediated cytotoxicity
and phagocytosis have yet to be fully elucidated in the context
of antibody mediated rejection.
Fourth, the factors regulating isotype and subclass spec-
ification are incompletely understood. Different routes of
allosensitization seem to stimulate distinct subclass reper-
toires,20 but the milieu of B cell activation during different
alloantigen exposures is uncharacterized. That de novo
donor-specific antibody production is highly associated
with medication nonadherence34,35 implies that current
T cell–directed immunosuppression does have some effect
on B cell activation and antibody production. In the studies
discussed earlier, each center used different induction thera-
pies. Little is known about how class switching might differ af-
ter induction therapies or under immunosuppressive regimens
that are used in transplant patients. More studies are needed to
describe the “natural history” of anti-HLA IgG subclasses in
the pretransplant and posttransplant periods and to under-
stand how they correlate with clinical outcome.
Finally, the measurement of all 4 IgG subclasses in addi-
tion to the generic SA assay is quite expensive. These costs
seem only be justified if the IgG subclass information pro-
vides substantial diagnostic and/or prognostic value beyond
the generic SA assay.
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