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Intramolecular T cell spreading in unprimed MRLlpr miceImportance of the U1-70k protein sequence 131151.

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Vol. 50, No. 10, October 2004, pp 3232–3238
DOI 10.1002/art.20510
© 2004, American College of Rheumatology
Intramolecular T Cell Spreading in Unprimed MRL/lpr Mice
Importance of the U1-70K Protein Sequence 131–151
Fanny Monneaux, Véronique Parietti, Jean-Paul Briand, and Sylviane Muller
Conclusion. This is the first study to demonstrate
that intramolecular T cell spreading effectively occurs
in MRL/lpr mice with lupus, and that region 131–151 is
important in the cascade of events observed in the
murine lupus response. This sequence might originate a
mechanism of tolerance spreading that leads to the
beneficial effect observed in MRL/lpr mice after treatment with the phosphorylated peptide 131–151.
Objective. To analyze spontaneous T cell spreading against determinants of the U1-70K protein in
young autoimmune MRL/lpr lupus mice, in comparison
with the T cell spreading occurring in normal BALB/c
mice immunized with peptide 131–151 of this protein.
Methods. Peripheral blood lymphocytes (PBLs)
from both unprimed MRL/lpr mice and immunized
BALB/c mice were tested for their ability to proliferate
ex vivo in response to 18 overlapping peptides of the
U1-70K spliceosomal protein, using assays for lymphocyte proliferation and secretion of interleukin-2.
Results. The proliferative response to peptides of
the U1-70K protein evolved rapidly in MRL/lpr mice
tested at different ages. At least 5 peptides were recognized by PBLs from 8-week-old autoimmune mice,
whereas a different peptide was recognized by PBLs
from MRL/lpr mice at 12 weeks of age. At 15 weeks, the
proliferative response was weak or negative when assessed with any of the test peptides. At least 2 major
peptides recognized by MRL/lpr PBLs were also recognized by PBLs generated in the BALB/c mice primed
with peptide 131–151. We further demonstrated that, in
preautoimmune MRL/lpr mice, repeated administration
of phosphorylated peptide 131–151 (called P140), which
was shown previously to be protective, transiently abolished T cell intramolecular spreading to other regions of
the 70K protein.
Autoimmunity toward the different components
of small nuclear RNP (snRNP) particles is typically
observed in certain autoimmune rheumatic diseases,
such as systemic lupus erythematosus (SLE), as well as
in experimental models of lupus. Whereas the so-called
anti–U1 RNP antibodies target the 70K, A, and C
proteins, which are present specifically within the U1
snRNP complex, anti-Sm antibodies predominantly react with the Sm B/B⬘ and D common proteins, which are
also present in the other spliceosomal snRNP particles
(1). The autoimmune response to snRNP observed in
human and murine lupus is complex in terms of its
specificity, and to date, the precise mechanism leading
to the broad diversity of antibody specificities is largely
It is now widely believed that this diversification
of the antibody response results from a typical phenomenon of B and T cell epitope spreading, which is
preceded by the initiation of immunity to a single or a
few self components (2–6). A model of intra- and
intermolecular diversification of the antibody response
has been proposed and is supported by several experimental studies in naive animals immunized with self
antigens (7–16). Longitudinal studies of sera from patients with lupus and from lupus mice have also demonstrated that, with time, the immune response matures in
an apparently ordered manner and gives rise to a
diversified, non–cross-reacting antibody population
Supported in part by a grant from the Association de Recherche sur la Polyarthrite. Dr. Monneaux’s work was supported by a
postdoctoral grant from the Centre National de la Recherche Scientifique and BioDelivery Systems (Wittelsheim, France).
Fanny Monneaux, PhD, Véronique Parietti, BSc, Jean-Paul
Briand, PhD, Sylviane Muller, PhD: Centre National de la Recherche
Scientifique, Strasbourg, France.
Address correspondence and reprint requests to Sylviane
Muller, PhD, Institut de Biologie Moléculaire et Cellulaire, UPR9021,
Centre National de la Recherche Scientifique, 15 rue René Descartes,
67084 Strasbourg, France. E-mail:
Submitted for publication November 27, 2003; accepted in
revised form June 4, 2004.
(5,17–19), thus reinforcing the concept of intra- and
intermolecular B cell epitope spreading.
In the prevalent view of the mechanisms involved
in epitope spreading, T helper cells play a central role.
Although spreading of the antibody response has been
extensively studied in immunized and lupus-prone animals, little is known concerning the spreading of the T
cell response in lupus. The only data suggesting the
existence of T cell diversification in the case of lupusassociated antigens were obtained in normal animals
immunized with peptide containing a single T helper cell
epitope from lupus autoantigens (20,21). To our knowledge, there is no study published, to date, in which the
natural T cell spreading that occurs in unprimed lupus
mice is discussed.
Our group has identified an epitope within the
131–151 sequence of the U1-70K spliceosomal protein,
which is recognized very early in the course of the
disease by CD4⫹ T cells from MRL/lpr and (NZB ⫻
NZW)F1 mice with lupus (22,23). Peptide 131–151 binds
to diverse murine and human class II major histocompatibility complex molecules and is recognized by IgG
antibodies from lupus mice and from patients with lupus.
Furthermore, we recently demonstrated that administration of peptide 131–151 phosphorylated on Ser140
(called peptide P140) in preautoimmune MRL/lpr mice
reduced the production of anti–double-stranded DNA
(anti-dsDNA) antibody, delayed the development of
proteinuria, and significantly prolonged survival (24).
The mechanism by which the phosphorylated
sequence 131–151 may restore tolerance in autoimmune
mice is not yet known. This P140 peptide encompasses a
conserved RNP1 motif in residues 139–151 that interacts
with RNA within the U1 RNP particle. This motif is also
found in other RNP and non-RNP proteins, and we
previously postulated that autoreactive T cells to this
candidate sequence might drive the diversification of the
autoimmune response at an early, asymptomatic stage of
the disease (14,25).
To investigate this possibility experimentally, we
tested the T cell immune response in MRL/lpr mice
longitudinally with a set of 18 overlapping peptides of
the U1-70K protein, and followed up the hierarchy of
the appearance and disappearance of T cell reactivity
over time. We compared the results with the T cell
reactivity in normal mice immunized with peptide 131–
151 administrated in adjuvant. The findings of the
present study support our model and emphasize the
importance of the region 131–151 in the cascade of
events observed in the murine lupus response.
Peptides. The eighteen 16- to 25-mer peptides corresponding to the murine sequence of the snRNP 70K protein
have been described previously (22,24). The homogeneity of
each purified peptide was checked by analytical highperformance liquid chromatography, and the identity of each
peptide was assessed by matrix-assisted laser desorption and
ionization time-of-flight (TOF) mass spectrometry using a
Protein TOF apparatus (Bruker Spectrospin, Bremen, Germany).
Mice. Female BALB/c (H-2d) and MRL/lpr (H-2k)
mice, 5–6 weeks old at the beginning of the experiments, were
purchased from Harlan (Gannat, France).
Lymphocyte proliferation assays and measurement of
interleukin-2 (IL-2) secretion. To test the immunogenicity of
peptide 131–151 in normal mice, BALB/c mice (7 per group)
were immunized subcutaneously at the base of the tail and
hindfoot pads with 100 ␮g of peptide 131–151 or peptide 21–41
(used as a control), which was dissolved in water and mixed
(volume/volume) with Freund’s complete adjuvant. After 13
days, blood was withdrawn from the BALB/c mice and lymphocytes were purified by density separation (Lympholyte-M,
density ⫽ 1.0875; Cedarlane, Hornby, Ontario, Canada). Peripheral blood lymphocytes (PBLs) were collected and washed
3 times in L-alanyl-L-glutamine–enriched RPMI 1640 medium
(Biomedia, Boussens, France) containing 10% fetal calf serum
(FCS; Biomedia), 10 ␮g/ml gentamycin, 10 mM HEPES, and
5 ⫻ 10⫺5M ␤-mercaptoethanol, and resuspended at 3 ⫻ 106
cells/ml in the above-described medium. The proliferative
response to the 18 overlapping peptides of the 70K protein was
measured in duplicate using 3 ⫻ 105 cells/well and a single
peptide concentration (120 ␮M).
After 72 hours, the cultures were pulsed for 18 hours
with tritiated thymidine (specific activity 6.7 Ci/mmole; 1
␮Ci/well) and DNA-incorporated radioactivity was measured
using a Matrix 9600 direct beta counter (Packard, Meriden,
CT). The SD value in duplicate cultures was always less than
20% of the mean value. Control tests were performed by
adding concanavalin A (100 ␮l/well; 5 ␮g/ml) to cells during
the time course (90 hours) of the culture. In some experiments,
proliferation and IL-2 secretion from lymph node cells were
measured 10 days after immunization, as previously described
To study the natural spreading occurring in autoimmune mice, blood was regularly withdrawn from the MRL/
lpr mice (at weeks 5, 8, 12, and 15), and lymphocyte proliferation was measured as described above for the BALB/c mice,
except that peptides of the 70K protein were tested at various
concentrations, rather than at a single concentration. In some
experiments, anti-CD4 monoclonal antibody GK1.5 (10 ␮g/ml;
PharMingen, San Diego, CA) was added to the culture.
To study the effect of P140 peptide administration on
spontaneous T cell spreading occurring in lupus-prone mice,
preautoimmune MRL/lpr mice were injected intravenously
(IV) with 100 ␮g of peptide P140 in saline (9 mice), or received
phosphate buffered saline (PBS) alone (10 mice), at weeks 5, 7,
9, and 13, as described previously (24). Blood was then
regularly obtained from the treated mice at 10, 12, or 14 weeks,
and each peptide (at a concentration of 80 ␮M) was tested in
the cultures for its ability to induce proliferation of PBLs.
Intramolecular T cell spreading in unprimed
MRL/lpr mice. Blood samples were obtained from 20
young, nonimmunized MRL/lpr mice at weeks 5, 8, 12,
and 15, and pooled PBLs were systematically tested for
their ability to proliferate in response to 2 concentrations (80 ␮M and 120 ␮M) of each of the 18 overlapping
16–25-mer peptides spanning the mouse U1-70K protein. Responses were considered to be positive only
when stimulation indices were higher than 3 in the
proliferation assays using either 80 ␮M or 120 ␮M of the
recall peptides, and a dose-dependent response was
As shown in Figure 1A, no proliferative response
of PBLs purified from 5-week-old MRL/lpr mice was
observed. In contrast, a major shift occurred at week 8
(Figure 1B). Eight peptides of the 70K protein, namely
peptides 57–77, 96–116, 131–151, P140, 219–238, 231–
250, 407–428, and 426–448, were found to be able to
stimulate proliferation of PBLs from 8-week-old MRL/
lpr mice.
At 12 weeks, most of the peptides that had shown
a positive response with the T cells from 8-week-old
mice failed to stimulate proliferation of MRL/lpr PBLs,
whereas one peptide, encompassing residues 183–202,
which had been unable to recall a proliferative response
with T cells from the 5- and 8-week-old mice, produced
a strongly positive response in the 12-week-old mice
(Figure 1C). Thus, at 12 weeks, T cells proliferated in
the presence of peptides P140 and 183–202 in the
cultures. Peptides 201–220 and 303–320 were considered
to yield negative results, since they did not produce a
positive dose-dependent response (this was confirmed in
another independent experiment in which both also
showed a negative response). Finally, at 15 weeks, 4
peptides, 96–116, P140, 183–202, and 303–320, were able
to stimulate proliferation with indices higher than 5, but
only at the high peptide concentration of 120 ␮M
(Figure 1D); therefore, in accordance with our criteria,
the results in the 15-week-old mice have to be regarded
as negative.
In these assays, T cell proliferation was dose
dependent (as illustrated with a few examples in Figure
2A) and was found to be inhibited by an anti-CD4
monoclonal antibody (Figure 2B). These results provide
strong evidence that the main proliferative cell population corresponds to CD4-positive T cells.
T cell spreading in BALB/c mice immunized with
peptide 131–151. We recently proposed a model in
which the RNP1 motif present in sequence 131–151 of
Figure 1. Spontaneous T cell spreading in unprimed MRL/lpr mice.
The proliferative response of peripheral blood lymphocytes from 5(A), 8- (B), 12- (C), and 15-week-old mice (D) was measured in the
presence of 80 ␮M and 120 ␮M of each of 18 overlapping peptides of
the U1-70K protein. The results are expressed as the mean and SEM
stimulation index, calculated as the cpm in cultures with peptide
divided by the cpm in cultures without peptide. A mean stimulation
index ⬎3 was considered to be positive (horizontal line). The average
tritiated thymidine incorporation in the absence of peptide was 100
cpm. This experiment is 1 of 2 individual experiments that showed
similar results.
the U1-70K protein could be responsible for the spreading of the autoimmune response directed against spliceosomal proteins (14,25). To evaluate this hypothesis
and perform a comparison with the findings obtained in
the unprimed lupus mice, we immunized BALB/c mice
subcutaneously with peptide 131–151 or the control
peptide 21–41 in Freund’s complete adjuvant, and 13
114–132 and 317–334 (at the limit of positivity) showed
a positive response, as defined by our stringent criteria,
in the proliferation assay (Figure 3A).
These results suggest a mechanism of T cell
spreading, rather than simply representing a crossreaction between the 3 peptides. First, sequence analysis, as shown in Figure 3A, revealed no overlapping
stretches of residues between peptides 57–77, 131–151,
and 219–238. The 3 peptides are hydrophilic, but
Figure 2. Dose-dependent proliferative response to U1-70K peptides
in unprimed MRL/lpr mice, with mediation by CD4 T cells. A, The
proliferative response of peripheral blood lymphocytes from 8-weekold MRL/lpr mice was measured in the presence of various concentrations of peptides 96–116 (solid diamond), 131–151 (open triangle),
P140 (open circle), and 219–250 (solid square) of the U1-70K protein.
The results are expressed as the mean ⫾ SEM stimulation index,
calculated as the cpm in cultures with peptide divided by the cpm in
cultures without peptide. A mean stimulation index ⬎3 was considered
to be positive (horizontal line). The average tritiated thymidine
incorporation in the absence of peptide was 100 cpm. This experiment
is 1 of 2 individual experiments that showed similar results. B,
Proliferative response to peptide P140 (120 ␮M) in 8-week-old MRL/
lpr mice measured in the presence (⫹) or absence (⫺) of neutralizing
anti-CD4 monoclonal antibodies (10 ␮g/ml).
days after administration, we studied the response of
PBLs to the 18 overlapping peptides of the U1-70K
When peptide 21–41 was used as the immunogen, no or very weak proliferation was measurable with
17 of the peptides, with the one exception being in
response to the homologous peptide (results not shown).
In contrast, when peptide 131–151 was used as the
immunogen, we found that not only did the P140
phosphorylated analog yield positive results, but also 2
of the other unrelated peptides of the U1-70K protein,
namely peptides 57–77 and 219–238, as well as peptides
Figure 3. T cell spreading in BALB/c mice immunized with peptide
131–151 of the 70K protein. A, The proliferative response of peripheral
blood lymphocytes from BALB/c mice immunized with peptide 131–
151 was measured 13 days after immunization, in the presence of 120
␮M of each of the 18 overlapping peptides of the U1-70K protein. The
results are expressed as the mean ⫾ SEM stimulation index. A mean
stimulation index ⬎3 was considered to be positive (horizontal line).
The average tritiated thymidine incorporation in the absence of
peptide was 120 cpm. The sequence of 3 strongly positive peptides is
shown (note that, in this experiment performed 13 days after immunization, the response to the homologous peptide is weak or negative).
B, Lymph node cells from BALB/c mice immunized with peptide
131–151 were recalled ex vivo after 10 days in the presence of
increasing concentrations of peptides 131–151 (solid diamond), 57–77
(open square), and 219–238 (open triangle). The average tritiated
thymidine incorporation in the absence of peptide and in the presence
of concanavalin A was 2,500 cpm and 14,000 cpm, respectively. A mean
stimulation index ⱖ2.0 in the proliferation test was considered to be
positive (horizontal line). Secretion of interleukin-2 (IL-2) was measured using CTL-L cells. Bars show the mean ⫾ SEM.
whereas peptide 131–151 is largely basic (ratio of positive charge to negative charges of 5), peptides 57–77 and
219–238 are rather acidic or neutral (ratio of 0.67 and
1.2, respectively). Second, as shown in Figure 3B, we
demonstrated unambiguously that lymph node T cells
generated against peptide 131–151 in BALB/c mice
cannot be activated ex vivo by either of the other 2
peptides, 57–77 and 219–238. Finally, we also showed
that whereas peptide P140 was strongly recognized by
PBLs from unprimed 15-week-old MRL/lpr mice, peptides 57–77 and 219–238 were weakly or not at all
recognized by the same cells (Figure 1D).
Effect of P140 therapy on spontaneous T cell
spreading. We recently demonstrated that IV administration of the phosphorylated peptide P140 to healthy
(preautoimmune) MRL/lpr mice reduced the production
of high titers of anti-dsDNA antibodies, delayed the
appearance of severe proteinuria, and significantly enhanced the survival of treated mice (24). The cellular
and molecular mechanisms involved in this apparent
restoration of tolerance are still unknown. It is particularly striking to observe that the IV administration of a
single 21-mer peptide of the U1 snRNP particle seems to
affect the autoimmune response to native DNA, as well
as important clinical features, leading to a substantial
improvement in the treated animals.
We have proposed several potential mechanisms,
which are presently under investigation in our laboratory, to explain these observations. We have used the
term “tolerance spreading,” as introduced by Kaliyaperumal et al (26), to describe the possible tolerogenic
effect of peptide P140 on autoimmune B and T cell
responses (24). With the intention of experimentally
investigating the effect of peptide P140 on spontaneous
T cell spreading, we studied the proliferative response of
PBLs collected serially from lupus mice that were first
subjected to P140 treatment. Preautoimmune MRL/lpr
mice received 4 IV injections of peptide P140 at weeks 5,
7, 9, and 11 and blood was withdrawn at weeks 10, 12,
and 14 (for review, see ref. 24). The capacity of a set of
selected peptides to recall the T cell response of PBLs ex
vivo was then tested (Figure 4).
As expected, at week 10, in the mice that received
3 administrations of PBS only, PBLs proliferated in
response to the 5 selected peptides 96–116, P140, 219–
238, 407–428, and 426–448 (Figure 4A). However, in the
mice that received 3 administrations of P140, we observed that, with the exception of peptide 96–116, the
proliferative response was significantly lower, and mean
stimulation indices were even under the level of positiv-
Figure 4. Effect of brief phosphorylated peptide P140 therapy on the
spontaneous T cell spreading in MRL/lpr mice. Mice either were
treated with peptide P140 (hatched bar), administrated intravenously
in saline at weeks 5, 7, 9, and 13 (as indicated by the arrows), or
received phosphate buffered saline (PBS) (solid bar) alone at the same
time points. The proliferative response of peripheral blood lymphocytes from 10- (A), 12- (B), and 14-week-old mice (C) was measured in
the presence of 6 selected peptides (80 ␮M). A mean stimulation index
⬎3 was considered to be positive (vertical line). Bars show the mean ⫾
SEM. Values adjacent to bars are the percentage reduction in treated
mice versus nontreated mice. NS ⫽ nonsignificant reduction; na ⫽ not
ity, with peptides P140, 219–238, 407–428, and 426–448
(31–51% decrease) (Figure 4A). The same drop in
proliferative response was observed in 12-week-old mice
with peptide 183–202 (85% decrease) (Figure 4B). At 14
weeks (1 week after the fourth and last injection), PBLs
from MRL/lpr mice that received PBS or P140 proliferated equally well in response to recall peptides P140 and
183–202 (Figure 4C).
In recent years, several laboratories have been
interested in the molecular and cellular mechanisms of
autoantibody diversification in response to SLE autoantigens. As methods are developed to rapidly screen
epitopes of self proteins with miniaturized test formats,
such studies will emerge that have the potential to
generate results with implications for the analysis of
patients’ sera. These previous studies led to the proposal
of a model of intra- and intermolecular diversification
based on the particle hypothesis introduced by Hardin
(27), which associates antigen-reactive B cells and T cells
providing help to these B cells.
Although the diversification of serum circulating
autoantibodies has been studied, particularly in murine
lupus, that of autoreactive T cells is largely unknown in
lupus. Nevertheless, in nonobese diabetic mice that
develop a type 1 diabetes mellitus–like disease, for
example, this phenomenon has been studied in detail,
and the results revealed the progression from a Th1 type
of response directed to a single determinant (peptide
524–543 of glutamic acid decarboxylase [GAD]) in
4-week-old mice to a diversified response to additional
GAD determinants as well as to other beta cell antigens
such as insulin B chain and the 65-kd heat shock protein
in 12-week-old mice (28). Such sequential spreading of T
cell autoreactivity has also been observed in patients
with multiple sclerosis during disease progression, as
well as in the experimental autoimmune encephalomyelitis (EAE) murine model.
The present study shows that in MRL/lpr mice,
the T cell repertoire evolves rapidly in a few weeks,
between weeks 7–8 and 12–13. The behavior of peripheral T cells then changes, and at week 15, when the
lupus disease is established in most MRL/lpr mice, T
cells become ignorant ex vivo in the presence of peptides
of the U1-70K protein. We have observed that certain
peptides are recognized very early by peripheral MRL/
lpr T cells, and then with the emergence of new reactivity, a regression of some, but not all, of the primary
reactivity occurs. Thus, as shown previously in EAE (29),
there is a cascade of primary and secondary determinants as well as more stable determinants that remain
during the progression toward lupus. Regression of T
cell autoreactivity with age, visualized ex vivo, may result
from different mechanisms, such as T cell exhaustion
due to a chronic stimulation in vivo, a modified balance
of the peripheral apoptotic cells associated with disease
onset, and in the MRL/lpr mouse model, the well-known
accumulation of CD4⫺/CD8⫺ double-negative T cells.
The maturation of MRL/lpr T cell reactivity
toward determinants of the U1-70K protein occurs
concomitantly with the autoantibody reactivity described
previously (25). It is notable that sequences 96–116 and
131–151 contain both a B cell epitope and an epitope
recognized by peripheral T cells. However, the peptide
21–41, which was recognized very early by IgG antibodies from 8-week-old mice and by antibodies from as
many as 9 of twenty 22-week-old mice tested serially
(25), was unable to activate peripheral MRL/lpr T cells
ex vivo. Conversely, with the exception of peptides
96–116 and 131–151, several peptides able to recall
peripheral MRL/lpr T cells ex vivo were not recognized
by antibodies. Furthermore, in contrast to the
emergence/regression type of T cell spreading observed
in the case of the 70K protein, B cell spreading arising in
8–22-week-old MRL/lpr mice in response to this antigen
was shown to progress without disappearance of existing
autoantibody subsets (25).
In the present study, we have shown that normal
mice that were immunized with peptide 131–151 in
adjuvant developed a peripheral T cell response revealed with the P140 peptide, but also with 2 peptides
that have no structural similarities with, and are located
at a distance from, the sequence 131–151. Most importantly, these 2 peptides were also strongly recognized by
peripheral T cells from MRL/lpr mice. Such a result
strongly supports our model of spreading based on the
RNA binding domain (RBD) motif called RNP1,
present in the sequence 131–151 of the U1-70K protein
as well as in other lupus common antigens such as the
U1A and hnRNPA2/B1 proteins (14,25). Recent observations made by Greidinger et al also support our
hypothesis (30). Using T cell clones derived from patients with SLE and mixed connective tissue disease,
those authors identified 5 T cell epitopes on the U1-70K
protein, and all 5 of these epitopes reside in the RBD
motif of the protein.
The most important finding in this report is the
effect of phosphorylated peptide analog P140 on the
diversification of the T cell response. We clearly demonstrated that administration of peptide P140 (in saline)
at least transiently abolishes the intramolecular spreading to other regions of the 70K protein. Although there
is no available information suggesting any putative role
of CD4⫹ T cell clones to RNP proteins in the pathogenic events shown in lupus, this result supports the view
that a single peptide can effectively modulate T cell
reactivity to other regions of the protein and potentially
to other RNP1⫹ and RNP1⫺ proteins of the same
particle (14), and thus can regulate the possible functions of autoreactive T cell clones on the B cell response
(26,31). This result, shown experimentally for the first
time, is crucial, since it demonstrates that epitope
spreading is not a severe limitation to the treatment of
autoimmune individuals with a single peptide, or with a
small cocktail of well-selected peptides. Therefore, at
this stage, the challenge is to define the nature of
pathogenic driving clones (not necessarily the initiating
clones [32]) and to pursue the identification and improvement of potent molecules able to immunoregulate
the functions of these driver clones.
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