Institute of Biochemistry, BIL Biomedical Research Centre, University of Lausanne, 1066 Epalinges, Switzerland
Correspondence to: P. Romagnoli, U395 INSERM, BP 3028, 31024 Toulouse Cedex 3, France
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Abstract |
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Keywords: glycosylphosphatidylinositol, paroxysmal nocturnal hemoglobinuria, TCR signaling
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Introduction |
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The function and membrane localization of p56lck and p59fyn are regulated by dual acylation (4). p56lck mutants that lack either myristate or palmitate moieties do not localize to the plasma membrane and do not function properly (5). Post-translational addition of lipids has been shown to target p56lck and p59fyn to a glycolipid-enriched membrane (GEM) compartment or detergent-insoluble glycolipid-enriched domain (6) or detergent-resistant membrane domain (7). This membrane compartment contains glycolipids, sphingolipids, cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins and signal transducing molecules, such as trimeric G protein, Ras and phosphoinositides (4). Interestingly, LAT, a recently identified critical substrate of PTK activated upon TCR engagement also localizes to this compartment (8). The specific enrichment in signaling components suggests that the GEM fraction represents a specialized signaling domain on the cell membrane. In agreement with this hypothesis, it has been reported that antibody cross-linking of GPI-anchored molecules leads to T cell proliferation and cytokine production. Interestingly, signaling through GPI-anchored molecules requires surface expression of a functional TCR chain (9,10) and expression of p59fyn kinase (11,12). Furthermore, in addition to p59fyn, also p56lck is also found in GPI-anchored molecule immunoprecipitates of T cells (13,14).
Collectively these results show that GPI-anchored molecules and the TCR complex share proximal components of the T cell signaling machinery (p56lck, p59fyn and TCR), and when engaged are able to elicit similar responses. It is thus of interest to study the relationship and cross-talk between these two signaling complexes.
Functional studies using T cells deficient in GPI biosynthesis suggest that GPI-anchored molecules may play a role in TCR-mediated activation. In patients affected with paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic disorder characterized by the presence of GPI-anchor deficient (GPI-deficient) hematopoietic cells, GPI-deficient peripheral T cells display a more naive phenotype as compared to wild-type controls (15). Furthermore, proliferative responses to allogeneic antigen-presenting cells (APC) are defective in mutant T cells (16). Similarly, ovalbumin-specific T cell hybridomas, lacking surface expression of several GPI-anchored molecules, were shown to have a decreased capacity to respond to antigen, concanavalin A and anti-CD3 ligation (17). In this regard we have recently shown that TCR engagement in five murine mutant cell lines with deficiencies in GPI biosynthesis fails to induce tyrosine phosphorylation of the TCR
chain and ZAP-70 (18). These data suggest that the connection between signaling through the TCR and GPI-anchored molecules may reside in the modulation of Src kinases, p56lck and p59fyn, known to be involved in tyrosine phosphorylation of the TCR
chain and ZAP-70.
To further investigate the role of GPI-anchored molecules in TCR-mediated T cell activation, TCR signaling events were analyzed in human T cells derived from two patients affected with PNH. PNH is caused by somatic mutations in the X-linked gene encoding a protein termed phosphatidylinositol glycan class A (PIG-A) necessary for the synthesis of the very early intermediates of GPI-anchor (19). The fact that only part of bone marrow-derived cells is affected allows for the isolation of wild-type and mutant (GPI-deficient) T cells from the same individual. Here we report that TCR engagement fails to optimally activate p56lck in GPI-deficient T cells, ultimately resulting in a defective calcium flux and proliferative response. Interestingly, decreased activation of p56lck in mutant T cells induces not only a quantitatively but also a qualitatively different pattern of tyrosine phosphorylated substrates. These findings could explain the high proportion of naive GPI-deficient T cells found in the peripheral blood of PNH patients and the depressed proliferative responses to allogeneic APC.
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Methods |
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Antibodies and antisera
The following antibodies were used for immunoprecipitations, immunoblotting, FACS analysis and confocal microscopy: anti-CD3 mAb OKT3 (ATCC, Rockville, MD); anti-TCR
mAb H146-968 (20) and 6B10.2 (Santa Cruz Biotechnology, Santa Cruz, NM); anti-DAF mAb Bric 216 (International Blood Group Reference Laboratory, Bristol, UK); phycoerythrin (PE)-conjugated anti-CD4 mAb, PE-conjugated anti-CD8 mAb, FITC-conjugated anti-CD45 RO mAb and FITC-conjugated anti-CD3
mAb (Becton Dickinson, San Jose, CA); anti-CD4 mAb 101.69 (21), kindly provided by Dr D. Rimoldi, Ludwig Institute for Cancer Research, Lausanne, Switzerland; anti-CD45 mAb, anti-phospholipase C-
1 antiserum (Santa Cruz Biotechnology); anti-p56lck antisera p56-1 and 2166 (5) (kindly provided by Dr S. C. Ley, Division of Cellular Immunology, National Institute for Medical Research, London, UK); anti-p59fyn antiserum (kindly provided by M. F. White, Harvard Medical School, Boston, MA); anti-PY mAb 4G10 (unlabeled and biotin-conjugated; Upstate Biotechnology, Lake placid, NY). Horseradish peroxidase-conjugated goat anti-mouse, goat anti-rat and goat anti-rabbit antisera were purchased from Sigma (Buchs, Switzerland). StreptavidinFITC, FITC-conjugated and Texas Red-conjugated donkey F(ab')2 fragment anti-mouse IgG and FITC-conjugated hamster F(ab')2 fragment anti-rabbit IgG were purchased from Jackson ImmunoResearch (West Groove, PA).
Proliferation assay
Round-bottom microplates were coated with different concentrations of anti-CD3 mAb (OKT3) and 5x103 T cells/well were added. Cells were stimulated for 2 days at 37°C and then pulsed with 1 µCi of [3H]thymidine. Incorporation of radiolabeled nucleotide was determined 16 h later. PHA responses were assessed by culturing 5x103 cells/well T cells with 5x103 irradiated allogeneic PBMC in IL-2 containing medium in flat-bottomed microplates. After 2 days cells were pulsed with 1 µCi of [3H]thymidine and harvested 16 h later. Alloreactivity was assessed by culturing 2x104 alloreactive T cells with increasing concentration of an irradiated EpsteinBarr virus-transformed B cell line derived from PBMC of the healthy donor in flat-bottomed microplates for 48 h; 1 µCi of [3H]thymidine was added during the last 16 h of incubation.
Surface staining
Cells were washed twice in PBS containing 2.5% FCS and 0.02% NaN3, and incubated on ice for 20 min with antibody at saturating concentration. Hybridoma culture supernatants or directly labeled antibodies were used. Where applicable, after two washes with PBS containing 2.5% FCS and 0.02% NaN3, cells were incubated with the indicated FITC-conjugated second antibodies for 20 min on ice (see figure legends). The stained cells were analyzed with a FACScan using Lysys II software (Becton Dickinson). Dead cells were excluded from analysis by appropriate gating on forward and side scatter.
Stimulation, precipitation and immunoblotting
Cells were washed twice in PBS and resuspended at a concentration of 2x107/ml. They were incubated for 20 min at 37°C and subsequently stimulated for the indicated amount of time by addition of 10 µg/ml (final concentration) of anti-CD3 mAb OKT3. After stimulation cells were rapidly washed with ice cold PBS containing 1 mM Na3VO4, and lysed in lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM Na3VO4, 5 µg/ml leupeptin, 10 µg/ml aprotinin and 2 mM PMSF) containing 0.51% Triton X-100 (as indicated in the figure legends). Lysates were immunoprecipitated using mAb pre-bound to Protein ASepharose beads or rabbit anti-mouse IgG-coated agarose beads. Eluted samples were resolved by SDSPAGE in reducing conditions, transferred to PVDF membranes and immunoblotted with the indicated antibody (see figure legends).
Immunofluorescence microscopy
Cells were washed twice in PBS and resuspended in PBS containing 2.5% FCS at a concentration of 5x106 cells/ml. Cells were subsequently incubated for 15 min on ice with either anti-CD3 (OKT3) or anti-CD4 mAb. After a quick wash, cells were incubated for 15 min on ice with Texas Red-conjugated donkey F(ab')2 fragment anti-mouse IgG. Cells were washed and either left on ice or incubated for indicated amount of time at 37°C. After incubation cells were rapidly washed with cold PBS containing 2% BSA. They were then fixed in 3% paraformaldehyde for 30 min at room temperature, and permeabilized for 4 min with PBS containing 0.1% Triton, 2.5% FCS and 10 mM Na3VO4. Non-specific staining was blocked by incubation in PBS containing 5% FCS and 10 mM Na3VO4. Cells were subsequently incubated with anti-p56lck rabbit antiserum p561 (1:400 diluted) or with biotinylated anti-phosphotyrosine mAb 4G10 (2 µg/ml) at room temperature for 45 min. After three washes with PBS, cells were incubated with either FITC-conjugated hamster F(ab')2 fragment anti-rabbit IgG or with streptavidinFITC for 45 min at room temperature, washed and mounted on slides with Fluor Save reagent (Calbiochem Novabiochem, La Jolla, CA). Cells were analyzed using a Zeiss Confocal Laser Scanning System attachment LSM (Zeiss, New York, NY) with the Axiovert 100 photomicroscope system.
In vitro kinase assays
Precipitated immune complexes were washed 3 times in lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM Na3VO4, 5 µg/ml leupeptin, 10 µg/ml aprotinin and 2 mM PMSF) containing 1% Brij 96 and once in lysis buffer without detergent. Complexes were resuspended in 15 µl of kinase buffer (25 mM HEPES, pH 7, 10 mM MnCl2, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM Na3VO4 and 10 µCi [-32P]ATP), and kinase reactions performed for 15 min at room temperature and subsequently terminated by the addition of sample buffer. Proteins were resolved by 10% SDSPAGE in reducing conditions. Gels were treated with 1 M KOH for 1 h at 58° C to remove [32P]Ser and [32P]Thr, washed, and [32P]Tyr content assayed by autoradiography.
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Results |
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Decreased proliferation in response to TCR engagement in GPI-deficient T cell lines and TLC
Dose-dependent responses to anti-CD3 mAb (OKT3) and to PHA were analyzed in wild-type and GPI-deficient T cell lines obtained from two PNH patients. A strongly reduced proliferative response to TCR ligation was observed in GPI-deficient T cell lines as compared to wild-type controls, while PHA responses were only marginally affected (Fig. 1a
).
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CD4+ TLC were isolated from PHA T cell lines of patient no. 1. Proliferative responses to anti-CD3 mAb of GPI-deficient CD4+ TLC were strongly impaired as compared to wild-type controls, while PHA responses where only marginally decreased (Fig. 1c
).
Collectively these experiments show that proliferative responses to TCR ligation are affected in GPI-deficient T cells. These results confirm and extend the previously reported weaker responses of GPI-deficient T cells to allogeneic B cells blasts (16). However, they are not in agreement with the reported similar proliferation to stimulation via CD3 in wild-type and GPI-deficient T cells (15). The discrepancy between our and the previously published results can be explained by the different experimental set-up. In previous experiments T cells were stimulated with a fixed concentration of antibody (15). In our study a dose-dependent response was analyzed, allowing us to identify the proliferative defect, that is most striking at low antibody concentrations. In contrast to anti-CD3 mAb-mediated proliferation, PHA-induced proliferation was not affected in GPI-deficient TLC, suggesting quantitative and/or qualitative (22) differences in the signaling pathways following anti-CD3
mAb or PHA stimulation.
We then proceeded to characterize the molecular basis of defective TCR signal transduction in GPI-deficient T cells, mainly using wild-type and GPI-deficient CD4+ TLC.
Wild-type and GPI-deficient TLC derived from a PNH patient express similar levels of proteins involved in TCR-proximal signaling events
First, we assessed the level of surface expression of the TCR complex on wild-type and GPI-deficient TLC. CD3 was found to be expressed at comparable levels in wild-type and GPI-deficient TLC (Fig. 2a
). TCR
chain is known to be involved in TCR signaling and its expression has been shown to be regulated independently from that of the rest of the TCR complex (20,23,24). Western blot analysis revealed that wild-type and GPI-deficient TLC expressed comparable amounts of TCR
(Fig. 2b
).
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Altered TCR-mediated signaling could result from differences in the expression of CD45, a phosphatase known to activate p56lck (29). However, we have observed no difference in the cell surface expression of the CD45 isoform expressed by antigen-experienced T lymphocytes (CD45RO) between wild-type and mutant TLC (Fig. 2a). Similar results were obtained analyzing GPI-deficient and wild-type T cell lines (data not shown).
In addition, Western blot analysis of the expression level of the Src kinases p56lck and p59fyn (both involved in the earliest intracellular signaling events following TCR-mediated activation), and of phospholipase C-1 (initiating the essential phosphoinositide pathway) did not reveal any difference between wild-type and GPI-mutant cells (Fig. 2b
).
Collectively these data indicate that impaired TCR-mediated responses of GPI-deficient cells are not caused by a reduced expression level of proteins implicated in early TCR signaling events.
Impaired calcium mobilization upon TCR ligation in GPI-deficient T cell lines and TLC
To examine TCR-mediated signaling events, changes in [Ca2+]i were analyzed in wild-type and GPI-deficient T cell lines (periodically re-stimulated with PHA) and clones by flow cytometry. After stimulation with anti-CD3 mAb, a rapid and sustained increase in [Ca2+]i was found in wild-type T cell lines and TLC, while a delayed and quantitatively decreased mobilization of [Ca2+]i was observed in wild-type GPI-deficient T cell lines and TLC (Fig. 3
).
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Quantitative and qualitative differences in the induction of phosphorylated substrates in wild-type and GPI-deficient TLC
To examine TCR proximal events in the signal transduction pathway, the induction of tyrosine phosphorylation was analyzed in total lysates of wild-type and mutant TLC by Western blot. Upon stimulation, an increase in tyrosine phosphorylation of low mol. wt proteins of 20, 28 and 3638 kDa was observed in wild-type TLC, which was not appreciable in GPI-deficient TLC (Fig. 4). These data reveal that the decreased responsiveness of GPI-deficient T cells upon anti-CD3
stimulation correlates with the reduction in CD3
-induced tyrosine phosphorylation.
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In conclusion, both quantitative and qualitative differences in the induction of phosphorylated substrates were observed between wild-type and GPI-deficient TLC.
Decreased tyrosine phosphorylation of the TCR chain upon TCR engagement in GPI-deficient TLC
One of the earliest events of TCR-mediated signaling is tyrosine phosphorylation of the TCR chain. To examine whether this early activation event was affected in human GPI-deficient TLC, lysates from unstimulated and anti-CD3
antibody stimulated T cells were immunoprecipitated with a mAb specific for the TCR
chain. Precipitates were resolved by SDSPAGE and immunoblotted with anti-phosphotyrosine or anti-TCR
mAb. In wild-type TLC, after 2 min of TCR-mediated stimulation an increase in TCR
chain phosphorylation was observed (Fig. 5a
). The GPI-deficient TLC showed a significant reduction in the level of TCR
chain phosphorylation as compared to wild-type cells. Normalization of the level of TCR
chain phosphorylation to the total amount of precipitated TCR
from wild-type and mutant cells (as detected by anti-TCR
chain mAb) reveals a 2- to 5-fold reduction in phospho-
. A quantitatively similar reduction in the induction of phospho-
was already detectable at earlier time points after TCR stimulation indicating that phosphorylation is quantitatively decreased and not simply delayed in mutant TLC (Fig. 5b
).
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Localization of p56lck in wild-type and GPI-deficient TLC
None of the components of the TCR contains intrinsic kinase activity. Rather, kinases are recruited to the TCR complex early upon activation. To test whether kinase recruitment to the TCR is affected by the PNH mutation, TCR capping was induced by anti-CD3 antibody cross-linking and p56lck redistribution was analyzed by confocal microscopy. Intracellular distribution of p56lck in unstimulated wild-type and mutant TLC was found to be similar, with a predominant membrane localization, as previously described for normal human T cells (30) (Fig. 6
). This result was confirmed by immunoblot analysis of cytosolic and membrane fractions isolated from wild-type and mutant TLC (data not shown).
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To investigate whether the delayed kinetics of capping and internalization of the TCR reflected a general defect in surface receptor mobilization of GPI-deficient TLC, the co-receptor CD4, was induced to cap using anti-CD4 mAb. CD4 caps formed with indistinguishable kinetics in GPI-deficient and wild-type TLC (Fig. 7).
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Activation of p56lck in wild-type and GPI-deficient TLC
Given the predominant role of p56lck in TCR phosphorylation (31), we next investigated whether an altered activation of p56lck could explain the signaling defect observed in GPI-deficient TLC. It has previously been demonstrated that upon TCR engagement a new form of p56lck appears with an apparent mol. wt of 60 kDa (p60lck) (32). To analyze p56lck activation in wild-type and mutant TLC we performed Western blot analysis on cell lysates using anti-p56lck antibodies. Upon TCR-mediated activation, p60lck induction is reproducibly decreased in TLC 26 as compared to wild-type cells (Fig. 8a
), indicating that activation of p56lck is impaired in GPI-deficient cells. Normalization of the level of p60lck to p56lck in wild-type and mutant cells (as detected by anti-Lck mAb) reveals a 1.6-fold reduction in p60lck induction in GPI-deficient TLC. An altered activation of p56lck was also observed in TLC 24 when compared to wild-type TLC (data not shown). In vitro kinase assay of Lck precipitates did not reveal any reproducible differences between wild-type and GPI-deficient TLC (Fig. 8b
), indicating that Lck is not defective in GPI-deficient cells.
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Discussion |
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In this report, we now provide evidence that TCR-mediated activation of p56lck is defective in GPI-deficient human TLC. The reduction in p56lck activation is accompanied by the induction of a quantitatively and qualitatively altered pattern of phosphorylated substrates in mutant cells, ultimately resulting in a strongly reduced proliferative response to TCR ligation. Interestingly although the extent of TCR cap formation and internalization is not dramatically affected in GPI-deficient TLC, they occur with delayed kinetics. These observations are consistent with the previously reported importance of the rate of TCR triggering in the onset of the response (34).
The defect in TCR signaling observed in GPI-deficient TLC probably reflects the importance of GPI-anchored proteins in TCR signal transduction. However, since in our system TCR ligation is achieved with soluble anti-CD3 mAb without engagement of GPI-anchored molecules, a specific role of their ectodomain seems unlikely.
p56lck plays an important role in signaling events mediated by both TCR (31) and GPI-anchored molecules (14). While p56lck does not associate directly with the TCR, it co-precipitates with GPI-anchored molecules (14,35). Intriguingly, p56lck (5) and GPI-anchored molecules (36) localize in GEM microdomains recently implicated in TCR signaling (37,38). It has been proposed that self-association of sphingolipids and cholesterol induces the formation of microdomains that separate from the more abundant glycerophospholipids (7). Strikingly, the majority of the proteins found in these domains are anchored in the membrane by lipid moieties. These include both GPI-anchored molecules and acylated signaling molecules such as p56lck, p59fyn (39), Ras (40) and LAT (8). Extensive mutagenesis studies have shown that the presence of fatty acids is very critical in GEM targeting and signaling function of all these molecules (4,8,41).
One of the proteins excluded from these domains is CD45 (42). CD45 exclusion from GEM may selectively regulate p56lck, by constituting a reservoir of enzyme hyperphosphorylated on tyrosine that can be readily activated. The importance of membrane compartmentalization in T cell activation may thus resides in concentrating certain signaling molecules, allowing the initiation of an effective TCR-mediated signaling cascade.
It has been recently reported that several other components of the T cell signaling machinery such as Cbl, Syk, Vav, ZAP-70, phospholipase C-1 and TCR
chain translocate to GEM upon TCR engagement (37,38). Interestingly, in murine thymocytes, it has been shown that Lck, ZAP-70 and TCR
chain are present in the same GEM vesicles as GPI-anchored molecules (38). A deficiency in GPI biosynthesis may affect this membrane microdomain and consequently TCR signal transduction. The large quantity of cells required to separate such domains has hampered so far their isolation from wild-type and GPI-deficient TLC.
At least two mechanisms can be envisaged on how surface expression of GPI-anchored molecules could affect TCR-mediated p56lck activation. GEM formation might be defective in GPI-deficient TLC, leading to an alteration in p56lck. The fact that CD4 cap formation is not affected in GPI-deficient TLC as compared to wild-type cells, argues against this possibility. CD4 localizes in GEM (35,43) and is directly associated with Lck (44). Taken together these results suggest that a deficiency in GPI biosynthesis does not compromise receptor activation and mobilization in GEM, suggesting that these domains are not grossly altered.
Alternatively, the interactions between proteins that localize in the glycerophospholipid membrane domains and proteins that reside in GEM could be affected in GPI-deficient T cells. Consistent with this hypothesis we observed a decreased activation of p56lck, and a delay in TCR cap formation and p56lck redistribution in mutant cells, suggesting that upon TCR engagement events leading to p56lck activation are impaired. In this regard it is important to remember that in epithelial cells two subcompartments of GEM have been identified: caveolae, enriched in signaling molecules such as Yes, Lyn and the subunit of G proteins, and a GPI-anchored molecule rich domain located proximally to the caveolae neck (45). If a similar structural organization is conserved in caveolin-negative cells, such as T cells, GPI-anchored molecules could play an important structural or functional role in connecting TCR and p56lck.
The data presented in this paper provide evidence for a role of GPI-anchored molecules in enhancing TCR-mediated Lck activation and indicate that expression of GPI-anchored molecules is required for optimal TCR signal transduction. These findings may explain the unusually large fraction of naive T cells found among GPI-deficient T cells in PNH patients (15), as well as their decreased response to alloantigens (16).
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Acknowledgments |
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Abbreviations |
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APC | antigen-presenting cell |
DAF | decay accelerating factor |
GEM | glycolipid-enriched membrane compartment |
GPI | glycosylphosphatidylinositol |
HRP | horseradish peroxidase |
PBMC | peripheral blood mononuclear cell |
PE | phycoerythrin |
PHA | phytohemagglutinin |
PNH | paroxysmal nocturnal hemoglobinuria |
PTK | protein tyrosine kinase |
TLC | T cell clone |
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Notes |
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Received 8 February 1999, accepted 10 May 1999.
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References |
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