Correspondence to: Gillian M. Griffiths, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK. Tel:44-1865-275571 Fax:44-1865-275515 E-mail:gillian.griffiths{at}path.ox.ac.uk.
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Abstract |
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Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.
Key Words: Rab27a, cytotoxic T lymphocyte, secretory lysosomes, immunological synapse, Arp2/3
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Introduction |
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Cytotoxic T lymphocytes (CTLs)1 destroy their target cells by regulated secretion from specialized secretory lysosomes known as lytic granules. Several other cell types possess secretory lysosomes, most of which are derived from the hemopoietic lineage. Several lines of evidence support the idea that the mechanisms regulating exocytosis of these lysosomal secretory compartments differ from those controlling conventional secretory granules (for review see
The regulated secretion of the lytic granules from CTLs is triggered by T cell receptor (TCR) recognition of a target cell. Recognition results in kinesin-driven lytic granule movement along microtubules to the point of membrane contact and release of perforin and granzymes which trigger rapid destruction of the target (for review see
Expression of the Ras-like GTPase Rab27a is particularly high in spleen, platelets, and melanocytes, consistent with a potential role in secretory lysosome secretion ( subunit of Rab geranylgeranyl transferase (RGGT) (
Rab27a is therefore a strong candidate as a regulator of lysosome secretion. To address this possibility, we have examined CTL secretion and the polarization of lytic granules at the immunological synapse in these two natural mouse mutants in which Rab27a activity is affected. Our findings demonstrate an important role for Rab27a in the final stages of secretion and indicate that other Rab proteins may be involved in granule polarization.
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Materials and Methods |
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Mice
Gunmetal mice (C57BL/6-gm/gm)were kindly supplied by Richard Swank (Roswell Park Cancer Institute, Buffalo, NY) (
Antibodies and Cells
The rabbit antiserum against cathepsin D was the generous gift of S. Kornfeld (Washington University, St. Louis, MO), the rat monoclonal recognizing granzyme A (7.1) was a gift from M. Simon (Max-Planck-Institute, Freiburg, Germany) (
Cell Culture
CTLs were derived by activation of 2.5 x 106 splenocytes from effector strains (C57Bl/6, +/ash, ash/ash, +/gm, and gm/gm) with 2.5 x 106 irradiated (3,000 rads) stimulator splenocytes from BALB/C mice in 10 ml Iscove's modified Dulbecco's medium (IMDM) containing 10% FCS and 2-mercaptoethanol. After 5 d in culture at 37°C with 5.5% CO2, CTLs were purified over Ficoll 1096 (Sigma-Aldrich), washed three times, and either assayed for lysis as described below or resuspended in culture medium supplemented with 100 U/ml interleukin 2. Cells were restimulated every 710 d for continued passage in culture for up to 7 wk. P815 mouse target cells were maintained in RPMI/10% FCS.
Immunoblotting
Frozen cell pellets were thawed, resuspended in lysis buffer (50 mM Hepes, pH 7.2, 10 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 5 µg/ml pepstatin, 5 µg/ml aprotinin and 5 µg/ml leupeptin) (Sigma-Aldrich), and mechanically disrupted by serial passages through a 21-gauge needle. The protein concentration of this homogenate was determined using Coomassie Plus Protein Assay Reagent (Pierce Chemical Co.). Equal amounts of total protein (13 µg) were subjected to SDS-PAGE on 12.5% acrylamide gels and transferred to PVDF membranes using a Hoeffer transfer apparatus (2 h at 500 mA). The membranes were allowed to dry and then blocked with 5% skimmed milk in PBS with 0.2% Tween 20 (PBST) for 1 h at room temperature. The membranes were then incubated with monoclonal antirat Rab27a antibody, 4B12 (
In Vitro Prenylation Reactions and Immunoprecipitation after Prenylation
In vitro prenylation assays were performed essentially as described previously (2 wk. The radioactive pattern, representing Rabs to which [3H]GGPP had been transferred in vitro, was visualized using a Cyclone PhosphorImaging system. The same membrane was then probed with anti-Rab27a 4B12 antibody as described above. For immunoprecipitation, gm/gm CTL cytosolic Rabs were prenylated in vitro as above in a 75-µl reaction and then incubated overnight at 4°C with protein Asepharose beads bound to either polyclonal anti-Rab27 antibody (N688) (
Cytotoxicity Assays
Cytotoxicity was assayed using a Cytotox 96 nonradioactive kit (Promega) following the instructions provided. In brief, Ficoll-purified T cells were plated in 96-well plates at the effector/target ratios shown using 104 P815 (H2d) target cells per well in a final volume of 100 µl per well using RPMI lacking phenol red. Lactate dehydrogenase release was assayed after 4 h incubation at 37°C by removal of 50 µl supernatant from each well and incubation with substrate provided for 30 min and the absorbance read at 490 nm using the Thermomax plate reader (Molecular Devices). Percentage cytotoxicity = (experimental effectorspontaneous - target spontaneous/targetmaximum - target spontaneous) x 100. All cytotoxicity assays were reproducible in at least three separate assays.
Secretion Assay
Round-bottomed 96-well plates (Falcon) were coated with the same batch of hamster antimouse CD3 antibody, 145.2C11 (+CD3), or left uncoated (-CD3) overnight at 4°C. Wells were then blocked with IMDM with 10% FCS for 1 h at room temperature. Cells were plated at 5 x 104/well in 50 µl serum-free IMDM. For measurement of total activity 20 µl of medium was replaced with 20 µl 0.1% Triton X-100, and the final sample was resuspended completely before 20 µl was assayed for enzyme activity. After 1 h, plates were spun briefly at 1,000 rpm to ensure that all cells were pelleted and 20-µl aliquots were removed to test for hexosaminidase and granzyme A activity. Hexosaminidase activity was assayed by adding 20 µl test supernatant to 20 µl substrate buffer (34 mg p-nitrophenyl N-acetyl-ß-D-glucosaminide (Sigma-Aldrich) in 20 ml 50 mM sodium citrate, pH 4.8, 0.2% Triton X-100). Samples were incubated at 37°C for 1 h before 200 µl stop buffer (33 mM glycine, 83 mM sodium carbonate, 67 mM sodium chloride, pH 10.7) was added. Absorbance at 415 nm was read. Granzyme A activity was assayed by incubation of 20-µl samples with 180 µl of substrate (PBS, 0.2 mM N-benzyloxy-carbonyl-L-lysine-thiobenzylester, 0.2 mM dithiobis-nitrobenzoic acid) for 30 min, and the absorbance was read at 415 nm. For both enzymes the percentage of total secretion was calculated as 100 x (ODsupernatant/ODlysate).
Immunofluorescence and Electron Microscopy
CTLs taken at 58 d after restimulation were purified over Ficoll and either used directly or incubated overnight with 2 mg/ml HRP added to the growth medium. Cells were washed one to three times in RPMI (GIBCO BRL) lacking FCS (RPMI-), resuspended at 5 x 106 cells/ml in RPMI-, and mixed 1:1 with either RPMI- alone or RPMI- containing 107 cells/ml P815 cells prewashed as above. Samples were left in suspension for 5 min then plated onto either uncoated glass multiwell slides (50 µl/well) or into 12-well tissue culture plates (Nunc) (1 ml/well) and incubated at 37°C for a further 3055 min.
Cells on slides were fixed with either methanol precooled to 20°C or 2% paraformaldehyde (Electron Microscopy Sciences) in PBS and processed for immunofluorescence as described previously (
Cells in 12-well tissue culture plates were fixed by adding 1 ml of 2x fixative (3% glutaraldehyde [Agar Scientific Ltd.], 4% paraformaldehyde) directly to the medium. After 1020 min, fixative was replaced with fresh 1x fixative (1.5% gluteraldehyde, 2% paraformaldehyde) for a further 1020 min. Samples were then processed for HRP cytochemistry, postfixed with reduced osmium, and embedded in Epon as described previously (
Online Supplemental Material
Online supplemental materials can be found at http://www.jcb.org/cgi/content/full/152/4/825/DC1. Supplementary material is provided to show granule polarization of CTL from +/gm and gm/gm mice conjugated to P815 target cells in three dimensions. All images are stained with rabbit anticathepsin D (green) and antitalin (red) antibodies as described above and shown in Fig 6, a and d. Optical sections (0.2 µm apart) were taken using the BioRad 1024 confocal microscope, and assembled using BioRad Lasersharp software.
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Results |
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Rab27a Is Absent in Ashen and At Least Partially Inactivated in Gunmetal CTLs
To determine whether Rab27a is expressed in CTLs and whether there is a difference in Rab27a expression in CTLs derived from ashen and gunmetal mice, we used a monoclonal antibody specific for the Rab27a isoform (4B12) to probe CTL lysates (Fig 1 A). The CTL lysates derived from +/ash, +/gm, and gm/gm mice show a band migrating just above 28 kD corresponding to endogenous Rab27a. However, ash/ash CTLs exhibit no detectable endogenous Rab27a. The same blot was probed with an antibody against calnexin (ER membraneassociated protein) to demonstrate that equivalent levels of protein had been loaded. These results demonstrate that Rab27a is normally expressed in CTLs, but that it is absent in ashen CTLs. The expression of Rab27b could not be detected in CTLs (data not shown).
As predicted, Rab27a is expressed at normal levels in gunmetal CTLs (Fig 1 A). However, the deficiency of RGGT activity in these mice may result in underprenylation of Rab proteins in CTLs. To test this prediction, we employed an in vitro prenylation assay whereby recombinant RGGT will transfer [3H]GGPP to any unprenylated Rab proteins present in cytosolic extracts. As shown in Fig 1 B, gm/gm CTL cytosol shows an excess of Rabs available for prenylation compared with heterozygote +ash/ash and wild-type CTLs. An immunoblot performed on the same membrane using the Rab27a-specific antibody 4B12 (Fig 1 C) reveals a band coinciding with the largest band of in vitro prenylated Rabs, suggesting that Rab27a is one of the few Rabs affected in gm/gm CTLs. No significant amount of Rab27a is detectable in either +/gm or +/+ CTL cytosol, indicating that the majority of Rab27a is properly prenylated and membrane associated in these strains. To further demonstrate that one of the bands observed after in vitro prenylation of gm/gm CTLs is Rab27a, we subjected the in vitro prenylation reaction to immunoprecipitation with a specific anti-Rab27 antibody. Fig 1 D shows that this antibody was able to precipitate a significant portion of the largest in vitro prenylated band. These data indicate that Rab27a is among several Rabs that appear underprenylated in gunmetal CTLs, thus remaining cytosolic and inactive.
CTL s from Ashen Mice Are Unable to Kill and Unable to Secrete Their Granule Contents
We next asked whether Rab27a is involved in CTL-mediated lysis by deriving CTLs from ash/ash and +/ash mice by stimulation with BALB/C spleens. This allowed us to assess the lytic ability of the T cells against the P815 target cell line, which shares the same class I major mistocompatibility complex as the BALB/C strain (H2d). 5 d after activation, CTLs derived from +/ash killed P815 targets effectively. However, ash/ash CTLs were completely unable to kill targets (Fig 2 A). These results were entirely reproducible in three separate experiments. The inability to kill arises from a lack of secretion of the lytic granules as shown by measuring the levels of the lysosomal hydrolase, hexosaminidase, and the CTL-protease, granzyme A, release in response to TCR cross-linking (Fig 2 B). Using an antibody to the CD3 chain of the TCR immobilized on plates, we were able to demonstrate that granzyme A and hexosaminidase are secreted effectively in response to stimulation in heterozygote mice (+/ash). However, in the ashen homozygous mice (ash/ash) no secretion above background of unstimulated cells was detectable even though the total intracellular enzyme activity in heterozygote and homozygote CTLs was comparable (data not shown). This demonstrates that the lytic granules of ash/ash mice are not able to secrete their contents in response to TCR triggering.
CTLs from Gunmetal Mice Show Reduced Levels of Killing but Granule Secretion Is Not Dramatically Impaired
As shown in Fig 1, gunmetal CTLs contain underprenylated Rab27a, among other Rabs. It was therefore possible that CTL-mediated killing might also be affected in gunmetal mice. Cytotoxicity was measured against P815 target cells and gm/gm CTLs possessed only weak killing activity compared with the parental strain, C57BL/6 (Fig 3 A). These results suggest that defects in the RGGT perturb killing in gunmetal mice.
The ability of CTLs generated from C57BL/6, +/gm, and gm/gm to secrete their granule contents in response to TCR cross-linking was compared by examining the release of granzyme A and hexosaminidase (Fig 3 B). The parental strain, C57BL/6, secrete both granzyme A and hexosaminidase in response to TCR cross-linking. Gunmetal heterozygous (+/gm) CTLs secrete at least as well as the parental strain (if not slightly better). Interestingly, the secretion from the homozygous mutants (gm/gm) is not significantly impaired, with secreted levels of granyzme A and hexosaminidase comparable to those of wild-type C57BL/6 CTLs. The levels of hexosaminidase secretion for +/gm and gm/gm CTLs are comparable at 15, 30, and 60 min, being 0% for gm/gm and 3% for +/gm at 15 min and 10% for both at 30 min. This indicates that there are no significant differences in the rates of secretion. Since TCR cross-linking triggers not only degranulation but also new protein synthesis (
The Lytic Granules from Ashen CTLs Polarize Correctly towards Target Cells
To determine at which step of secretion Rab27a functions, we examined CTLtarget cell conjugates by immunofluorescence. We used antibodies against cathepsin D or granzyme A to detect the lytic granules and talin and the Arp2/3 complex to assess the formation of the immunological synapse.
In conjugates between +/ash and ash/ash CTLs and their targets (P815), we find that talin forms a ring at the site of target cell contact with a hole in the center. We find that Arp2/3 is focussed at the center of the talin ring and that in +/ash, the lytic granules are also focussed tightly within this ring (Fig 4, ac) very close to the contact site membrane. In CTLs derived from ash/ash mice, talin also forms a ring with Arp2/3 focussed in the center (Fig 4 f). All of the lytic granules are found clustered towards the point of contact with the target, demonstrating that the lytic granules can move along microtubules to the point of membrane contact and polarization is not impaired (Fig 4d and Fig e). Only two differences between +/ash and ash/ash conjugates are evident at this level of resolution. First, the granules always stain much more strongly at the synapse of ash/ash, consistent with the loss of content from +/ash but not ash/ash granules; second, that the ash/ash granules are not as tightly focussed within the central talin hole (for example, Fig 4d and Fig e). These results show that granule polarization is normal in ash/ash mice lacking Rab27a and demonstrate that Rab27a is not required for microtubule-mediated movement to the point of contact. The observation that ash/ash granules are not tightly clustered in the central talin hole at the immunological synapse suggests that movement from the ends of the microtubules to the plasma membrane may be impaired in ashen mutants.
To address this possibility, we examined the localization of granules in +/ash and ash/ash CTLs conjugated to targets by EM. Fig 5 shows the immunological synapse in +/ash (Fig 5, ac) and ash/ash (Fig 5, df) conjugates. The Golgi complex is seen polarized very close to the site of membrane contact (Fig 5b, Fig c, Fig e, and Fig f). Intriguingly, a central cleft is formed between two areas of tight membrane contact in both ash/ash and +/ash conjugates. Two important differences are observed between wild-type and mutant conjugates. First, in +/ash conjugates, granules are found docked at the plasma membrane (Fig 5 a), whereas in ash/ash CTL conjugates, the granules do not dock at the membrane but rather line up behind the Golgi complex (Fig 5 d). Second, the cleft at the contact site is filled with electron-dense and vesicular material in the +/ash conjugates consistent with the secretion of granule contents into this space. However, the cleft is devoid of content in the ash/ash conjugates (Fig 5e and Fig f), indicating that the granule contents are not secreted. Curiously, endosomes from the target cell also appear to be recruited to the target side of the contact site in both strains (Fig 5, a and d).
Granule Polarization Is Impaired in gm/gm CTLs
Our data shows that although killing by gm/gm CTLs is reduced, secretion is not (Fig 3). We therefore examined granule polarization in CTLtarget cell conjugates in order to determine whether synapse formation or polarization is impaired. In both +/gm and gm/gm, talin and Arp2/3 concentrate at the synapse between the cells, with talin forming an outer ring and Arp2/3 focussing in the center (Fig 6c and Fig f). In +/gm CTLtarget conjugates, the granules are tightly clustered at the center of the talin ring. However, in gm/gm conjugates the granules are incompletely polarized and many granules are left scattered around the perimeter of the cell (Fig 6d and Fig e). This differs markedly from unpolarized cells in which the granules are scattered throughout the cell rather than just at the periphery. Only 12% of conjugates showed a tight polarization of the majority of the granules at the talin synapse in gm/gm compared with 86% in +/gm and >90% in ash/ash and +/ash. These results demonstrate that granule polarization is partially impaired in gm/gm CTLs. Since CTLs from ash/ash mice are able to polarize their granules completely, we conclude that Rab27a is not involved. As the defect in polarization observed here must be due to reduced prenylation, it may well be the result of the reduced activity of the other two Rab proteins which are underprenylated in the gm/gm CTLs (Fig 1).
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Discussion |
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This study describes several novel findings concerning the mechanisms that regulate the secretion of the lytic granules from CTLs. First, we demonstrate a critical role for Rab27a in the exocytosis of the lytic granules during killing. Second, we show that Rab27a is not involved in granule polarization to the immunological synapse but other Rab proteins are required for polarization. Third, we characterize the fusion of the lytic granules at the immunological synapse and demonstrate that the granules focus at the center of the talin ring where the actin polymerizing protein, Arp2/3, also polarizes.
The expression of Rab27a has been shown to be high in spleen, platelets, and melanocytes, suggesting a specialized role for this Rab GTPase. The recent report showing mutations in Rab27a in a subset of Griscelli syndrome patients in which CTL activity is impaired supports a role for Rab27a in CTL activity (
We also show here that there is no major defect in polarization of lytic granules in the CTLs of ashen mice (Fig 4). All of the lytic granules in ashen CTLs appear in the vicinity of the immunological synapse, although we were unable to find granules focussed in the center of the talin ring as observed in heterozygous ashen CTLs. This raises the intriguing possibility that the defect in secretion could arise from an inability to move into a docking position at the plasma membrane before secretion. Our EM studies of the conjugates support this idea. Fig 5 shows that in +/ash conjugates the granules can be found polarized at the membrane, but in ash/ash mutants, the granules line up near the membrane but do not dock. This is reminiscent of the Rab3 mutants in Caenorhabditis elegans in which the synaptic vesicles are less tightly clustered around the presynaptic density (
Recent reports suggested that Rab27a might be cooperating with myosinVa in the movement of vesicles, namely melanosomes in melanocytes. Mutations in either Rab27a or myosinVa genes give rise to similar phenotypes in mice (ashen and dilute) and in human Griscelli disease (
CTLs derived from gunmetal show reduced killing but curiously are not significantly defective in the ability to secrete their lytic granule contents (Fig 3). Different possibilities could account for this. First, since several different Rab GTPases are affected by the loss of RGGT activity in gunmetal CTLs, it is possible that Rabs other than Rab27a are involved in correct sorting of lytic proteins to the granules. Alternatively, the C57BL/6 strain from which gunmetal are derived may simply be much better at responding to TCR cross-linking than the C3H strain from which ashen mice are derived. Our previous studies have shown that TCR cross-linking not only results in degranulation but also in rapid de novo synthesis of granule proteins, a large proportion of which are then secreted via the constitutive secretory pathway. This hypothesis is supported by the increased maximal values of secretion observed in C57BL/6 (32%) (Fig 3 b) compared with C3H/He (17%) mice (Fig 2 b). It is possible that a slight decrease in regulated secretion might be masked. Three-dimensional reconstruction of conjugates shows tight polarization of granules at the synapse in +/gm CTLs (see http://www.jcb.org/cgi/content/full/152/4/825/DC1). In some +/gm CTL conjugates, the granules are absent, consistent with secretion from the synapse. However, in gm/gm CTLs two types of conjugates are seen: (a) those with some granules polarized and others dispersed around the periphery; and (b) those with no granules at the synapse but some left around the periphery. These observations suggest that the unpolarized granules in gm/gm CTL conjugates are unable to fuse with the plasma membrane and secrete their contents.
Immunofluorescence on CTLtarget cell conjugates from gunmetal mice supports the idea that granules are secreted from gm/gm CTLs since a fraction of the granules are polarized towards the site of contact and some granules are seen within the talin ring. We show that although Rab27a prenylation is decreased in CTLs, the decreased killing could reflect the incomplete polarization of granules as well as the decrease in Rab27a activity. Since granules polarize completely in ashen mice, the polarization defects observed in gunmetal cannot be attributed to Rab27a and are likely to be the result of decreased prenylation of one or more other Rab GTPases. The identity of the Rab GTPase(s) involved in polarization is not known.
This study is the first to describe the fusion of the granules at the immunological synapse of CTLs. Kupfer's initial studies on T cell conjugates with target cells used CTLs and showed the concentration of talin, the microtubule organizing center, and Golgi complex at the contact site (
This study demonstrates the effects of the ashen and gunmetal mutations on the exocytosis of the secretory lysosomes (lytic granules) of CTLs. These are not the only cell types affected in these mice. Notably, melanosomes are affected since both mutants show hypopigmented coat colors. Also, platelet secretion has been shown to be impaired in gunmetal mice (for review see
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: CHS, Chediak-Higashi syndrome; CTL, cytotoxic T lymphocyte; IMDM, Iscove's modified Dulbecco's medium; RGGT, Rab geranylgeranyl transferase; TCR, T cell receptor; WASP, Wiskott-Aldrich syndrome protein.
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Acknowledgements |
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We thank Dick Swank for gunmetal mice and Stuart Kornfeld for anticathepsin D antisera.
This work was supported by a Wellcome Trust Senior Fellowship to G.M. Griffiths (040825), a Medical Research Council Component grant, and a Wellcome Trust Programme Grant to M.C. Seabra. D.C. Barral was supported by a PhD studentship, grant PRAXIS XXI from Fundacao Ciência e Tecnologia of Portugal.
Submitted: 9 November 2000
Revised: 21 December 2000
Accepted: 28 December 2000
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References |
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