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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 331-335

The detection and significance of T cells in nasopharyngeal carcinoma patients


1 Department of Laboratory, Hebei Daopei Hospital, Langfang, China
2 Department of Head and Neck Oncology, Cancer Hospital of Guizhou Province, Guiyang, China
3 Central Laboratory, Affiliated Hospital of Guiyang Medical College, Guiyang, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Li Ma
Central Laboratory, Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.235350

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 > Abstract 

Aim of Study: Nasopharyngeal carcinoma (NPC) is by far the most common malignant tumor of the nasopharynx and is suggested to be related to immune system dysfunction. T cells play a central role in the cell-mediated immunity. However, how the T cells vary during the NPC treatment is still unclear.
Materials and Methods: We divided the NPC patients into previously untreated, partial remission, complete remission, and relapse groups. Healthy controls were those without any autoimmune diseases, cancer, or recent infection. We used flow cytometry to detect the changes in T cell subsets.
Results: We found the quantity (%) of CD4+ CD25+ CD127low/− Treg regulatory T cells and CD8+ CD28 T cells were obviously increased in NPC previously untreated, partial remission, and relapse groups. There was no difference in these two subsets between complete remission groups and healthy controls. In addition, the quantity (%) of CD3+ CD4+ and CD8+ CD28+ effector T cells were reduced in NPC previously untreated, partial remission, and relapse groups. There was no difference in these two subsets between complete remission groups and healthy controls.
Conclusions: Our research determines the changes of T cell subsets in different stages of NPC.

Keywords: CD4+CD25+CD127low/− Treg regulatory T cells, CD8+CD28 T cells, CD8+CD28+ effector T cells, nasopharyngeal carcinoma


How to cite this article:
Chen M, Jin F, Ma L. The detection and significance of T cells in nasopharyngeal carcinoma patients. J Can Res Ther 2018;14, Suppl S2:331-5

How to cite this URL:
Chen M, Jin F, Ma L. The detection and significance of T cells in nasopharyngeal carcinoma patients. J Can Res Ther [serial online] 2018 [cited 2019 Jul 23];14:331-5. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/331/235350


 > Introduction Top


Nasopharyngeal carcinoma (NPC) is by far the most common malignant tumor of the nasopharynx.[1] It is vastly more common in certain regions of the East Asia and Africa than elsewhere,[2],[3] with viral, dietary and genetic factors implicated in its causation.[4],[5] NPC may be associated with the immune escape caused by the tolerance of tumor cells themselves.[6],[7] NPC can be treated by surgery, chemotherapy, or radiotherapy.[8],[9],[10],[11]

T cells, also called T lymphocytes, are a type of lymphocyte that plays a central role in cell-mediated immunity.[12],[13],[14] The several subsets of T cells each have a distinct function.[15],[16],[17] The CD4+ CD25+ CD127low/− Treg regulatory T cells and CD8+ CD28 T cells have suppressor function.[18] They are crucial for the maintenance of immunological tolerance.[19] Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus.[19] CD3+ CD4+ and CD8+ CD28+ T cells have effector function.[12] However, how the T cells vary during the treatment of NPC process is still unclear.

Here we divided the NPC patients into previously untreated, partial remission, complete remission, and relapse groups. We found the quantity (%) of CD4+ CD25+ CD127low/− Treg regulatory T cells and CD8+ CD28 T cells were increased in NPC previously untreated, partly partial remission, and relapse groups. There was no difference in these two subsets between complete remission groups and healthy controls. Moreover, the quantity (%) of CD3+ CD4+ and CD8+ CD28+ effector T cells was reduced in NPC previously untreated, partial remission, and relapse groups. There was no difference in these two subsets between complete remission groups and healthy controls. Our research suggests that the detection of T cells subsets can be an efficient diagnose during the NPC therapy.


 > Materials and Methods Top


Case selection

This research included 87 nasopharyngeal carcinoma cases admitted Epstein-Barr virus antibody-positive from a Head and Nasopharyngeal Carcinoma Tumor of one hospital. The age of them ranged from 15 to 73 years, and the mean age was 46.59 ± 12.15 years. All cases had been diagnosed histologically and staged in line with the 2003 version of the American Association of Cancer and the International Union Against Cancer tumor node metastasis staging of NPC. All the cases referenced to the response evaluation criteria in solid tumors were divided into previously untreated, partial remission, complete remission, and relapse groups.

The healthy control groups included 22 cases without any autoimmune diseases, cancer, or recent infection from the medical center of the same hospital, aged from 22 to 73 years old, and the mean age was 45.95 ± 12.34 years. The healthy control groups were age and sex matched with the NPC patients.

Flow cytometry

The quantity (%) of CD4+ CD25+ CD127low/− Treg regulatory T cells, CD8+ CD28 T cells, and CD3+ CD4+ and CD8+ CD28+ effector T cells were measured through flow cytometry (FCM). Each test included control and positive experimental groups. The positive experimental group used 100 μl peripheral blood and were added 10 μl containing a mixture of human anti-mouse monoclonal antibody CD4-FITC, CD127-APC, and CD25-PE-cy7 as positive antibody of CD4+ CD25+ CD127low/− Treg regulatory T cells, human anti-mouse monoclonal antibody CD3-FITC and CD4-PE as positive antibody of CD3+ CD4+ T cells or human anti-mouse monoclonal antibody CD8-FITC and CD28-PE as CD8+ CD28 T cells. The control group were added 10 μl containing a mixture of human anti-mouse monoclonal antibody CD4-FITC, IgG1 k-PE-Cy7, and IgG2a-APC (CD4+ CD25+ CD127low/− Treg regulatory T cells) or human anti-mouse monoclonal antibody IgG1-FITC and IgG2a-PE (CD3+ CD4+, CD8+ CD28, and CD8+ CD28+ T cells). All the samples were incubated without light for 15 min. Then 1000 μl 1 × hemolysin was added to lyse the red blood cells for 10 min. It was then centrifuged at 1000 rpm/min for 5 min, and the supernatant was removed after using phosphate-buffered saline (PBS) wash twice, 200 μl PBS was added to resuspend the cells. FCM was used to measure the quantity of the cells.

Flow cytometry analysis

BD FACSDiva software was used to analyze the results of the FCM. The leukocyte can be divided into three groups: Lymphocytes, mononuclear cells, and granulocytes, according to the size and nucleus complexity [Figure S1]. The lymphocytes [Figure S1]a, CD4 positive cells [Figure S1]b, and CD4 double positive cell used IgG2a-APC and IgG1 k-PE-Cy7 antibody as a control to adjust the voltage and compensation [Figure S1]c. Then the percentage of CD4+ CD25+ CD127low/− Treg regulatory T cells in peripheral blood CD4+ T cells can be obtained [Figure S1]d.
Figure S1: The flow diagram of analyzing the expression of CD4+CD25+CD127low/− Treg T cell. (a) Gating out of lymphocytes (b) gating out of CD4+ T cell (c) isotype control (d) CD4+CD25+CD127low/− Treg T cell

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The percentage of CD3+ CD4+, CD8+ CD28, and CD8+ CD28+ T cells in lymphocytes can also obtain using human antibody IgG1-FITC, IgG2a-PE as a control to adjust the voltage and compensation [Figure S2]a and [Figure S2]b.
Figure S2: The flow diagram of analyzing the expression of CD8+CD28+/CD8+CD28 and CD3+CD4+ T cells. (a) CD8+CD28+/CD8+CD28 T cell (b) CD3+CD4+ T cell

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Statistical analysis

Statistical significance was assessed by software SPSS 11.5 (Student's T test was used to test the significant between every groups). Gender analysis used the Chi-square test statistic. The amount (%) between the two groupsCD4+ CD25+ CD127low/− regulatory T cells, CD8+ CD28+ T cells, CD8+ CD28 T cells, or CD3+ CD4+ T cells were compared using a single factor analysis of variance, correlation analysis using bivariate correlation analysis. All measurement data used mean ± standard deviation, and P < 0.05 was considered statistically significant.


 > Results Top


The quantity changes of regulatory and suppressor T cell

NPC is suggested to be related to immune system dysfunction.[1],[4] The level of T cells could reflect the function of the immune system.[7] The level of CD4+ CD25+ CD127low/− Treg regulatory T cells and CD8+ CD28 T cells were tested firstly. We found the percentage of CD4+ CD25+ CD127low/− Treg regulatory T cells and CD8+ CD28 T cells were increased in NPC previously untreated, partial remission, and relapse groups [Table 1]. There were significant differences between these three groups and healthy controls (P < 0.01). During the treatment process, the levels of regulatory and suppressor T cells were reduced in partial remission and complete remission groups compared to the previously untreated group [Table 1]. There were significant differences between these two groups and previously untreated group (P < 0.01). And the levels of regulatory and suppressor T cells in relapse group were increased compared to partial remission, complete remission, and healthy controls [Table 1].
Table 1: Flow cytometry results of CD4+CD25+CD127low/- Treg and CD8+CD28- T cells in peripheral blood of nasopharyngeal carcinoma patients (%)

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We also found that there was a downward trend of CD4+ CD25+ CD127low/− Treg regulatory T cells and CD8+ CD28 T cells in previously untreated, partial remission, and complete remission groups [Table 1], trending F value was 34.734 and 49.693]. However, in relapse group, there was an increase in the quantity (%) of the CD4+ CD25+ CD127low/− Treg regulatory T cells and CD8+ CD28 T cells [Table 1]. After correlation analysis, there was no association with T cell changes in treatment progress.

The quantity changes of effector T cell

Next the quantity (%) of CD3+ CD4+ and CD8+ CD28+ effector T cells were measured. We found the percentage of CD3+ CD4+ and CD8+ CD28+ effector T cells were reduced in NPC previous untreated, partial remission, and relapse groups [Table 2]. There were significant differences between these three groups and healthy controls (P < 0.01). During the treatment process, the levels of effector T cells were increased in partial remission and complete remission groups compared to the previously untreated group [Table 2]. There were significant differences between these two groups and previously untreated groups (P < 0.01). And the levels of effector T cells in relapse groups were reduced compared to partial remission, complete remission, and healthy controls [Table 2].
Table 2: Flow cytometry results of CD3+CD4+ and CD8+CD28+ T cells and expression of TLR3 of peripheral blood stem cell in peripheral blood of nasopharyngeal carcinoma patients (%)

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After further analysis, we found that there was an upward trend of CD3+ CD4+ and CD8+ CD28+ effector T cells in previously untreated, partial remission, and complete remission groups [Table 2], trending F value was 115.794 and 13.257]. However in the relapse group, there was a reduction in the quantity (%) of the CD3+ CD4+ and CD8+ CD28+ effector T cells [Table 2]. After correlation analysis, there was no association with T cell changes in treatment progress.


 > Discussion Top


Recent research has shown that in the tumor microenvironment the amount of the CD4+ CD25+ CD127low/ Treg regulatory T cells increases and the role of it in the tumor immunity and immune escape has been further confirmed.[20],[21],[22] CD4+ CD25+ CD127low/ Treg T cell is regarded as the key component of the formation of tumor immune tolerance,[23],[24] and an important immunosuppressive tumor cell.[25],[26] In the tumor environment, Treg cells can secrete a variety of immunosuppressive cytokines, including interleukin (IL)-10, and transforming growth factor-beta (TGF-β)[27],[28] and inhibit the function of effector T cell through IL-10. TGF-β secreted by Treg cells in the peripheral blood of patients with head and neck tumor can significantly inhibit the antitumor immunity, and secreted by Treg cells in tumor tissue can inhibit the function of lymphokine activated killer cells (lymph-induced killer cells).[29] Meanwhile, Treg cells can interfere with cell metabolism,[30] differentiation and function of the effector T cells, and ultimately affect the body's antitumor immunity.[31] CD8+ T cells are be divided into cytotoxic T cells (CD8+ CD28+, CTL) and suppressor T cells (CD8+ CD28) based on the expression of CD28.[32] CD8+ CD28 suppressor T cell can inhibit antibody production, activate, and proliferate of CD4+ T cells, increase the depletion of CTL, and then affect the immune surveillance function.[7] This leads to no immune effect to the new antitumor cell, which increase the tumor cells immune escape and eventually lead to the tumorigenesis and development.

The results of our study showed that in the initial stage of NPC, the quantity (%) of Treg cells increases. This result is consistent with that found by Sui in 2009.[33] Treg cells can affect the body's antitumor immunity, and make the body in tumor immune tolerance state, resulting in the escape of tumor cells. The detection of Treg cells can help in the tumor finding.

The amount of peripheral blood CD8+ CD28 T cells in previously untreated and relapse NPC patients was higher than in other groups. The accumulation of CD8+ CD28 T cells is a sign of decline of T cell function, and can result in tumor recurrence and relapse.[4],[5] So the detection of CD8+ CD28 T cells can diagnose the occurrence and relapse of NPC.

Our results also showed that the amounts of Treg cells and CD8+ CD28 T cells in partial remission and complete remission NPC patients were reduced. And with the treatment, the amounts of Treg cells and CD8+ CD28 T cells decreased. This suggests that the immune inhibition caused by the Treg cells and CD8+ CD28 T cells lifted, the body's immune tolerance eased, with an increase in the antitumor immunity. The dynamic detection of the amount of Treg cells and CD8+ CD28 T cells can reflect the immunization and treatment status of NPC patients.

The amount of Treg cells and CD8+ CD28 T cells in relapse NPC patients increased again and had no difference with the previously untreated group. This suggests the body re-enters immune tolerance to the tumor. Tumor can escape from the immune surveillance. So the increase of Treg cells promotes tumor relapse. The detection of Treg cells and CD8+ CD28 T cells is important to control the relapse of NPC.

Cell immune mediated by CD8+ and CD4+ T cell plays an important role in the tumor growth control and killing tumors. CD3+ CD4+ T cell is a common immune cell subset. In the tumor environment, cell proliferation and effector ability of this type T cell will change. CD8+ CD28+ T cells can generate IL-2 under the activation signal, and release of granule enzymes perforin cytotoxic substance through the discharge granulation. This can dissolve the target cell. It can also induce apoptosis through the release of several cytokines.

The quantity (%) of CD3+ CD4+ and CD8+ CD28+ T cells is higher in those NPC patients in partial remission and complete remission. And with the process of the treatment, the quantity (%) of CD3+ CD4+ and CD8+ CD28+ T cells increased, and there was no difference between complete remission NPC patients and healthy controls. However, the quantity in the relapse group is lower than the others. This suggests that the quantity (%) D3+ CD4+ and CD8+ CD28+ T cells relate to the recovery of the NPC. Our research determines the changes of T cell subsets in different stages of NPC and this may provide an effective method to detect the stage of NPC.


 > Conclusions Top


Our research suggested that during the NPC therapy, the quantity of T cells subsets was different in each stage. This could make NPC therapy process more accurate.

Acknowledgment

We thank Wei Wang, Department of Labortary, Cancer Hospital of Guizhou Province, for the help in the samples collecting. This work was supported by the Affiliated Hospital of Guiyang Medical College Hospital Foundation (1-2012-25).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

1.
Chou J, Lin YC, Kim J, You L, Xu Z, He B, et al. Nasopharyngeal carcinoma – Review of the molecular mechanisms of tumorigenesis. Head Neck 2008;30:946-63.  Back to cited text no. 1
[PUBMED]    
2.
Cheah SK, Lau FN, Yusof MM, Phua VC. Treatment outcome with brachytherapy for recurrent nasopharyngeal carcinoma. Asian Pac J Cancer Prev 2014;14:6513-8.  Back to cited text no. 2
[PUBMED]    
3.
Huang TR, Zhang SW, Chen WQ, Deng W, Zhang CY, Zhou XJ, et al. Trends in nasopharyngeal carcinoma mortality in China, 1973-2005. Asian Pac J Cancer Prev 2012;13:2495-502.  Back to cited text no. 3
[PUBMED]    
4.
Licitra L, Bernier J, Cvitkovic E, Grandi C, Spinazzé S, Bruzzi P, et al. Cancer of the nasopharynx. Crit Rev Oncol Hematol 2003;45:199-213.  Back to cited text no. 4
    
5.
Li JX, Huang SM, Wen BX, Lu TX. Prognostic factors on overall survival of newly diagnosed metastatic nasopharyngeal carcinoma. Asian Pac J Cancer Prev 2014;15:3169-73.  Back to cited text no. 5
[PUBMED]    
6.
Fogg M, Murphy JR, Lorch J, Posner M, Wang F. Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein-Barr virus associated nasopharyngeal carcinoma. Virology 2013;441:107-13.  Back to cited text no. 6
[PUBMED]    
7.
Basso S, Zecca M, Merli P, Gurrado A, Secondino S, Quartuccio G, et al. T cell therapy for nasopharyngeal carcinoma. J Cancer 2011;2:341-6.  Back to cited text no. 7
[PUBMED]    
8.
Widesott L, Pierelli A, Fiorino C, Dell'oca I, Broggi S, Cattaneo GM, et al. Intensity-modulated proton therapy versus helical tomotherapy in nasopharynx cancer: planning comparison and NTCP evaluation. Int J Radiat Oncol Biol Phys 2008;72:589-96.  Back to cited text no. 8
    
9.
Krespi YP. Cancer surgery of the skull base. Clin Plast Surg 1985;12:389-92.  Back to cited text no. 9
[PUBMED]    
10.
Slevin NJ, Wilkinson JM, Filby HM, Gupta NK. Intracavitary radiotherapy boosting for nasopharynx cancer. Br J Radiol 1997;70:412-4.  Back to cited text no. 10
[PUBMED]    
11.
Dizman A, Coskun-Breuneval M, Altinisik-Inan G, Olcay GK, Cetindag MF, Guney Y. Reirradiation with robotic stereotactic body radiotherapy for recurrent nasopharyngeal carcinoma. Asian Pac J Cancer Prev 2014;15:3561-6.  Back to cited text no. 11
[PUBMED]    
12.
Poletaev A, Boura P. The immune system, natural autoantibodies and general homeostasis in health and disease. Hippokratia 2011;15:295-8.  Back to cited text no. 12
[PUBMED]    
13.
Zhu JY, Vereshchagina N, Sreekumar V, Burbulla LF, Costa AC, Daub KJ, et al. Knockdown of Hsc70-5/mortalin induces loss of synaptic mitochondria in a Drosophila Parkinson's disease model. PLoS One 2013;8:e83714.  Back to cited text no. 13
[PUBMED]    
14.
Kedzierska K, Sporniak-Tutak K, Kolasa A, Domanski L, Domanski M, Sindrewicz K, et al. The effect of immunosuppressive therapy on renal cell apoptosis in native rat kidneys. Histol Histopathol 2015;30:105-16.  Back to cited text no. 14
    
15.
Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med 2011;17:796-808.  Back to cited text no. 15
[PUBMED]    
16.
Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8:299-308.  Back to cited text no. 16
[PUBMED]    
17.
Zhang Y, Sun R, Liu B, Deng M, Zhang W, Li Y, et al. TLR3 activation inhibits nasopharyngeal carcinoma metastasis via downregulation of chemokine receptor CXCR4. Cancer Biol Ther 2009;8:1826-30.  Back to cited text no. 17
[PUBMED]    
18.
Hong H, Gu Y, Zhang H, Simon AK, Chen X, Wu C, et al. Depletion of CD4+CD25+ regulatory T cells enhances natural killer T cell-mediated anti-tumour immunity in a murine mammary breast cancer model. Clin Exp Immunol 2010;159:93-9.  Back to cited text no. 18
[PUBMED]    
19.
McHugh RS, Shevach EM. The role of suppressor T cells in regulation of immune responses. J Allergy Clin Immunol 2002;110:693-702.  Back to cited text no. 19
[PUBMED]    
20.
Lim KP, Chun NA, Ismail SM, Abraham MT, Yusoff MN, Zain RB, et al. CD4+CD25hiCD127low regulatory T cells are increased in oral squamous cell carcinoma patients. PLoS One 2014;9:e103975.  Back to cited text no. 20
[PUBMED]    
21.
Drennan S, Stafford ND, Greenman J, Green VL. Increased frequency and suppressive activity of CD127(low/-) regulatory T cells in the peripheral circulation of patients with head and neck squamous cell carcinoma are associated with advanced stage and nodal involvement. Immunology 2013;140:335-43.  Back to cited text no. 21
[PUBMED]    
22.
Zhu X, Ma LL, Ye T. Expression of CD4(+)CD25(high) CD127(low/-) regulatory T cells in transitional cell carcinoma patients and its significance. J Clin Lab Anal 2009;23:197-201.  Back to cited text no. 22
[PUBMED]    
23.
Cao X. Regulatory T cells and immune tolerance to tumors. Immunol Res 2010;46:79-93.  Back to cited text no. 23
[PUBMED]    
24.
Field EH, Gao Q. CD4 regulatory cells in immune tolerance. J Lab Clin Med 1998;132:91-6.  Back to cited text no. 24
[PUBMED]    
25.
Han Y, Wu J, Bi L, Xiong S, Gao S, Yin L, et al. Malignant B cells induce the conversion of CD4+CD25- T cells to regulatory T cells in B-cell non-Hodgkin lymphoma. PLoS One 2011;6:e28649.  Back to cited text no. 25
[PUBMED]    
26.
Lee WC, Wu TJ, Chou HS, Yu MC, Hsu PY, Hsu HY, et al. The impact of CD4+CD25+ T cells in the tumor microenvironment of hepatocellular carcinoma. Surgery 2012;151:213-22.  Back to cited text no. 26
    
27.
Jost NH, Abel S, Hutzler M, Sparwasser T, Zimmermann A, Roers A, et al. Regulatory T cells and T-cell-derived IL-10 interfere with effective anti-cytomegalovirus immune response. Immunol Cell Biol 2014;92:860-71.  Back to cited text no. 27
[PUBMED]    
28.
Liu Y, Zhang P, Li J, Kulkarni AB, Perruche S, Chen W. A critical function for TGF-beta signaling in the development of natural CD4+CD25+Foxp3+ regulatory T cells. Nat Immunol 2008;9:632-40.  Back to cited text no. 28
[PUBMED]    
29.
Larmonier N, Marron M, Zeng Y, Cantrell J, Romanoski A, Sepassi M, et al. Tumor-derived CD4(+) CD25(+) regulatory T cell suppression of dendritic cell function involves TGF-beta and IL-10. Cancer Immunol Immunother 2007;56:48-59.  Back to cited text no. 29
[PUBMED]    
30.
Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell 2008;13:472-82.  Back to cited text no. 30
[PUBMED]    
31.
Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer 2010;127:759-67.  Back to cited text no. 31
[PUBMED]    
32.
Weng NP, Akbar AN, Goronzy J. CD28(-) T cells: their role in the age-associated decline of immune function. Trends Immunol 2009;30:306-12.  Back to cited text no. 32
[PUBMED]    
33.
Ren YX, Sui J, Song X, Wong GW, Ma J, Yao H, et al. Correlation between CD4+CD25+Treg Cells and CCR4 in Nasopharyngeal Carcinoma, Clin Oncol Cancer Res 2011;8:106-13.  Back to cited text no. 33
    


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