Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması

çetinalp demircan, pınar

Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması

Pınar ÇETİNALP DEMİRCAN (İstanbul Bilim Üniversitesi, Tıbbi Biyokimya Anabilim Dalı, İstanbul, Türkiye)

Fırat Tıp Dergisi

19 2

Yıl: 2019 Cilt: 24 Sayı: 4 Sayfa Aralığı: 177 - 185 Metin Dili: Türkçe İndeks Tarihi: 08-10-2020

Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması

Öz:

Amaç: Bu çalışmada, CD3+T-hücrelerini fitohemaglutinin (phytohemagglutinin,PHA), İnterlökin-2(interleukin-2,IL-2) ve PHA+IL-2 ile birlikteuyararak, farklı uyarıcılarla uyarılmanın CD3+T-hücre çoğalım ve aktivasyon molekülleri ekspresyonuna etkisinin belirlenmesi amaçlandı.Gereç ve Yöntem: Sağlıklı 15 kadının herbirinden 6 mL periferik kan örneği alınarak RosetteSep yöntemiyle 3x106 adet CD3+T-hücre izole edildi.CD3+T-hücreleri faz-kontrast mikroskopta sayıldı ve izolasyon saflıkları immunfloresan ve akım sitometrik yöntemlerle belirlendi. 12 kuyucuklukültür kabının 3’er kuyucuğuna 2x105 adet paylaştırılan hücrelere 10 μg/mL PHA, 13μL/mL IL-2 ve 10 μg/mL PHA+13μL/mL IL-2 uygulanarak 24saat inkübatörde inkübe edildi. Uyarıcıyla uyarılmamış CD3+T-hücreleriyle (kontrol grubu) karşılaştırılan uyarılmış CD3+T–hücre gruplarının;çoğalımları spektrofotometrik yöntemle WST-1 testi ve akım sitometride CFSE testi ile aktivasyon moleküllerinin ekspresyonlarıCD25,CD38,CD69,CD71 ve HLA-DR antikorları ile akım sitometride ölçüldü.Bulgular: WST-1 ve CFSE test değerleri gruplar arasında istatistiksel olarak karşılaştırıldığında; PHA-CD3+T-hücrelerinin (p <0.001) ve PHA+IL-2-CD3+T-hücrelerinin (p <0.001) IL-2-CD3+T-hücrelerinden anlamlı olarak daha fazla çoğaldıkları yani uyarıldıkları tespit edildi. Akım sitometrideölçülen aktivasyon moleküllerinin düzeyleri gruplar arasında istatistiksel olarak karşılaştırıldığında; IL-2-CD3+T-hücrelerine kıyasla, PHA-CD3+Thücrelerindeve PHA+IL-2-CD3+T-hücrelerinde CD25 (p <0.001), CD38 (p <0.001), CD71 (p <0.001) ve HLA-DR (p <0.001) % ekspresyon düzeylerindeanlamlı artış tespit edildi.Sonuç: PHA ve PHA+IL-2 ile uyarılan CD3+T-hücrelerinde hücre proliferasyonunun ve aktivasyon molekülleri düzeylerdinin benzer olduğu ve IL-2ile uyarılan hücrelere kıyasla fazla olduğu tespit edildiğinden, invitro çalışmalarda CD3+T-hücre uyarıcısı olarak PHA kullanılmasının başka biruyarıcıya gerek duyulmaksızın tek başına etkin olduğu ve CD3+T-hücrelerde IL-2’ye kıyasla daha fazla çoğalımı ve uyarılmayı sağladığı sonucunavarıldı.

Anahtar Kelime:

Comparison of the Effect of Phytohemagglutinin and Interleukin-2 on Human-T-Cells

Öz:

Objective: In this study, it is aimed to determine the effect of stimulation with different stimuli on CD3+T-cells proliferation and activation molecules by stimulating phytohemagglutinin (PHA), Interleukin-2 (IL-2) and PHA+IL-2 together. Material and Method: Six mL peripheral blood samples were obtained from each 15 healthy women and their 3x106 CD3+T-cells were isolated by the RosetteSep method. CD3+T-cells were counted on a phase-contrast microscope and their isolation purity were determined by immunofluorescence and flow cytometric methods. 2x105 cells were apportioned in a 3-well dish of a 12-well culture dish, applied 10μg/mL PHA, 13μL/mL IL-2 and 10μg/mL PHA+13 μL/mL IL-2 and incubated in incubator for 24 hours. Cells groups of stimulated CD3+T-cells compared with CD3+T-cells(control group) not stimulated with any stimulants; their proliferations measured by spectrophotometric method using WST-1 test and flow cytometry by CFSE test, their expressions of activation molecules measured by flow cytometry with CD25, CD38, CD69, CD71 and HLA-DR antibodies. Results: When WST-1 and CFSE test values were compared statistically between groups; It was determined that PHA-CD3+T-cells (p <0.001) and PHA + IL-2-CD3+T-cells (p <0.001) were significantly more proliferated, in other words activated than IL-2-CD3+T-cells. When levels of activation molecules evaluated by flow cytometry were compared statistically between groups; in comparison to IL-2-CD3+T-cells, significantly increased % expression values of CD25 (p <0.001), CD38 (p <0.001), CD71 (p <0.001) and HLA-DR (p <0.001) were detected in PHA-CD3+T-cells and PHA+IL-2-CD3+T-cells. Conclusion: Since the proliferation and activation molecule levels in CD3+T-cells stimulated by PHA and PHA + IL-2 were similar and more than than stimulated by IL-2, it was concluded that the use of PHA as a CD3+T-cell stimulant in invitro studies alone was effective with no need for other stimulants and provided more proliferation and stimulation compared to IL-2 in CD3+T-cells.

Anahtar Kelime:

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

1. Germain RN. T cell development and the CD4- CD8 lineage decision. Nat Rev Immunol 2002; 2: 309-22.
2. Palmer E. Negative selection-clearing out the ad apples from the T cell repertoire. Nat Rev Immunol 2003; 3: 383-91.
3. Williams MA, Bevan MJ. Effector and memory CTL differentiation. Annu Rev Immunol 2007; 25: 171.
4. Camcıoğlu Y, Deniz G. Temel immünoloji, immün sistemin işlev ve bozuklukları: immün sisteme giriş. İstanbul Tıp Kitapevi 1. Baskı İstanbul 2007: 1-21.
5. Dardalhon V, Awasthi A, Kwon H, et al. IL-4 inhibits TGF-b-induced Foxp3+T cells and, together with TGF-β, generates IL-9+ IL-10+ Foxp3(-) effector T cells. Nat Immunol 2008; 9: 1347-55.
6. Wan YY, Flavell RD. How diverse- CD4 effector T cells and their functions. J Mol Cell Biol 2009; 1: 20-36.
7. Zhu J, Paul WE. Peripheral CD4+ T-cell differentiation regulated by networks of cytokines and transcription factors. Immunol Rev 2010; 238: 247-62.
8. Blanchard N1, Lankar D, Faure F, et al. TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex. J Immunol 2002; 168: 3235-41.
9. Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology. 7/E Elseiver Saunders. Philadelphia, USA 2009: 173-203.
10. Başkan EB. T hücre immünitesi. Türkderm 2013; 47: 18-23. 11. DüzgünN.İmmünSistemTanıtımı.İchastaliklarirom atoloji.medicine.ankara.edu.tr/files/2014; 97-122.
12. Katzen D, Chu E, Terhost C, et al. Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells. J Immunol 1985; 135: 1840-45.
13. Rothwell, L, Hamblin A, Kaiser P. Production and characterisation of monoclonal antibodies specific for chicken interleukin-2. Vet Immunol Immunopathol 2001; 83: 149-60.
14. Lawson S, Rothwell L, Lambrecht B, Howes K, Venugopal K, KaiserP. Turkey and chicken interferon-γ, which share high sequenceidentity, are biologically cross-reactive. Develop Compar Immunol 2001; 25: 69-82.
15. Zhou Z-H, Karnaukhova E, Rajabi M, Kozlowski S. Oversulfated chondroitin sulfate binds to chemokines and inhibits stromal cell- derived factor-1 mediated signaling in activated T cells. PLoS ONE, DOI: 10.1371/journal. 2014; 9: 9: e94402.
16. Mire-Sluis AR, Wickremasinghe RG, Hoffbrand AV, Timms AM, Francis GE. Human T lymphocytes stimulated by phytohaemagglutinin undergoa single round of cell division without a requirement for interleukin-2 or accessory cells. Immunology 1987; 60: 7-12.
17. Herre J1, Marshall AS, Caron E, et al. Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages. Blood 2004; 104: 4038-45.
18. Ma J1, Becker C, Lowell CA, Underhill DM. Dectin-1-triggered Recruitment of Light Chain 3 Protein to Phagosomes Facilitates Major Histocompatibility Complex Class II Presentation of Fungal-derived Antigens. J Biol Chem 2012; 287: 34149-56.
19. Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR. Lymphocyte stimulation: a rapid multiparameter analysis. 1976; Proceedings of the National Academy of Sciences of the United States of America 73: 2881-4.
20. Ko HS, Fu SH, Winchester RJ, Yu DTY, Kunkel HG: Ia determinants on stimulated human T lymphocytes. J Exp Med 1979; 150: 246-55.
21. Judd W, Poodry CA, Strominger JL: Novel surface antigen expressed on dividing cells but absent from nondividing cells. J Exp Med 1980; 152: 1430–35.
22. Cebrian M, Yague E, Rincon M, Lopez-Botet M, Landazurl MO, Sanchez-Madrid E. Triggering of T-cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J Exp Med 1988; 168: 1621-37.
23. Fraser JD, Strauss D, Weiss A: Signal transduction events leading to T-cell lymphokine gene expression. Immunol Today 1993; 14: 357-62.
24. Caruso A, Licenziati S, Corulli M, et al. Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation. Cytometry 1997; 27: 71-6.
25. Demircan PC, Sariboyaci AE, Unal ZS, Gacar G, Subasi C, Karaoz E. Immunoregulatory effects of human dental pulp-derived stem cells on T cells: comparison of transwell co-culture and mixedlymphocyte reaction systems. Cytotherapy 2011; 13: 1205-20.
26. Bitgen N, Altuntaş DH, Hamurcu Z, Demirtaş H, Ozturk F. Fitohemaglütinin ile uyarılmış insan Tlenfositlerindeki AgNOR motiflerinin T-lenfosit alt grupları ile karşılaştırmalı olarak incelenmesi. Erciyes Med J 2012; 34: 132-6.
27. Sariboyaci AE, Demircan PC, Gacar G, Unal ZS, Erman G, Karaoz E. Immunomodulatory properties of pancreatic islet-derived stem cells cocultured with T cells: Does it contribute to the pathogenesis of type 1 diabetes. Exp Clin Endocrinol Diabetes 2014; 122: 1-11.
28. Karaoz E, Demircan PC, Erman G, Güngörürler E, Sariboyacı AE. Comparative analyses of immune-suppressive characteristics of bonemarrow, Wharton's jelly and adipose-tissue derived human MSCs. Turk J Hematol 2017; 34: 213-25.
29. Walling BL and Kim M. LFA-1 in T cell migration and differentiation. Front Immunol 2018; 9: 1-10.
30. Triebel F, DeRoquefeuil S, Blanc C, Charron DJ, Debre P. Expression of MHC class II and Tac antigens on IL-2 activated human T-cell clones that can stimulate in MLR, AMLR, PLT, and can present antigen. Human Immunol 1986; 15: 302- 8.
31. Waldmann TA. The structure, function, and expression of interleukin-2 receptors on normal and malignant lymphocytes. Science 1986; 232: 727-32.
32. Siegal JP, Sharon M, Smith PL, Leonard WJ. The IL-2 receptor B chain (p70): Role in mediating signal for LAK, NK, and proliferative activities. Science 1987; 238: 75-8.
33. Nakamura S, Sung SSJ, Bjorndahl JM, Fu SM. Human T-cell activation IV. T-cell activation and proliferation via the early activation antigen EA-1. J Exp Med 1989; 169: 677-89.
34. Leroux M, Schindler L, Braun R, Doerr HW, Geissen HP, Kirchner H. A whole blood lymphoproliferation assay for measuring cellular immunity against herpes viruses. J Immunol Methods 1989; 79: 251-7.
35. Maino VC, Suni MA, Ruitenberg JJ. Rapid flow cytometric method for measuring lymphocyte subset activation. Cytometry 1995; 20: 127-33.
36. Krowka JF, Cuevas B, Maron DC, Steimer KS, Ascher MS, Sheppard HW. Expression of CD69 after in vitro stimulation: a rapid method for quantitating impaired lymphocyte responses in HIV-infected individuals. J Acquir Immune Defic Syndr 1996; 11: 95-104.
37. Sandoval-Montes C1, Santos-Argumedo L. CD38 is expressed selectively during the activation of a subset of mature T cells with reduced proliferation but improved potential to produce cytokines. J Leukoc Biol 2005; 77: 513-21.
38. Cooper David and Pellis NR. Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C. Journal of Leukocyte Biology 1998; 63: 550-62.
39. Pesoa S, Martín A, Mariani AL, Vullo C, Serra H. Interleukin 2 induction of proliferation in resting T lymphocytes requires contact with monocytes. Medicina (B Aires) 2000; 60: 202-10.
40. Bocharov1 G, Luzyanina T, Cupovic3 J, Burkhard L. Asymmetry of cell division in CFSE-based lymphocyte proliferation analysis. Front Immunol 2013; 4: 1-7.
41. Ahluwalia B, Magnusson MK, Isaksson S, Larsson F, Öhman L. Effects of Aloe barbadensis Mill. extract (AVH200®) on human blood T cell activity in

APA	çetinalp demircan p (2019). Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. , 177 - 185.
Chicago	çetinalp demircan pınar Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. (2019): 177 - 185.
MLA	çetinalp demircan pınar Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. , 2019, ss.177 - 185.
AMA	çetinalp demircan p Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. . 2019; 177 - 185.
Vancouver	çetinalp demircan p Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. . 2019; 177 - 185.
IEEE	çetinalp demircan p "Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması." , ss.177 - 185, 2019.
ISNAD	çetinalp demircan, pınar. "Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması". (2019), 177-185.

APA	çetinalp demircan p (2019). Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. Fırat Tıp Dergisi, 24(4), 177 - 185.
Chicago	çetinalp demircan pınar Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. Fırat Tıp Dergisi 24, no.4 (2019): 177 - 185.
MLA	çetinalp demircan pınar Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. Fırat Tıp Dergisi, vol.24, no.4, 2019, ss.177 - 185.
AMA	çetinalp demircan p Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. Fırat Tıp Dergisi. 2019; 24(4): 177 - 185.
Vancouver	çetinalp demircan p Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması. Fırat Tıp Dergisi. 2019; 24(4): 177 - 185.
IEEE	çetinalp demircan p "Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması." Fırat Tıp Dergisi, 24, ss.177 - 185, 2019.
ISNAD	çetinalp demircan, pınar. "Fitohemaglutinin ve İnterlökin-2’nin İnsan-T-Hücrelerine Etkisinin Karşılaştırılması". Fırat Tıp Dergisi 24/4 (2019), 177-185.