1. bookVolume 28 (2020): Issue 1 (January 2020)
Journal Details
License
Format
Journal
eISSN
2284-5623
First Published
08 Aug 2013
Publication timeframe
4 times per year
Languages
English
access type Open Access

Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

Published Online: 07 Feb 2020
Volume & Issue: Volume 28 (2020) - Issue 1 (January 2020)
Page range: 75 - 89
Received: 02 Oct 2019
Accepted: 25 Nov 2019
Journal Details
License
Format
Journal
eISSN
2284-5623
First Published
08 Aug 2013
Publication timeframe
4 times per year
Languages
English
Abstract

Peripheral blood lymphocytes (PBL) are able to synthesize various cytokines that play key roles in the immune response and intercellular signaling. Since alterations in cytokine production and/or activity occur in many pathological processes, the study of cytokine synthetic capacity of PBL is a valuable tool for assessing the immune profile. In this paper, we aimed to investigate the variability of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) synthetic capacity of CD4+/CD8+ T-cells stimulated ex-vivo in healthy subjects, by means of a commercial intracellular cytokine staining (ICS) protocol. Peripheral blood mononuclear cells were isolated from 16 healthy subjects by Ficoll gradient centrifugation and activated ex-vivo with PMA/Ionomycin/Brefeldin-A for 4 hours. Activated PBL were surface-stained for CD3/CD4/CD8, fixed and permeabilized. ICS was performed using anti-human IL-2/TNF-α/IFN-γ and samples were analyzed on a BD-FACSAria-III flow cytometer. We recorded high post-isolation and post-activation mean viabilities: 82.1% and 82.4% respectively, p=0.84. Both CD4+/CD8+ subpopulations were found to partially produce each of the three cytokines, but in different proportions. On average, a significantly greater percentage of CD4+ cells was shown to produce IL-2 and TNF-α, compared with CD8+ cells (61.5%+/-5.8 vs. 25%+/-5.6 and 26.9%+/-11 vs. 7.5%+/-3.3 respectively, p---lt---0.0001 for both). Contrarily, IFN-γ was produced by a higher proportion of CD8+ cells (8.4%+/-3.9 vs. 6.8%+/-3.2, p=0.01). These results show that the employed ICS protocol elicits a satisfactory and consistent cytokine response from PBL of healthy subjects. The collected data may be used to outline a preliminary reference range for future studies on both healthy/pathological subjects.

Keywords

1. Mesko B, Poliska S, Nagy L. Gene expression profiles in peripheral blood for the diagnosis of autoimmune diseases. Trends Mol Med. 2011;17(4):223-33 DOI: 10.1016/j.molmed.2010.12.00410.1016/j.molmed.2010.12.00421388884Search in Google Scholar

2. Shi G, Zhang Z, Li Q. New Biomarkers in Autoimmune Disease. Hindawi, J Immunol Res. 2017;2017:8702425. DOI: 10.1155/2017/870242510.1155/2017/8702425540558528491876Search in Google Scholar

3. Masucci GV, Cesano A, Hawtin R, Janetzki S, Zhang J, Kirsch I, et al. Validation of biomarkers to predict response to immunotherapy in cancer: Volume I -pre-analytical and analytical validation. J Immunother Cancer. 2016 Nov 15;4:76. DOI: 10.1186/s40425-016-0178-110.1186/s40425-016-0178-1510974427895917Search in Google Scholar

4. Yuan J, Hegde PS, Clynes R, Foukas PG, Harari A, Kleen TO, et al. Novel technologies and emerging biomarkers for personalized cancer immunotherapy. J Immunother Cancer. 2016 Jan 19;4:3. DOI: 10.1186/s40425-016-0107-310.1186/s40425-016-0107-3471754826788324Search in Google Scholar

5. Rahmoune H, Guest PC. Studies of Isolated Peripheral Blood Cells as a Model of Immune Dysfunction. Methods Mol Biol. 2018;1735:221-9. DOI: 10.1007/978-1-4939-7614-0_1210.1007/978-1-4939-7614-0_1229380315Search in Google Scholar

6. Končarević S, Lößner C, Kuhn K, Prinz T, Pike I, Zucht HD. In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics. Int J Proteomics. 2014;2014:129259. DOI: 10.1155/2014/12925910.1155/2014/129259395866524724028Search in Google Scholar

7. Friberg D, Bryant J, Shannon W, Whiteside TL. In vitro cytokine production by normal human peripheral blood mononuclear cells as a measure of immunocompetence or the state of activation. Clin Diagn Lab Immunol. 1994;1(3):261-810.1128/cdli.1.3.261-268.19943682467496960Search in Google Scholar

8. Jason J, Archibald LK, Nwanyanwu OC, Byrd MG, Kazembe PN, Dobbie H, et al. Comparison of serum and cell-specific cytokines in humans. Clin Diagn Lab Immunol. 2001;8(6):1097-103. DOI: 10.1128/CDLI.8.6.1097-1103.200110.1128/CDLI.8.6.1097-1103.20019623211687446Search in Google Scholar

9. Chang S, Kohrt H, Maecker HT. Monitoring the immune competence of cancer patients to predict outcome. Cancer Immunol Immunother. 2014;63(7):713-19 DOI: 10.1007/s00262-014-1521-310.1007/s00262-014-1521-3405835824487923Search in Google Scholar

10. Osnes LT, Nakken B, Bodolay E, Szodoray P. Assessment of intracellular cytokines and regulatory cells in patients with autoimmune diseases and primary immunodeficiencies - Novel tool for diagnostics and patient follow-up. Autoimmun Rev. 2013 Aug;12(10):967-71. DOI: 10.1016/j.autrev.2013.02.00310.1016/j.autrev.2013.02.00323541481Search in Google Scholar

11. Killestein J, Den Drijver BF, Van der Graaff WL, Uitdehaag BM, Polman CH, Van Lier RA. Intracellular cytokine profile in T-cell subsets of multiple sclerosis patients: different features in primary progressive disease. Mult Scler. 2001 Jun;7(3):145-50. DOI: 10.1177/13524585010070030210.1177/13524585010070030211475436Search in Google Scholar

12. Guo H, Qiao Z, Zhu L, Wang H, Su L, Lu Y, et al. Th1/Th2 cytokine profiles and their relationship to clinical features in patients following nonmyeloablative allogeneic stem cell transplantation. Am J Hematol. 2004 Feb;75(2):78-83. DOI: 10.1002/ajh.1044310.1002/ajh.1044314755372Search in Google Scholar

13. Kim TK, St John LS, Wieder ED, Khalili J, Ma Q, Komanduri KV. Human late memory CD8+ T cells have a distinct cytokine signature characterized by CC chemokine production without IL-2 production. J Immunol. 2009 Nov 15;183(10):6167-74. DOI: 10.4049/jimmunol.090206810.4049/jimmunol.090206819841187Search in Google Scholar

14. Hamzaoui K, Hamzaoui A, Guemira F, Bessioud M, Hamza M, Ayed K. Cytokine profile in Behçet’s disease patients. Relationship with disease activity. Scand J Rheumatol. 2002;31(4):205-10. DOI: 10.1080/03009740232031838710.1080/03009740232031838712369651Search in Google Scholar

15. Rocha AM, Souza C, Rocha GA, de Melo FF, Clementino NC, Marino MC, et al. The levels of IL-17A and of the cytokines involved in Th17 cell commitment are increased in patients with chronic immune thrombocytopenia. Haematologica. 2011 Oct;96(10):1560-4. DOI: 10.3324/haematol.2011.04641710.3324/haematol.2011.046417318632121972211Search in Google Scholar

16. Chang DM, Su WL, Chu SJ. The expression and significance of intracellular T helper cytokines in systemic lupus erythematosus. Immunol Invest. 2002 Feb;31(1):1-12. DOI: 10.1081/IMM-12000321710.1081/IMM-12000321711990459Search in Google Scholar

17. Holland N, Dong J, Garnett E, Shaikh N, Huen K, Harmatz P, et al. Reduced intracellular T-helper 1 interfer-on-gamma in blood of newly diagnosed children with Crohn’s disease and age-related changes in Th1/Th2 cytokine profiles. Pediatr Res. 2008 Mar;63(3):257-62. DOI: 10.1203/PDR.0b013e318163a89710.1203/PDR.0b013e318163a897325204618287963Search in Google Scholar

18. Vitale S, Strisciuglio C, Pisapia L, Miele E, Barba P, Vitale A, et al. Cytokine production profile in intestinal mucosa of paediatric inflammatory bowel disease. PLoS One. 2017 Aug 10;12(8):e0182313. DOI: 10.1371/journal.pone.018231310.1371/journal.pone.0182313555223028797042Search in Google Scholar

19. Card CM, Keynan Y, Lajoie J, Bell CP, Dawood M, Becker M et al. HIV controllers are distinguished by chemokine expression profile and HIV-specific T-cell proliferative potential. J Acquir Immune Defic Syndr. 2012 Apr 15;59(5):427-37. DOI: 10.1097/QAI.0b013e-3182454fcdSearch in Google Scholar

20. Kannanganat S, Ibegbu C, Chennareddi L, Robinson HL, Amara RR. Multiple-Cytokine-Producing Antiviral CD4 T Cells Are Functionally Superior to Single-Cytokine-Producing Cells. J Virol. 2007 Aug;81(16):8468-76. DOI: 10.1128/JVI.00228-0710.1128/JVI.00228-07195137817553885Search in Google Scholar

21. Sutherland JS, Adetifa IM, Hill PC, Adegbola RA, Ota MO. Pattern and diversity of cytokine production differentiates between Mycobacterium tuberculosis infection and disease. Eur J Immunol. 2009 Mar;39(3):723-9. DOI: 10.1002/eji.20083869310.1002/eji.20083869319224636Search in Google Scholar

22. Zhang XL, Komada Y, Chipeta J, Li QS, Inaba H, Azuma E, et al. Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia. Cancer Immunol Immunother. 2000 Jun;49(3):165-72. DOI: 10.1007/s00262005061610.1007/s00262005061610881696Search in Google Scholar

23. Gallego A, Vargas JA, Castejón R, Citores MJ, Rome-ro Y, Millán I, et al. Production of intracellular IL-2, TNF-alpha, and IFN-gamma by T cells in B-CLL. Cytometry B Clin Cytom. 2003Nov;56(1):23.9. DOI: 10.1002/cyto.b.1005210.1002/cyto.b.1005214582134Search in Google Scholar

24. Karanikas V, Lodding J, Maino VC, McKenzie IF. Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy. Clin Cancer Res. 2000 Mar;6(3):829-37.Search in Google Scholar

25. Rossmann ED, Lewin N, Jeddi-Tehrani M, Osterborg A, Mellstedt H. Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B-CLL), Eur J Haematol. 2002 May;68(5):299-306. DOI: 10.1034/j.1600-0609.2002.01612.x10.1034/j.1600-0609.2002.01612.x12144536Search in Google Scholar

26. Nakayama H, Kitayama J, Muto T, Nagawa H. Characterization of intracellular cytokine profile of CD4(+) T cells in peripheral blood and tumor-draining lymph nodes of patients with gastrointestinal cancer. Jpn J Clin Oncol. 2000 Jul;30(7):301-5. DOI: 10.1093/jjco/hyd07810.1093/jjco/hyd07811007162Search in Google Scholar

27. Penner RE, Ionescu L, Mital S, Foster B, Birk P, Phan V, et al. - A Novel Tool to Quantify Immune Suppression after Pediatric Transplantation: Flow Cytometric Activation Assays. The Journal of Heart and Lung Transplantation. 2019 April;38(4):S86. DOI: 10.1016/j. healun.2019.01.197Search in Google Scholar

28. Meesing A, Abraham RS, Razonable RR. Clinical Correlation of Cytomegalovirus Infection With CMV-specific CD8+ T-cell Immune Competence Score and Lymphocyte Subsets in Solid Organ Transplant Recipients. Transplantation. 2019 Apr;103(4):832-8. DOI: 10.1097/TP.000000000000239610.1097/TP.000000000000239630086091Search in Google Scholar

29. Molina-Ortega A, Martín-Gandul C, Mena-Romo JD, Rodríguez-Hernández MJ, Su-er M, Bernal C, et al. Impact of pretransplant CMV-specific T-cell immune response in the control of CMV infection after solid organ transplantation: a prospective cohort study. Clin Microbiol Infect. 2019 Jun;25(6):753-8. DOI: 10.1016/j. cmi.2018.09.019Search in Google Scholar

30. Şerban GM, Mănescu IB, Manu DR, Dobreanu M. Optimization of a density gradient centrifugation protocol for isolation of peripheral blood mononuclear cells. Acta Medica Marisiensis 2018;64(2):83-90. DOI: 10.2478/amma-2018-001110.2478/amma-2018-0011Search in Google Scholar

31. http://www.bdbiosciences.com/ds/ab/others/559302_Book_Website.pdfSearch in Google Scholar

32. Ai W, Li H, Song N, Li L, Chen H. Optimal method to stimulate cytokine production and its use in immunotoxicity assessment. Int J Environ Res Public Health. 2013;10(9):3834-42. DOI: 10.3390/ijerph1009383410.3390/ijerph10093834379951623985769Search in Google Scholar

33. Baran J, Kowalczyk D, Ozóg M, Zembala M. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin Diagn Lab Immunol. 2001;8(2):303-13. DOI: 10.1128/CDLI.8.2.303-313.200110.1128/CDLI.8.2.303-313.20019605411238213Search in Google Scholar

34. Boulougouris G, McLeod JD, Patel YI, Ellwood CN, Walker LS, Sansom DM. Positive and Negative Regulation of Human T Cell Activation Mediated by the CTLA-4/CD28 Ligand CD80. 1998 Oct 15;161(8):3919-24Search in Google Scholar

35. Hou H, Zhou Y, Yu J, Mao L, Bosco MJ, Wang J, et al. Establishment of the Reference Intervals of Lymphocyte Function in Healthy Adults Based on IFN-γ Secretion Assay upon Phorbol-12-Myristate-13-Acetate/Ionomycin Stimulation. Front Immunol. 2018 Feb 7;9:172. DOI: 10.3389/fimmu.2018.0017210.3389/fimmu.2018.00172580831629467761Search in Google Scholar

36. Kallas EG, Gibbons DC, Soucier H, Fitzgerald T, Treanor JJ, Evans TG. Detection of intracellular antigen-specific cytokines in human T cell populations. J Infect Dis. 1999 May;179(5):1124-31. DOI: 10.1086/31470210.1086/31470210191213Search in Google Scholar

37. Ollsen I, Sollid LM. Pitfalls in determining the cytokine profile of human T cells. J Immunol Methods. 2013 Apr 30;390(1-2):106-12. DOI: 10.1016/j.jim.2013.01.01510.1016/j.jim.2013.01.015Search in Google Scholar

38. Brignall R, Cauchy P, Bevington SL, Gorman B, Pisco AO, Bagnall J, et al. Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells. J Immunol. 2017 Oct 15;199(8):2652-67. DOI: 10.4049/jimmunol.160203310.4049/jimmunol.1602033Search in Google Scholar

39. Zlei M, Grigore G, Peptanariu D, Constantinescu D, Cianga C, Carasevici E et al. Alternative Fixation Method Improves Flow Cytometry-Assisted Phospho-Detection Competence. Rev Romana Med Lab. 2010 Dec;18(4):55-65.Search in Google Scholar

40. https://www.bdbiosciences.com/us/resources/s/cytokinesfcaSearch in Google Scholar

41. Macián F, García-Cózar F, Im SH, Horton HF, Byrne MC, Rao A. Transcriptional mechanisms underlying lymphocyte tolerance. Cell. 2002 Jun 14;109(6):719-31. DOI: 10.1016/S0092-8674(02)00767-510.1016/S0092-8674(02)00767-5Search in Google Scholar

42. Glisic-Milosavljevic S, Waukau J, Jana S, Jailwala P, Rovensky J, Ghosh S. Comparison of apoptosis and mortality measurements in peripheral blood mono-nuclear cells (PBMCs) using multiple methods. Cell Prolif. 2005 Oct;38(5):301-11. DOI: 10.1111/j.1365-2184.2005.00351.x10.1111/j.1365-2184.2005.00351.xSearch in Google Scholar

43. Jeurink PV, Vissers YM, Rappard B, Savelkoul HF. T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production. Cryobiology. 2008 Oct;57(2):91-103. DOI: 10.1016/j.cryobiol.2008.06.00210.1016/j.cryobiol.2008.06.002Search in Google Scholar

44. Chaplin DD. Overview of the immune response. J Allergy Clin Immunol. February 2003 Feb;111(2 Suppl):S442-59. DOI: 10.1067/mai.2003.12510.1067/mai.2003.125Search in Google Scholar

45. Cox MA, Harrington LE, Zajac AJ. Cytokines and the inception of CD8 T cell responses. Trends Immunol. 2011;32(4):180-6. DOI: 10.1016/j.it.2011.01.00410.1016/j.it.2011.01.004Search in Google Scholar

46. Mehta AK, Gracias DT, Croft M. TNF activity and T cells. Cytokine. 2018;101:14-18. DOI: 10.1016/j. cyto.2016.08.003Search in Google Scholar

47. Nelson BH. IL-2, Regulatory T Cells, and Tolerance. J Immunol. 2004 Apr 1;172(7):3983-8. DOI: 10.4049/jimmunol.172.7.398310.4049/jimmunol.172.7.3983Search in Google Scholar

48. Seder RA. Acquisition of lymphokine-producing phenotype by CD4+ T cells. J Allergy Clin Immunol. 1994 Dec;94(6 Pt 2):1195-202. DOI: 10.1016/0091-6749(94)90332-810.1016/0091-6749(94)90332-8Search in Google Scholar

49. Chaplin DD. Overview of the immune response. J Allergy Clin Immunol. 2010 Feb;125(2 Suppl 2):S3-23. DOI: 10.1016/j.jaci.2009.12.98010.1016/j.jaci.2009.12.980292343020176265Search in Google Scholar

50. Decker ML, Grobusch MP, Ritz N. Influence of Age and Other Factors on Cytokine Expression Profiles in Healthy Children - A Systematic Review. Front Pediatr. 2017;5:255. DOI: 10.3389/fped.2017.0025510.3389/fped.2017.00255573514129312902Search in Google Scholar

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