Characterization of CD4+ and CD8+ T Cell Subsets and Interferon Regulatory Factor 4 (IRF4) in MS Patients Treated with Fingolimod (FTY-720): A Follow-up Study

  • Bahare Laribi Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Mohammad Ali Sahraian MS Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran
  • Mehdi Shekarabi Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
  • Rahimeh Emamnejad Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran
  • Mohsen Marzban Department of Neurosciences, Tehran University of Medical Sciences, Tehran, Iran
  • Shokufeh Sadaghiani MS Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran
  • Maryam Izad Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran AND MS Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran
Keywords: Fingolimod, IFN regulatory factor 4, Multiple sclerosis, T cell


Fingolimod is a novel immunomodulatory drug used in patients with relapsing multiple sclerosis (MS) which reversibly inhibits egress of lymphocytes from lymph nodes. In this longitudinal study, the frequency of Interferon- gamma (IFN-γ)+, IL4+, IL17+ and IL10+ CD4+ and CD8+ T cell subsets were measured in Fingolimod treated patients before and after 12 months’(12M) therapy using flow cytometry and compared to those of naive, Betaferon treated MS patients and healthy individuals. Additionally, the level of transcription factor IRF4 and IL-6, IL-23, TGF-β1 cytokines, required for differentiation of IL-17+ T cells, were assessed by RT-PCR and ELISA, respectively. In Fingolimod treated MS patients, we observed a significant decrease in the percentage of IFN-γ+/IL17+ CD4+ and CD8+ T cell subsets. In contrast, Fingolimod increased IL10+ CD4+ T cells. We also showed that IFN-γ+IL17+ co-producing CD8+ T cells were reduced in patients under fingolimod therapy. furthermore, Fingolimod could reduce the expression level of IRF4 in patients while IL6 was increased in the supernatant of cultured peripheral blood mononuclear cells. Our data showed that Fingolimod treatment alters CD4+ and CD8+ T cell subsets and reduces expression of IRF-4, which affects the proportion of pathogenic memory T cells in peripheral blood.


1. Zipp F. A new window in multiple sclerosis pathology: non-conventional quantitative magnetic resonance imaging outcomes. J Neurol Sci 2009; 287(Suppl):S24-S9.

2. De Carli M, D'Elios MM, Zancuoghi G, Romagnani S, Del Prete G. Human Th1 and Th2 cells: functional properties, regulation of development and role in autoimmunity. Autoimmunity 1994; 18(4):301-8.

3. Benvenuto R, Paroli M, Buttinelli C, Franco A, Barnaba V, Fieschi C, et al. Tumour necrosis factor-alpha synthesis by cerebrospinal-fluid-derived T cell clones from patients with multiple sclerosis. Clin Exp Immunol 1991; 84(1):97-102.

4. Bettelli E, Oukka M, Kuchroo VK. TH-17 cells in the circle of immunity and autoimmunity. Nat Immunol 2007; 8(4):345-50.

5. Durelli L, Conti L, Clerico M, Boselli D, Contessa G, Ripellino P, et al. T-helper 17 cells expand in multiple sclerosis and are inhibited by interferon-β. Ann Neurol 2009; 65(5):499-509.

6. Kebir H, Kreymborg K, Ifergan I, Dodelet-Devillers A, Cayrol R, Bernard M, et al. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat Med 2007; 13(10):1173-5.

7. Montes M, Zhang X, Berthelot L, Laplaud D-A, Brouard S, Jin J, et al. Oligoclonal myelin-reactive T-cell infiltrates derived from multiple sclerosis lesions are enriched in Th17 cells. Clin Immunol 2009; 130(2):133-44.

8. Chen Z, Tato CM, Muul L, Laurence A, O'Shea JJ. Distinct regulation of interleukin-17 in human T helper lymphocytes. Arthritis Rheum 2007; 56(9):2936-46.

9. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, et al. Phenotypic and functional features of human Th17 cells. J Exp Med. 2007; 204(8):1849-61.

10. Kebir H, Ifergan I, Alvarez JI, Bernard M, Poirier J, Arbour N, et al. Preferential recruitment of interferon‐γ–expressing TH17 cells in multiple sclerosis. Ann Neurol 2009; 66(3):390-402.

11. Cosmi L, Cimaz R, Maggi L, Santarlasci V, Capone M, Borriello F, et al. Evidence of the transient nature of the Th17 phenotype of CD4+CD161+ T cells in the synovial fluid of patients with juvenile idiopathic arthritis. Arthritis Rheum 2011; 63(8):2504-15.

12. Delfs MW, Furukawa Y, Mitchell RN, Lichtman AH. CD8+ T Cell Subsets Tc1 and Tc2 Cause Different Histopathologic Forms of Murine Cardiac Allograft Rejection. Transplantation 2001; 71(5):606-10.

13. Dobrzanski MJ, Reome JB, Hollenbaugh JA, Dutton RW. Tc1 and Tc2 Effector Cell Therapy Elicit Long-Term Tumor Immunity by Contrasting Mechanisms That Result in Complementary Endogenous Type 1 Antitumor Responses. J Immunol 2004; 172(3):1380-90.

14. Wong MT, Ong DEH, Lim FSH, Teng KWW, McGovern N, Narayanan S, et al. A high-dimensional atlas of human T cell diversity reveals tissue-specific trafficking and cytokine signatures. Immunity 2016; 45(2):442-56.

15. Tzartos JS, Friese MA, Craner MJ, Palace J, Newcombe J, Esiri MM, et al. Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am J Pathol 2008; 172(1):146-55.

16. Huber M, Heink S, Pagenstecher A, Reinhard K, Ritter J, Visekruna A, et al. IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis. J Clin Invest 2013; 123(1):247-60.

17. Wang HH, Dai YQ, Qiu W, Lu ZQ, Peng FH, Wang YG, et al. Interleukin-17-secreting T cells in neuromyelitis optica and multiple sclerosis during relapse. J Clin Neurosci 2011; 18(10):1313-7.

18. Salehi Z, Doosti R, Beheshti M, Janzamin E, Sahraian MA, Izad M. Differential frequency of CD8+ T cell subsets in multiple sclerosis patients with various clinical patterns. PloS one 2016; 11(7):e0159565.

19. Huber M, Lohoff M. IRF4 at the crossroads of effector T‐cell fate decision. Eur J Immunol 2014; 44(7):1886-95.

20. Brüstle A, Heink S, Huber M, Rosenplänter C, Stadelmann C, Yu P, et al. The development of inflammatory TH-17 cells requires interferon-regulatory factor 4. Nat Immunol 2007; 8(9):958-66.

21. Mudter J, Amoussina L, Schenk M, Yu J, Brüstle A, Weigmann B, et al. The transcription factor IFN regulatory factor–4 controls experimental colitis in mice via T cell–derived IL-6. J Clin Invest 2008; 118(7):2415-26.

22. Mudter J, Yu J, Zufferey C, Brüstle A, Wirtz S, Weigmann B, et al. IRF4 regulates IL‐17A promoter activity and controls RORγt‐dependent Th17 colitis in vivo. Inflamm Bowel Dis 2011; 17(6):1343-58.

23. Chen Q, Yang W, Gupta S, Biswas P, Smith P, Bhagat G, et al. IRF-4-binding protein inhibits interleukin-17 and interleukin-21 production by controlling the activity of IRF-4 transcription factor. Immunity 2008; 29(6):899-911.

24. Cohen JA, Barkhof F, Comi G, Hartung HP, Khatri BO, Montalban X, et al. Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis. N Engl J Med 2010; 362(5):402-15.

25. Kappos L, Radue EW, O'Connor P, Polman C, Hohlfeld R, Calabresi P, et al. A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med 2010; 362(5):387-401.

26. Gräler MH, Goetzl EJ. The immunosuppressant FTY720 down-regulates sphingosine 1-phosphate G-protein-coupled receptors. FASEB J 2004; 18(3):551-3.

27. Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, et al. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004; 427(6972):355-60.

28. Gholamnezhadjafari R, Falak R, Tajik N, Aflatoonian R, Ali Keshtkar A, Rezaei A. Effect of FTY720 (fingolimod) on graft survival in renal transplant recipients: a systematic review protocol. BMJ Open 2016; 6(4):e010114.

29. Mehling M, Brinkmann V, Antel J, Bar-Or A, Goebels N, Vedrine C, et al. FTY720 therapy exerts differential effects on T cell subsets in multiple sclerosis. Neurology 2008; 71(16):1261-7.

30. Mehling M, Lindberg R, Raulf F, Kuhle J, Hess C, Kappos L, et al. Th17 central memory T cells are reduced by FTY720 in patients with multiple sclerosis. Neurology 2010; 75(5):403-10.

31. Groves A, Kihara Y, Chun J. Fingolimod: direct CNS effects of sphingosine 1-phosphate (S1P) receptor modulation and implications in multiple sclerosis therapy. J Neurol Sci 2013; 328(1-2):9-18.

32. Blumenfeld S, Staun-Ram E, Miller A. Fingolimod therapy modulates circulating B cell composition, increases B regulatory subsets and production of IL-10 and TGFβ in patients with Multiple Sclerosis. J Autoimmun 2016; 70:40-51.

33. Song Z-Y, Yamasaki R, Kawano Y, Sato S, Masaki K, Yoshimura S, et al. Peripheral blood T cell dynamics predict relapse in multiple sclerosis patients on fingolimod. PloS one 2015; 10(4):e0124923.

34. Claes N, Dhaeze T, Fraussen J, Broux B, Van Wijmeersch B, Stinissen P, et al. Compositional Changes of B and T Cell Subtypes during Fingolimod Treatment in Multiple Sclerosis Patients: A 12-Month Follow-Up Study. PLOS ONE 2014; 9(10):e111115.

35. Johnson TA, Evans BL, Durafourt BA, Blain M, Lapierre Y, Bar-Or A, et al. Reduction of the Peripheral Blood CD56<sup>bright</sup> NK Lymphocyte Subset in FTY720-Treated Multiple Sclerosis Patients. J Immunol 2011; 187(1):570-9.

36. Serpero LD, Filaci G, Parodi A, Battaglia F, Kalli F, Brogi D, et al. Fingolimod modulates peripheral effector and regulatory T cells in MS patients. J Neuroimmune Pharmacol 2013; 8(5):1106-13.

37. Sato DK, Nakashima I, Bar-Or A, Misu T, Suzuki C, Nishiyama S, et al. Changes in Th17 and regulatory T cells after fingolimod initiation to treat multiple sclerosis. J Neuroimmunol 2014; 268(1-2):95-8.

38. Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria. Ann Neurol 2011; 69(2):292-302.

39. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4):402-8.

40. Kowarik MC, Pellkofer HL, Cepok S, Korn T, Kumpfel T, Buck D, et al. Differential effects of fingolimod (FTY720) on immune cells in the CSF and blood of patients with MS. Neurology 2011; 76(14):1214-21.

41. Soliven B, Miron V, Chun J. The neurobiology of sphingosine 1-phosphate signaling and sphingosine 1-phosphate receptor modulators. Neurology 2011;76(Suppl3):S9-S14.

42. Chun J, Hartung HP. Mechanism of action of oral fingolimod (FTY720) in multiple sclerosis. Clin Neuropharmacol 2010; 33(2):91-101.

43. Rudnicka J, Czerwiec M, Grywalska E, Siwicka-Gieroba D, Walankiewicz M, Grafka A, et al. Influence of fingolimod on basic lymphocyte subsets frequencies in the peripheral blood of multiple sclerosis patients - preliminary study. Cent Eur J Immunol 2015; 40(3):354-9.

44. Peelen E, Thewissen M, Knippenberg S, Smolders J, Muris AH, Menheere P, et al. Fraction of IL-10+ and IL-17+ CD8 T cells is increased in MS patients in remission and during a relapse, but is not influenced by immune modulators. J Neuroimmunol 2013; 258(1-2):77-84.

45. Man K, Miasari M, Shi W, Xin A, Henstridge DC, Preston S, et al. The transcription factor IRF4 is essential for TCR affinity-mediated metabolic programming and clonal expansion of T cells. Nat Immunol 2013; 14(11):1155-65.

46. Raczkowski F, Ritter J, Heesch K, Schumacher V, Guralnik A, Höcker L, et al. The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8+ T cells. Proc Natl Acad Sci U S A 2013; 110(37):15019-24.

47. Yao S, Buzo BF, Pham D, Jiang L, Taparowsky EJ, Kaplan MH, et al. Interferon regulatory factor 4 sustains CD8(+) T cell expansion and effector differentiation. Immunity 2013; 39(5):833-45.

48. Wullschleger A, Kapina V, Molnarfi N, Courvoisier DS, Seebach JD, Santiago-Raber M-L, et al. Cerebrospinal fluid interleukin-6 in central nervous system inflammatory diseases. PloS one 2013; 8(8):e72399.

49. Krei K, Fredrikson S, Fontana A, Link H. Interleukin-6 is elevated in plasma in multiple sclerosis. J Neuroimmunol 1991; 31(2):147-53.

50. Chen Y-C, Yang X, Miao L, Liu Z-G, Li W, Zhao Z-X, et al. Serum level of interleukin-6 in Chinese patients with multiple sclerosis. J Neuroimmunol 2012; 249(1):109-11.

51. Fu S, Zhang N, Yopp AC, Chen D, Mao M, Chen D, et al. TGF-β Induces Foxp3 + T-Regulatory Cells from CD4 + CD25 − Precursors. Am J Transplant 2004; 4(10):1614-27.

52. Santarlasci V, Maggi L, Capone M, Frosali F, Querci V, De Palma R, et al. TGF-β indirectly favors the development of human Th17 cells by inhibiting Th1 cells. Eur J Immunol 2009; 39(1):207-15.

How to Cite
Laribi B, Sahraian MA, Shekarabi M, Emamnejad R, Marzban M, Sadaghiani S, Izad M. Characterization of CD4+ and CD8+ T Cell Subsets and Interferon Regulatory Factor 4 (IRF4) in MS Patients Treated with Fingolimod (FTY-720): A Follow-up Study. Iran J Allergy Asthma Immunol. 17(4):346-360.
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