Immune Netw.  2015 Aug;15(4):199-205. 10.4110/in.2015.15.4.199.

Anti-proliferative Activity of T-bet

• Oh YJ
• Shin JH
• Won HY
• Hwang ES

• KMID: 2168047
• DOI: http://doi.org/10.4110/in.2015.15.4.199

Abstract

T-bet is a critical transcription factor that regulates differentiation of Th1 cells from CD4+ precursor cells. Since T-bet directly binds to the promoter of the IFN-gamma gene and activates its transcription, T-bet deficiency impairs IFN-gamma production in Th1 cells. Interestingly, T-bet-deficient Th cells also display substantially augmented the production of IL-2, a T cell growth factor. Exogenous expression of T-bet in T-bet deficient Th cells rescued the IFN-gamma production and suppressed IL-2 expression. IFN-gamma and IL-2 reciprocally regulate Th cell proliferation following TCR stimulation. Therefore, we examined the effect of T-bet on Th cell proliferation and found that T-bet deficiency significantly enhanced Th cell proliferation under non-skewing, Th1-skewing, and Th2-skewing conditions. By using IFN-gamma-null mice to eliminate the anti-proliferative effect of IFN-gamma, T-bet deficiency still enhanced Th cell proliferation under both Th1- and Th2-skewing conditions. Since the anti-proliferative activity of T-bet may be influenced by IL-2 suppression in Th cells, we examined whether T-bet modulates IL-2-independent cell proliferation in a non-T cell population. We demonstrated that T-bet expression induced by ecdysone treatment in human embryonic kidney (HEK) cells increased IFN-gamma promoter activity in a dose dependent manner, and sustained T-bet expression considerably decreased cell proliferation in HEK cells. Although the molecular mechanisms underlying anti-proliferative activity of T-bet remain to be elucidated, T-bet may directly suppress cell proliferation in an IFN-gamma- or an IL-2-independent manner.

Keyword

T-bet; Proliferation; IFN-gamma; IL-2; Ecdysone; Th cells

MeSH Terms

Animals
Cell Proliferation
Ecdysone
Humans
Interleukin-2
Kidney
Mice
Th1 Cells
Transcription Factors
Ecdysone
Interleukin-2
Transcription Factors

Figure

• 

  Figure 1 Effect of T-bet on Th cell proliferation. CD4+ Th cells were isolated from WT and T-bet KO mice in the absence (-, A) or presence (+, B) of exogenous rhIL-2 and stimulated with different amounts of anti-CD3 under non-skewing (NS) conditions for 3 days. Cells were seubsequently incubated with 3H-thymidine and subjected to liquid scintillation counting using Micro Beta and TopCount equipment (Perkin Elmer). Y-axes represent count per minute (CPM). Three independent experiments were performed using 6 mice for each experiment. Data are expressed as the average ±SD of 6 mice. *p<0.05, **p<0.005.

• 

  Figure 2 Effect of T-bet on Th1 and Th2 cell proliferation. CD4+ Th cells isolated from WT and T-bet KO mice were activated using anti-CD3 and differentiated into Th1 and Th2 cells in the presence (+) or absence (-) of exogenous rhIL-2. Developing Th1 and Th2 cells were incubated with 3H-thymidine to determine cell proliferation rate. Y-axes represent count per minute (CPM). A total of 6 mice per group were analyzed and data are presented as the average ±SD of 3 experiments. *p<0.05, **p<0.005, ***p<0.0005.

• 

  Figure 3 IFN-γ independence of T-bet's anti-proliferation activity. CD4+ Th cells were isolated from IFN-γ KO mice and IFN-γ/T-bet double KO mice and stimulated with anti-CD3 in the either presence (+) or absence (-) of exogenous rhIL-2. Cells were cultured for an additional 2 to 3 days under Th1-skewing (A) or Th2-skewing (B) conditions. Cell proliferation rates of developing Th cells from either T-bet WT or T-bet KO mice established IFN-γ-deficient background were determined using a thymidine incorporation assay. A total of 5 mice per group were used for analysis and data are expressed as the average ±SD. **p<0.005, ***p<0.0005.

• 

  Figure 4 IL-2 suppression and IFN-γ induction by T-bet in Th cells. Spleens and lymph node tissues were collected from WT and DTg/KO mice and CD4+ Th cells were isolated. Isolated Th cells were stimulated with anti-CD3 (2 µg/mL) in the either presence (+) or absence (-) of doxycycline (0.5 µg/mL) for 3 days. (A) Cell pellets were harvested and subjected to protein analysis by immunoblotting with Ab against T-bet or actin. (B) Cell supernatants were harvested for IL-2 and IFN-γ analysis by ELISA. Five independent experiments were performed and data are expressed as the average ±SD for 5 mice per group. *p<0.05; ***p<0.0005.

• 

  Figure 5 Ecdysone-inducible expression of functional T-bet in non-T cells. EcR-HEK cells were transiently transfected with T-bet expression vector and subsequently treated with PonA for 24 hours. (A) Whole cell protein extracts were resolved by SDS-PAGE and subjected to immunoblotting with Ab against Flag, T-bet (4B10), or actin. (B) EcR-HEK cells were transiently transfected with T-bet expression vector together with pIFN-γ-luciferase reporter gene and pCMVβ as an internal control. After treatment with different concentrations of PonA, cells were harvested for determination of luciferase activity. At least three independent reporter assays were performed and data are given as the average ±SD. ***p<0.0005.

• 

  Figure 6 Suppression of cell proliferation by T-bet. Stable T-bet expressing cell clones were established in EcR-HEK cells and maintained in DMEM containing G418 and Zeocin. (A) Stable cells were treated with PonA for 24 h. Protein extracts were prepared from each stable cell clone and analyzed by immunoblotting with the T-bet (4B10) Ab. (B) Stable cell clones were cultured with or without PonA at the indicated concentrations. Cells were counted by Trypan blue exclusion assay every 2 days in triplicate. Data are expressed as the average±SD from 3 separate experiments. ns, not significant; *p<0.05; **p<0.005; ***p<0.0005.

Reference

1. Lazarevic V, Glimcher LH, Lord GM. T-bet: a bridge between innate and adaptive immunity. Nat Rev Immunol. 2013; 13:777–789.

2. Hoyler T, Connor CA, Kiss EA, Diefenbach A. T-bet and Gata3 in controlling type 1 and type 2 immunity mediated by innate lymphoid cells. Curr Opin Immunol. 2013; 25:139–147.

3. Miller SA, Weinmann AS. Molecular mechanisms by which T-bet regulates T-helper cell commitment. Immunol Rev. 2010; 238:233–246.

4. Chen Z, Lin F, Gao Y, Li Z, Zhang J, Xing Y, Deng Z, Yao Z, Tsun A, Li B. FOXP3 and RORgammat: transcriptional regulation of Treg and Th17. Int Immunopharmacol. 2011; 11:536–542.

5. Ziegler SF, Buckner JH. FOXP3 and the regulation of Treg/Th17 differentiation. Microbes Infect. 2009; 11:594–598.

6. Liao W, Lin JX, Leonard WJ. IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol. 2011; 23:598–604.

7. Lan RY, Selmi C, Gershwin ME. The regulatory, inflammatory, and T cell programming roles of interleukin-2 (IL-2). J Autoimmun. 2008; 31:7–12.

8. Letourneau S, Krieg C, Pantaleo G, Boyman O. IL-2- and CD25-dependent immunoregulatory mechanisms in the homeostasis of T-cell subsets. J Allergy Clin Immunol. 2009; 123:758–762.

9. Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell. 2000; 100:655–669.

10. Lighvani AA, Frucht DM, Jankovic D, Yamane H, Aliberti J, Hissong BD, Nguyen BV, Gadina M, Sher A, Paul WE, O'Shea JJ. T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells. Proc Natl Acad Sci U S A. 2001; 98:15137–15142.

11. Mullen AC, High FA, Hutchins AS, Lee HW, Villarino AV, Livingston DM, Kung AL, Cereb N, Yao TP, Yang SY, Reiner SL. Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science. 2001; 292:1907–1910.

12. Finotto S, Neurath MF, Glickman JN, Qin S, Lehr HA, Green FH, Ackerman K, Haley K, Galle PR, Szabo SJ, Drazen JM, De Sanctis GT, Glimcher LH. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science. 2002; 295:336–338.

13. Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, Glimcher LH. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science. 2002; 295:338–342.

14. Werneck MB, Lugo-Villarino G, Hwang ES, Cantor H, Glimcher LH. T-bet plays a key role in NK-mediated control of melanoma metastatic disease. J Immunol. 2008; 180:8004–8010.

15. Hwang ES, Szabo SJ, Schwartzberg PL, Glimcher LH. T helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science. 2005; 307:430–433.

16. Park JW, Min HJ, Sohn JH, Kim JY, Hong JH, Sigrist KS, Glimcher LH, Hwang ES. Restoration of T-box-containing protein expressed in T cells protects against allergen-induced asthma. J Allergy Clin Immunol. 2009; 123:479–485.

17. Lee K, Min HJ, Jang EJ, Hong JH, Hwang ES. In vivo tumor suppression activity by T cell-specific T-bet restoration. Int J Cancer. 2010; 127:2129–2137.

18. Hwang ES, Hong JH, Glimcher LH. IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508. J Exp Med. 2005; 202:1289–1300.

19. Lazarevic V, Chen X, Shim JH, Hwang ES, Jang E, Bolm AN, Oukka M, Kuchroo VK, Glimcher LH. T-bet represses T(H)17 differentiation by preventing Runx1-mediated activation of the gene encoding RORgammat. Nat Immunol. 2011; 12:96–104.

20. Koch MA, Thomas KR, Perdue NR, Smigiel KS, Srivastava S, Campbell DJ. T-bet(+) Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor beta2. Immunity. 2012; 37:501–510.

21. McPherson RC, Turner DG, Mair I, O'Connor RA, Anderton SM. T-bet Expression by Foxp3(+) T Regulatory Cells is Not Essential for Their Suppressive Function in CNS Autoimmune Disease or Colitis. Front Immunol. 2015; 6:69.

22. Hwang ES. Transcriptional regulation of T helper 17 cell differentiation. Yonsei Med J. 2010; 51:484–491.

23. Jang EJ, Park HR, Hong JH, Hwang ES. Lysine 313 of T-box is crucial for modulation of protein stability, DNA binding, and threonine phosphorylation of T-bet. J Immunol. 2013; 190:5764–5770.

24. Oh S, Hwang ES. The role of protein modifications of T-bet in cytokine production and differentiation of T helper cells. J Immunol Res. 2014; 2014:589672.

25. Zamorano J, Wang HY, Wang R, Shi Y, Longmore GD, Keegan AD. Regulation of cell growth by IL-2: role of STAT5 in protection from apoptosis but not in cell cycle progression. J Immunol. 1998; 160:3502–3512.

26. Ben-Sasson SZ, Le GG, Conrad DH, Finkelman FD, Paul WE. IL-4 production by T cells from naive donors. IL-2 is required for IL-4 production. J Immunol. 1990; 145:1127–1136.

27. Hwang ES, White IA, Ho IC. An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines. Proc Natl Acad Sci U S A. 2002; 99:13026–13030.