The fate of regulatory T cells: survival or apoptosis

The fate of regulatory T cells: survival or apoptosis

1_{Department of Medical Biotechnology and Laboratory Science and Graduate}

Institute of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.

2_{National Institute of Infectious Diseases and Vaccinology, National Health Research}

Institutes, Miaoli, Taiwan.

3_{Graduate Institute of Immunology, China Medical University, Taichung, Taiwan.}

Corresponding to: Hsin-Wei Chen, No. 35, Keyan Road, Zhunan Town, Miaoli

Foxp3+ _{regulatory T (Treg) cells are unique in their immunosuppressive abilities and}

contribute to immune regulation. However, the homeostatic processes and survival programs that maintain the Treg population remain unclear. Here we highlight the recent study by Liston et al.1_{, who have dissected the regulatory mechanisms of Treg} homeostasis and survival. By utilizing the transgenic models, they provided evidence to support that peripheral Treg cells are able to alter their proliferative and apoptotic rates to restore numerical deficit rapidly through both the interleukin 2 and costimulation-dependent pathways.

Regulatory T cells (Tregs) are characterized by their capabilities in modulating immune responses and can usually be identified with the specific expression of Foxp3, the transcription factor which endows T cells with regulatory functions2, 3_. Tregs are critical and indispensible for maintaining peripheral tolerance and there's an active balance between the regulatory and effective immune responses under the steady state. Deterioration of the balance between regulatory and effective immune responses could lead to the induction of several kinds of diseases. For instance, the effective immune responses are often hindered by the excessive numbers of Tregs in the tumor microenvironment4 _{whereas the decline of the numbers and functionalities} of Tregs are seen in many autoimmune diseases or inflammatory conditions5, 6, 7_.

Given that Tregs are postulated valuable target for immune therapies against tumor and autoimmune diseases, it is therefore an urgent need to understand both the cellular and molecular mechanisms contributing to Tregs homeostasis. Unlike effector T cells, Tregs are considered a group of anergic and quiescent cell supported by in vitro studies8_{. On the contrary, Tregs undergo homeostatic expansion and proliferate} vigorously especially in lymphopenic host9_{, suggesting that they are vivacious under} such circumstances and an intact immune system may help guarding Treg homeostasis, and vice versa.

Programmed cell death (apoptosis) is the predominant underlying mechanism for maintaining T cell homeostasis10, 11_{. Immune responses against foreign antigens start} robustly with effector T cell activation and proliferation, and then the population of activated T cells are restrained and controlled through apoptosis to circumvent excessive immune responses. Apoptosis itself is a delicate process and it can be initialized by either extrinsic or intrinsic signaling cascade12_{. Bcl-2 protein family} including Bcl-2, Bcl-XL, Mcl-1, Blf-1 and A1 are antiapoptotic proteins and they are able to suppress the activation of apoptotic regulators, Bax and Bak. On the other hand, antiapoptotic Bcl-2 protein family can be antagonized by pro-apoptotic BH3-only proteins including Bim, Bik, Puma, and Bad13_{. In T cells, the reliance of the}

apoptotic-related proteins varies in different developmental stages14, 15, 16, 17_.

The regulation of Treg homeostasis and survival is dissected by a study recently reported in Nature Immunology by Liston et al.. They utilized transgenic model to study the cellular and molecular mechanisms contributing to Tregs population homeostasis1_{. To assess if the Treg population is stable and quiescent, they} administrated BrdU to identify the proliferative profile of Tregs in vivo. They found that Tregs actually proliferate more rapidly than conventional T cells under static condition indicating that Tregs population is dynamic. By using transgenic female mice heterozygous for Thy1.1 and DTR (diphtheria toxin receptor) in the Foxp3 locus of X chromosome (Foxp3DTR/Thy1.1_{), the authors demonstrated that Thy1.1}+ _Tregs (which constitutes 50% of the Tregs population due to X chromosome inactivation) proliferate vigorously and fulfill the Treg population shortly after DTR+ _{Tregs were} removed by DT administration. Signals provided by IL-2 are essential for maintenance of Tregs in the periphery and IL-2 appears to be critical during the niche-filling process due to IL-2 acts as a mediator along with co-stimulatory signals to stimulate the proliferation and to reduce the apoptosis of Tregs.

proliferative rate was inversely correlated with apoptotic rate during the niche-filling process, the authors further substantiated the roles of apopotosis in Tregs homeostasis. Specifically knockout Bak and Bax in Tregs resulted in peripheral accumulation of Tregs in the host, indicating that integral intrinsic apoptosis pathway is required for peripheral Tregs homeostasis. The interactions between anti-apoptotic proteins, pro-apoptotic proteins and apoptosis regulators determine the fate of cells. Among the anti-apoptotic proteins, the role of Bcl-2 and Bcl-XL in Tregs homeostasis was ruled out via hematopoietic reconstitution and specific knockout, respectively. In addition, the authors utilized fate-mapping strategy by crossing mice knocked in human CD4 as a reporter to one anti-apoptotic Mcl-1 allele flanked with loxp sequence with mice bearing CD127 promoter drove Cre recombinase (CD127Cre_Mcl1CD4/+_{), resulting in}

human CD4 along with Mcl-1 expression in all T subsets to understand the importance of Mcl-1 during T cell development. Higher levels of Mcl-1 expression were identified in DP (CD4+_CD8+ _{double positive) T cells and in CD4}+_Foxp3+ _{T cells} but not in CD8+ _{or CD4}+_Foxp3- _{T cells. The host with specific ablation of Mcl-1 in} Tregs forfeited immune homeostasis implicated by decreased Tregs population and the development of scurfy-phenotype. Furthermore, the essential role of Mcl-1 for Treg survival was assessed by hematopoietic chimeras. Mcl-1 or Bcl-2 was ablated via tamoxifen-inducible Cre in 50% of the bone marrow cell transferred, and only the

Tregs population without functional Mcl-1 dropped soon after tamoxifen administration, strongly suggesting that Mcl-1 is the dominant anti-apoptotic member in Tregs in maintaining Tregs homeostasis.

Pro-apoptotic BH3-only proteins are able to antagonize the anti-apoptotic effects of anti-apoptotic bcl-2 family proteins13_{. Elevated percentage of CD4}+_Foxp3+ _{T cells was} identified in mice with Tregs specifically ablated Bim though the increment is not as obvious as that in mice with Bax and Bak double knockout Tregs. IL-2 is crucial for maintaining Tregs especially in periphery. To substantiate the correlation between IL-2 and Mcl-1 expression, the authors first crossed CD1IL-27Cre_Mcl1CD4/+ _{with FoxP3}DTR/+

and found Mcl-1 expression raised during homeostatic expansion of Tregs. Since serum IL-2 as well as IL-2 secreting CD4+ _{T cells increased during the process, IL-2} plus anti-IL-2 antibody complex, usually causes rapid expansion of Tregs, was introduced to CD127Cre_Mcl1CD4/+ _{mice. Mcl-1 expression (as was reflected by human}

CD4 expression) increased specifically in Tregs population receiving IL-2 plus IL-2 antibody complex.

To sum up, Tregs proliferate when their homeostatic population is disturbed through an IL-2 and co-stimulatory signal dependent mechanism, which in turn

down-regulates apoptosis and facilitates the restoration of Tregs population. In addition, Tregs population is constrained through Bax and Bak mediated intrinsic apoptosis pathway. Moreover, molecular mechanisms involved in the progress including the signals provided by IL-2, which elevates anti-apoptotic Mcl-1 expression during expansion while Bim is the major antagonist of Mcl-1 in Tregs.

In steady state, Tregs are a stable but dynamic population owing to their high turnover rate. However, Tregs tend to loss their suppressive function under inflammatory/ disease conditions. Take multiple sclerosis for example, Treg/TH17 ratio decreased in patients with MS and the ratios were negatively correlated with disease severity18_{. The} importance of cytokines is also implied in the study demonstrated by Liston et al.. Neutralizing IL-2 by antibodies cannot completely antagonize Tregs homeostatic expansion1_{. Their results suggested that cytokines other than IL-2 may synchronize} with the co-stimulatory signals to sustain Tregs homeostatic expansion and population. Indeed, TGF-β, IL-35 and IL-4 have also been proved related to Tregs homeostasis19, 20, 21_{. It is therefore important to take all the factors related to Tregs} homeostasis including cytokines, co-stimulatory signals and molecular balance between pro- and anti-apoptotic responses into consideration to achieve better effects while designing Treg based therapeutic strategies.

Acknowledgments

Disclosure: The authors declare that they have no conflicts of interest and

acknowledge the financial support of grants from the National Health Research Institutes, National Science Council 101-2320-B-182-027-MY3 and Chang Gung Memorial Hospital (CMRPD3B0052 and BMRP440). CRS and WCY contribute equally in this work.

Figure 1. A proposed regulatory mechanism of Treg homeostasis and survival.

In steady state, the interactions between pro-apoptotic Bim, anti-apoptotic Mcl-1 and apoptotic-regulator Bax and Bak actively maintains the homeostatic population of Tregs. When the homeostatic population of Tregs is disturbed, signals providing by IL-2 along with co-stimulatory signal up-regulate anti-apoptotic Mcl-1 expression, which in turn inhibit Bak and Bax mediated intrinsic apoptosis pathway and subsequently allows Tregs to proliferate during the niche-filling process.

Treg

Numeric deficit

Naïve T cells

Effector T

cells

Disturb

Treg

population

Niche filling: Apoptosis↓ Proliferation↑

Bi

m

Mcl-1

Ba

x

Ba

k

Apopt

osis

(Suppressive capabilities ↓)

IL

-2

IL-2

1. Pierson W, Cauwe B, Policheni A, Schlenner SM, Franckaert D, Berges J, et al. Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3+ _{regulatory T cells. Nature immunology 2013; 14: 959-965.}

2. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+_CD25+ _{regulatory T cells. Nature immunology 2003; 4:} 330-336.

3. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299: 1057-1061.

4. Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer 2010; 127: 759-767.

5. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of functional suppression by CD4+_CD25+ _{regulatory T cells in patients with multiple} sclerosis. The Journal of experimental medicine 2004; 199: 971-979.

6. Wan S, Xia C, Morel L. IL-6 produced by dendritic cells from lupus-prone mice inhibits CD4+_CD25+ _{T cell regulatory functions. J Immunol 2007; 178: 271-279.} 7. Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M,

Ashby M, et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nature immunology 2009;

10: 1000-1007.

8. Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, et al. Immunologic self-tolerance maintained by CD25+_CD4+ _{naturally anergic and} suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol 1998; 10: 1969-1980.

9. Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, Barbosa TC, Cumano A, Bandeira A. CD25+_CD4+ _{T cells regulate the expansion of peripheral CD4 T cells} through the production of IL-10. J Immunol 2001; 166: 3008-3018.

10. Shi Y, Radvanyi LG, Sharma A, Shaw P, Green DR, Miller RG, et al. CD28-mediated signaling in vivo prevents activation-induced apoptosis in the thymus and alters peripheral lymphocyte homeostasis. J Immunol 1995; 155: 1829-1837.

11. Swat W, Ignatowicz L, von Boehmer H, Kisielow P. Clonal deletion of immature CD4+₈+ _{thymocytes in suspension culture by extrathymic antigen-presenting} cells. Nature 1991; 351: 150-153.

12. Kurokawa M, Kornbluth S. Caspases and kinases in a death grip. Cell 2009;

138: 838-854.

13. Huang DC, Strasser A. BH3-Only proteins-essential initiators of apoptotic cell death. Cell 2000; 103: 839-842.

14. Bouillet P, O'Reilly LA. CD95, BIM and T cell homeostasis. Nat Rev Immunol 2009; 9: 514-519.

15. Hernandez JB, Newton RH, Walsh CM. Life and death in the thymus--cell death signaling during T cell development. Curr Opin Cell Biol 2010; 22: 865-871.

16. Hildeman D, Jorgensen T, Kappler J, Marrack P. Apoptosis and the homeostatic control of immune responses. Curr Opin Immunol 2007; 19: 516-521.

17. Marsden VS, Strasser A. Control of apoptosis in the immune system: Bcl-2, BH3-only proteins and more. Annu Rev Immunol 2003; 21: 71-105.

18. Jamshidian A, Shaygannejad V, Pourazar A, Zarkesh-Esfahani SH, Gharagozloo M. Biased Treg/Th17 balance away from regulatory toward inflammatory phenotype in relapsed multiple sclerosis and its correlation with severity of symptoms. J Neuroimmunol 2013: in press.

19. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral CD4+_CD25- _{naive T cells to CD4}+_CD25+ _{regulatory T cells by} TGF-beta induction of transcription factor Foxp3. The Journal of experimental

20. Collison LW, Chaturvedi V, Henderson AL, Giacomin PR, Guy C, Bankoti J, et al. IL-35-mediated induction of a potent regulatory T cell population. Nature

immunology 2010; 11: 1093-1101.

21. Dorsey NJ, Chapoval SP, Smith EP, Skupsky J, Scott DW, Keegan AD. STAT6 Controls the Number of Regulatory T Cells In Vivo, Thereby Regulating Allergic Lung Inflammation. J Immunol 2013; 191: 1517-1528.