Foxp3: Important regulatory gene for the development of regulatory T-cells

Regulatory T-cells overview:

All T-cells come from progenitor cells in the bone marrow, but their fate is determined in the thymus. Regulatory T-cells (Treg) suppress immune activities at the end of an immune reaction and also prevent autoimmunity.  Treg cells are able to suppress over enthusiastic immune responses.  It is very important to maintain an immune system response balance between the inflammatory responses and defense against self-reactivity (Zhang et al. 2007).  A disruption in this balance has been observed in many autoimmune diseases.  Treg cells play an essential role in maintaining this balance by suppressing effector T-cell activity (Zheng et al. 2007).

Natural Treg cells can be divided into 2 main groups, naturally occurring Tregs (n Tregs), and induced Tregs, which are derived from naïve T cells.

The most studied Tregs express the proteins CD4, CD25, and Fox3p. Naturally occurring Treg cells originate in the thymus and are different from other T-cells because they contain the FOXP3 protein (Watanabe et al. 2005).  CD4 and CD25 act as identification markers of the cell (Figure 1).

Regulatory T- cell

Figure 1: Figure 1 shows a T-cell and its various markers as well as the transcriptional repressor, FOXP3 (Milojevic. 2008).

Regulatory T-cell Development Stages:

Double Negative Stage – All T-cells are originally CD4-CD8-.  Individual cells will uniquely rearrange their T-cell receptor genes.  Cells thrive and begin to express both CD4 and CD8.

Double Positive Stage – The T-cells are now CD4+CD8+. Here cells begin transcription of FOXP3, and become Treg cells.

Single Positive Stage – The T-cells are now either CD4+ or CD8+.  Cells begin to express FOXP3 and are now functional Treg cells (Watanabe et al. 2005).

Video on T-cell Development

Natural and Inducible Treg cells

Figure 2: Figure 2 is a depiction of how natural regulatory T-cells mature in the thymus and express the cell-surface markers CD25, CD4 and the transcriptional repressor FOXP3 (Mills. 2004).

Regulatory protein (FOXP3) overview

FOXP3 is part of the FOX protein family, and is important in immune system reactivity.  FOXP3 works as a regulator in the development and function of regulatory T-cells.  This protein is a forkhead/winged-helix transcriptional regulator, and is encoded by the FOXP3 protein (National Institute of Health, 2012).

Function of FOXP3 in development of regulatory T-cells

The FOXP3 protein binds to regions of DNA, therefore changing the expression of immune system regulating genes.  Therefore, the FOXP3 protein is a transcription factor. FOXP3 is essential for the production and regular function of Treg cells, which play an important role in preventing self-reactivity (eBioscience.  2011).  The FOXP3 protein is found primarily in the thymus, where regulatory T cells are also primarily found (Shohei et. Al 2003).

Use of Epigenetic Modification to Induce FOXP3 Expression in Naïve T Cells

Hypothesis: “Epigenetic modification agents can induce FOXP3 expression, promoting the conversion of naïve T cells to Tregs” (Moon et al. 2009).

Because the Treg population is so small, but they play such a crucial role in immunity, numerous researchers have attempted to find a technique to convert naïve T-cells into Tregs.  Ex vivo induction of Tregs may be a useful therapeutic technique to develop tolerance in organ transplant patients (Moon et al. 2009).

Epigenetic modification can be done using DNA demethylation and histone protein acetylation of the FOXP3 gene locus.  This would induce FOXP3 expression in naïve T cells, which would cause them to convert to Treg cells (Moon et al.  2009).

Methods:

The spleens of multiple eight-week-old inbred albino mice were harvested.

Monocytes from the spleens were stained.

Isolated CD4+/CD25- T-cells were cultured in a nurturing environment.  TCRs were stimulated and 72 hours later, cells were either allowed to continue to propagate without treatment (control), or were treated with 5AzaD, a DNA-methyltransferase inhibitor, or TSA, a histone protein deacetylase (HDAC) inhibitor.

Flow cytometry was used to investigate the phenotypic purity of the isolated T cells.  FACS analysis was used to obtain data on expression levels of Treg markers.

Cells were stained, and the FOXP3 expression level in n Tregs, naïve T cells, and induced Tregs were measured by Western blot analysis.

The ability of the induced Treg cells to suppress the inflammatory immune response was assessed (Moon et al.  2009).

Results:

Treatment of naïve T cells with 5AzaD or TSA 72 hours after TCR stimulation produced cells that expressed the CD4+ /CD25+ / FOXP3+ phenotype of Treg cells.

These induced Tregs expressed greater amounts of the FOXP3 protein than either the control or the natural Tregs (Figure 3).

FOXP3 Expression

Figure3: Figure 3 shows FOXP3 expression in naïve T cells and in natural regulatory T cells (left). It also shows the expression of FOXP3 in naïve T cells treated with 5AzaD and TSA after TCR stimulation (Moon et al. 2009).

The induced Treg cells also demonstrated T-cell suppression.  In 5AzaD- or TSA treated cells, when the number of repressor cells was extremely high compared to responders, responder proliferation was much lower than compared to that of those in the absence of induced Treg cells.

In the absence of induced Treg cells, around 50% of responder cells proliferated among the groups treated with 5AzaD or TSA.  When the number of Treg cells was many times that on the responders, 1-7% of responders proliferated among these groups (Moon et al.  2009).

Conclusions:

These data demonstrate that epigenetic modification agents can prompt FOXP3 expression, bringing about the conversion of naïve T cells to Tregs.

Treatment of naïve T cells with 5AzaD or TSA 72 hours after TCR stimulation produced cells that expressed the CD4+ /CD25+ / FOXP3+ phenotype of Treg cells.  This suggested that 5AzaD and TSA caused epigenetic modifications that converted naïve T cells to FOXP3 carrying Tregs.

This way of epigenetically inducing FOXP3 expression is the first successful conversion of naïve T cells to functional regulatory T cells (Moon et al.  2009).

Strengths/Weaknesses:

This research was ground breaking in the formation of functional induced Treg cells from naïve T-cells.  Cultures were grown at optimal temperature and concentration for increased proliferation.

FOXP3 Mutations in mice:

Mutations of the FOXP3 gene can prevent regulatory T-cell development.  In mice, deletion of the FOXP3 gene is responsible for ‘Scurfy’, which is lethal in males with only a single copy of the gene.  These mice exhibit over proliferation of CD4+ T-cells.  Scurfy is thought to be caused by an inability to accurately regulate CD4+ T-cell activity (Shohei et. Al 2003).

FOXP3 Mutations

Figure 4: Mutations in the gene FOXP3, have been found in IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked syndrome) and XLAAD (X-Linked Autoimmunity-Allergic Disregulation) patients as well as scurfy mice. TReg cells inhibit CD4+ causing the prevention of these disorders (O’Garra et al. 2003).

Future:

Scientists hope that in the future, cell therapy with FOXP3 positive T-cells may lead to treatments or cures to autoimmune diseases like diabetes and asthma and aid during recovery from organ transplantation (T-cell Modulation Group.  2009).

Video about Regulatory T-cells in Disease

References:

Mills, Kingston H. G.  Regulatory T-cells: Friend of Foe in immunity to infection.  Nature Reviews Immunology 4:841-855.  2004.  Image

Milojevic, Diana et al.  Regulatory T-cells and their Role in Rheumatic diseases: a potential target for novel therapeutic development.  Pediatric Rheumatology 6(1).  2008.  Image

Moon C. et al.  Use of Epigenetic Modification to Induce FOXP3 Expression in Naïve T Cells.  Transplantation Proceedings (41): 1848–1854.  2009.  Full Text

O’Garra, Anne and Vieira, Paulo.  Twenty-first Century FOXP3.  Nature Immunology 4:304 – 306.  2003.  Full Text

Shohei Hori, Takashi Nomura, Shimon Sakaguchi.  Control of Regulatory T Cell Development by the Transcription Factor Foxp3. Science 299 (1057).  2003.  Full Text

Watanabe N, Wang YH, Lee HK, Ito T, Wang YH, Cao W, Liu YJ. “Hassall’s corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus”. Nature 436 (7054): 1181–5.  2005.  Full Text

Zhang L, Zhao Y. The regulation of Foxp3 expression in regulatory CD4(+)CD25(+)T cells: multiple pathways on the road. J. Cell. Physiol. 211 (3): 590–597.  2007.  Full Text

Zheng, Ye et al.  Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate.  Nature (463): 808-812. 2010. Full Text

Unknown.  2009.  Beginners Guide to T-cells.  T-cell Modulation Grouphttp://www.tcells.org/beginners/tcells/

Unknown.  2012.  FOXP3.  National Institute of Healthhttp://ghr.nlm.nih.gov/gene/FOXP3

Unknown.  2011.  FOXP3.  eBiosciencehttp://www.ebioscience.com/knowledge-center/antigen/foxp3.htm

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