A new mechanism of regulatory T cell generation
to suppress excessive immune responses―Non-coding DNA sequences indispensable for immune tolerance were discoveredーKeyword：Regulatory T cell (Treg)、Foxp3、Non-coding DNA sequences、Autoimmune disease

Key points
 IFReC team identified non-coding DNA sequences*1 indispensable for regulatory T cell (Treg) *2generation.
 These DNA sequences are distributed into the two separated regions near the Foxp3 gene*3 locus, themaster transcription factor of Tregs. Defect of these elements developed severe autoimmune-diseases*4.
 Controlling the development and function of Tregs holds promise for the new therapies for
immunological diseases.

AbstractIFReC Experimental Immunology research team (Shimon Sakaguchi Lab.) investigated thegenomic regions related to Treg development in the thymus. As a result, they found specific DNA
elements indispensable for Treg development and the maintenance of immune homeostasis.

Background
The immune system maintains immune tolerance and prevents excessive immune response totolerable antigens such as self-derived molecules, foods, and symbiotic bacterial antigens. Regulatory
T cells (Tregs) are one of the most significant cells in the maintenance of immune tolerance. Tregs work
to maintain homeostasis of the body in various situations such as prevention of autoimmune diseases,
termination of immune responses and damaging tissue repairing, by braking excessive immuneresponses.
Tregs are generated from a portion of antigen-stimulated CD4+ T cells. The transcription factor
Foxp3 is important for the suppressive function. The detailed mechanisms of Treg generation in theepigenetic and physical three-dimensional chromatin contexts are largely unknown.

1The details of the present study
Treg cells are differentiated from CD4-positive T cells that encounter antigens in the thymus andperiphery; e.g. intestinal tissues. Large numbers of Tregs are produced in the thymus during childhood.
In order to clarify the developmental mechanism of Tregs, the research group used next-generation DNA
sequencing technology*5 to investigate the DNA regions that are activated during the differentiation ofimmature cells into Tregs in the mouse thymus.
As a result, two DNA elements were found as activated regions from the early stage of T celldifferentiation scattered around the Foxp3 gene region, which is a transcription factor important for thedifferentiation and function of regulatory T cells. These two regions are located on the X chromosome
and are small DNA regions of 400 base pairs and 200 base pairs, respectively, called the CNS0 andCNS3 regions. Importantly, CNS0 and CNS3 are non-coding DNA regions that do not have any
sequence information for protein translation. However, the research team hypothesized that CNS0 andCNS3 may be involved in the development of regulatory T cells due to significant conservation of thesesequences was found in mammalian genomes including humans.

In this study, mice lacking either CNS0 or CNS3, or both, were generated using a genome-editing
technology called the CRISPR/Cas9 system*6, and the effects on the differentiation and function ofregulatory T cells were examined. Mice lacking only one of CNS0 or CNS3 showed only a slight decrease
in regulatory T cells, but the individuals developed normally. On the other hand, mice lacking both of thetwo sites simultaneously failed to differentiate into normal regulatory T cells in the thymus and developed
serious autoimmune diseases in various organs of the body. These experiments demonstrated that theregulation of Foxp3 expression through non-coding DNA regions, plays an important role in thedevelopment of Treg cells and the establishment of immune tolerance.
CNS0 and CNS3 regions each harbored DNA sequences that were recognized by various nuclear
proteins involved in regulatory T cell differentiation and function. However, these protein species did notnecessarily overlap, suggesting that they are not functionally redundant. Furthermore, these regions areindependently activated during T cell differentiation and form steric interactions between DNA regions
around the Foxp3 gene. These results collectively suggest that CNS0 and CNS3 regions physicallyinteract with the promoter region of the Foxp3 gene during the differentiation of regulatory T cells thereby
control Foxp3 gene expression.

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Significance of this study
In many cases, there is no universal prevention or treatment regimens for diseases that damage
the body by hyper-activation of the immune system, such as autoimmune diseases and new coronavirusrelated diseases that are currently prevalent worldwide. This study revealed the significance of DNA
elements for the function and development of Treg cells, which is a major step forward in understanding
the mechanism of their development. This will enable us to develop methods to convert over-active T
cells into Treg cells or create an environment in the body that facilitates Treg generation, which leads tothe development of fundamental prevention and treatment methods for various immune diseases.
These results were published in an Elsevier's journal Immunity in collaboration with the Institute for
Frontier Life and Medical Sciences, Kyoto University and Stanford University.

Terminology＊１： Non-coding sequence
Genomic DNA regions that are not used as information for protein translation. They occupy most of
the total eukaryotic genome although the majority of their functions are unclear.
＊２： Regulatory T cell; Treg
A subset of CD4-positive helper T cell that specializes in immunosuppression.
＊３： Foxp3 geneA transcription factor that is important for the function of regulatory T cells. It controls the expressionof various genes and provides T cells with immunosuppressive properties.
＊４： Autoimmune disease
A term for various diseases that occur when the immune system misrecognizes to attack self.
＊５： Next-generation DNA sequencing technology
Recently advanced high-throughput DNA sequencing technologies that enable to rapid andcomprehensively obtain information on the complete nucleotide sequence of a particular species'
genome and gene expression.
＊６： Genome-editing
A technology that targets and modifies specific DNA sequences in the genome.

Publication
Title: “Distinct Foxp3 enhancer elements coordinate development, maintenance and function ofregulatory T cells”
Authors: Ryoji Kawakami*, Yohko Kitagawa*, Kelvin Y. Chen, Masaya Arai, Daiya Ohara, Yamami
Nakamura, Keiko Yasuda, Motonao Osaki, Norihisa Mikami, Caleb A. Lareau, Hitomi Watanabe, GenKondoh, Keiji Hirota, Naganari Ohkura, and Shimon Sakaguchi**(*; contributed equally, **; corresponding)
Immunity Apr 29, 2021
DOI:https://doi.org/10.1016/j.immuni.2021.04.005

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Acknowledgements
This achievement is a part of the "Study of the function and development of regulatory T cells." Specially
Promoted Research of the Japan Society for the Promotion of Science (JSPS) and the "Leading
Advanced Projects for medical innovation" of the Japan Agency for Medical Research and Development(AMED).

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