Regulatory T Cells: Function of CD4, CD25 T Cells, and FOXP3 Treg Cells

September 2021

The human immune system is made up of many different cells–some of which are responsible for identifying unfamiliar pathogens, some of which recognize familiar pathogens, and some that attack anything harmful to the host. There are many moving parts that must cooperate to properly defend against foreign particles and diseases. These interactions between cells and pathogens are maintained and controlled by specialized lymphocytes called regulatory T cells.

What Are Regulatory T Cells?

Regulatory T Cells, or Tregs, are T cells that control or suppress other cells in the immune system. Treg cells play a vital role in the upkeep of peripheral immunities to tumors and pathogens. By regulating other cells, Tregs combat harmful foreign particles while simultaneously preventing the body from attacking itself.

Individuals or animals without regulatory T cells can experience dangerous autoimmune diseases in which the body attacks its own cells. Studying regulatory T cells can provide insight into the treatment of autoimmune diseases and potentially some forms of cancer. Tumors are the result of unregulated cell growth and reproduction. Tregs regulate everything from immune cell growth to function, which could help limit this dangerous and unmoderated cell proliferation.

A regulatory T cell’s function is mainly dictated by Treg cytokines and its surface molecules. The function of a Treg is decided when Treg differentiation occurs.

Treg Differentiation

There are two primary categories of regulatory T cells–Natural Tregs and adaptive Tregs. As a regulatory T cell matures, it is exposed to certain antigens in the body that determine its function and specificities based on the co-stimulation that occurs. Treg cell development that occurs within the thymus produces natural T cells. Adaptive Tregs are differentiated from naïve T cells in the periphery outside of the thymus. Both cells contain the same fundamental components and strive to prevent the immune system from overreacting.

Components of Regulatory T Cells

To qualify as a healthy regulatory T cell, a cell must express the CD4 T cell co-receptor, the IL-2 component CD25, and nuclear transcription factor Forkhead box P3 (FOXP3). Tregs are always CD4+ and CD25+, with the added help of FOXP3 to determine specific development and function. Although a variety of other surface molecules can be found on Tregs, and these components can be found elsewhere on other cells, the presence of these three components is necessary for a regulatory T cell to function properly.

CD4 T Cells

The CD4 T cell co-receptor is best known for proliferating into CD4+ helper T cells. These CD4+ cells communicate with other white blood cells to fight off harmful pathogens and tailor the immune response to specific diseases. Natural CD4+ Tregs that express CD25 are capable of regulating CD4+ and CD8+ cells, also known as effector cells. CD8+ cytotoxic T cells are primarily tasked with seeking out and eliminating harmful pathogens. With the help of other proteins and co-receptors, naturally occurring CD4+ Tregs can signal both helper cells and cytotoxic cells to activate or deactivate depending on the desired immune response.

This ability creates an immune response that can be catered to fight off unfamiliar pathogens without causing damage to the host. CD8+ T cells can be especially dangerous if not suppressed after carrying out their function. Without the CD25 surface molecule, effector T cells could cause a significant amount of damage.

CD25 T Cells

CD25 is the alpha chain of interleukin-2 (IL-2), a polypeptide chain widely responsible for T cell growth and proliferation. This specific co-receptor is highly expressed on both activated effector T cells and Treg cells. In regulatory T cells, CD25 mediates survival and induces the suppression of other T cells through the secretion of IL-2. IL-2 can act as both a catalyst for cell growth as well as a suppressor. Leveraging this protein is how natural Tregs can increase and decrease the immune response as needed.

CD25 is also expressed by a multitude of other cells. This could suggest that a variety of cells contribute to the manipulation of effector T cell homeostasis. More research is needed to understand the complex mechanisms of IL-2 and CD25. However, in the scope of regulatory T cells, CD25 is a necessary and sufficient component when it comes to activating and suppressing other T lymphocytes.

FOXP3 Treg Cells

Forkhead box P3, also called FOXP3, acts as a specific marker for regulatory T cells that is both necessary and sufficient for them to carry out their suppressive functions. FOXP3 Treg functions as a nuclear transcription factor, which means that it helps control the rate at which DNA can be transcribed to messenger RNA. This makes it an essential part of moderating the antibody-antigen relationship with vital host cells. Both humans and mice suffer from life-threatening auto-immune diseases when they experience a mutation in their FOXP3 gene. For humans, the disease is called immune dysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome, or IPEX for short, and involves self-reactive lymphocytes that attack the host’s immune system. FOXP3 being identified as a Treg transcription factor has progressed research on the development and function of Treg cells.

What Do Regulatory T Cells Do?

Naturally occurring CD4+CD25+ regulatory T cells play a vital role in suppressing immune functions and controlling the action of other immune cells. Tregs also allow the immune system to learn specific protocols and build up a tolerance against familiar pathogens. The ability of Tregs to identify a threat and signal other effector cells allows the body to perform a swift and specific treatment strategy.

In studies performed with mice that lacked natural CD4+CD25+ regulatory T cells, the mice developed autoimmune and inflammatory diseases. These issues could be easily solved by the reintroduction of natural CD4+CD25 Treg cells.

Regulatory T Cells and Immune Tolerance

Perhaps the most important function of regulatory T cells is to maintain unresponsiveness to self-antigens and suppress excessive immune response. When a foreign pathogen enters the host, a variety of different T lymphocytes will spring into action to attack the infected cells or unknown particles. Once the problem is eliminated, those effector cells would continue to attack and destroy other valuable cells if not for Tregs. Treg cells signal other cells to cease functioning in an effort to protect fragile systems in the human body.

Regulatory T Cell Isolation

With Tregs playing such a pivotal role in dictating our immune systems, they can be of great benefit to cell researchers. Studying Treg cells allows scientists to identify key processes in the way we respond to harmful pathogens, diseases, and tumors. To properly study regulatory T cells, they must be purified and isolated into a homogenous cell population.

Though this can be done in a variety of ways, it is important to choose a cell separation method that works quickly, effectively and maintains cell health and physiology. Traditional methods such as flow cytometry and magnetic-based cell isolation require expensive equipment that must be stored and properly maintained. These cell sorting cycles can also take long amounts of time and require multiple attempts for large quantities. Not to mention that the harsh physical and magnetic forces involved in sortation can damage the cell membranes resulting in cell death. This cell death could lead to inaccurate downstream results.

Leveraging Innovative Microbubble Technology from Akadeum Life Sciences for Regulatory T Cell Isolation

Because current isolation techniques can be time-consuming and induce stress on cells, the development of a gentle and effective method to obtain a high purity of a small target cell population for downstream analysis is of the utmost importance.

Akadeum’s Buoyancy-Activated Cell Sorting (BACS™) microbubble technology represents a breakthrough in cell separation. The core of each microbubble is a hollow microsphere which allows researchers to leverage the power of gravity for quick and gentle removal of captured cells from a complex biological mixture. With gravity as the driving force powering microbubble-based separation, microbubbles provide consistent results and eliminate the need for extraneous equipment and consumables like magnets and columns, while enabling reliable separations regardless of sample volume or container shape, or size. Akadeum has taken technology that traditionally requires various equipment and consumables to perform and put it into a single container for self-separation that is exceptionally gentle on delicate cells.

Because Tregs express CD4, a sample preparation step using Akadeum’s CD4+ T Cell Microbubbles allows for fast, easy, and exceptionally gentle enrichment prior to cell sorting. The unwanted cells are first labeled using Akadeum’s optimized biotinylated antibody cocktail. The microbubbles are then added to the sample, where they seek out and bind to the labeled cells, floating them to the surface for removal. An optional centrifugation step pellets the unlabeled CD4+ T cells, and the microbubbles, bubble-bound cells, and supernatant are removed using vacuum aspiration – leaving a highly enriched population of happy, healthy CD4+ T cells for downstream use and analysis.

Learn more about using Microbubbles for Treg Isolation 

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