T Cell Separation Using the CD45 Marker

What is CD45?

The protein antigens present on the surface of a cell play a role in its function. Since some of these functions are specific to an individual cell type, the presence of those antigens helps to identify a given cell. 

CD45 is an antigen found on the surface of all nucleated hematopoietic cells. A given cell is said to be “CD45 positive” if an isoform of the CD45 antigen is present on its surface; the cell is said to be “CD45 negative” if no isoforms of CD45 are detected on its surface. In this way, the expression of CD45 is commonly used as a “white blood cell marker.”  

The CD45 antigen is a type of protein tyrosine phosphatase (PTP), which is a group of biomolecules that regulate cellular processes such as cell growth and differentiation. A variety of isoforms of CD45 can be expressed depending on the type, developmental stage, and function of the blood cell in question.

CD45 T cells

T cells are a type of white blood cell that play a variety of critical roles in the body’s immune system. T cells are coated with T cell Receptors (TCRs) that recognize and bind to antigens presented to them by other immune cells called antigen presenting cells (APCs). These foreign antigens are acquired by the APCs from foreign cells and pathogens. Binding to a foreign antigen can activate a naïve T cell to become a cytotoxic CD8+ T cell that can kill pathogens, a helper CD4+ T cell that secretes cell signaling molecules called cytokines that summon other immune cells (such as macrophages or cytotoxic T cells) to the area, or regulatory T cells that prevent other T cells from attacking healthy host cells.

CD45 on the surface of T cells is critical to the proper function of the TCRs. CD45 helps to regulate TCR costimulation by controlling the activation state of another protein called Lck that increases T cell activity. This allows for fine-tuning of T cell to proliferation, differentiation, or production of effector cytokines.

The expression of CD45 on the surface of all white blood cells is strong, and CD45 antigens can make up as much as 10% of the proteins present on the surface of T cells. For a given T cell, the specific isoform of CD45 and its expression level can be used to identify which T cell subset the cell belongs to, its state of maturation, and whether it has previously encountered its cognate antigen. 

So far, six different isoforms of CD45 have been identified. These isoforms of CD45 are categorized based on the exons contained in their mRNA; in humans, the six CD45 isoforms are RA, RO, RB, RAB, RBC, and RABC:

• CD45 isoforms contain different combinations of exons in the CD45 gene. In addition, while they play similar regulatory roles in T cell function, the particular combination of the exons expressed changes along with the activation and differentiation state of the T cell in question.
• CD45RA is typically found on naïve T cells that are mature but have not yet been activated in the periphery.
• CD45RO is most commonly expressed on the surface of memory T cells, as well as other lymphocytes such as B cells. CD45RO facilitates signaling-mediated activation of T.
• CD45RB plays a key role in TCR signaling. As T cells progress from naïve to memory T cells, CD45RB expression is downregulated. Thus, the expression level of CD45RB in relation to other CD45 isoforms can be used to identify naive, effector, and memory stages of T cell development. 

CD45RA vs CD45RO

CD45RA is a long isoform of CD45 typically found on naïve T cells that have yet to encounter antigens. Meanwhile, CD45RO is a shorter isoform and is typically expressed on memory T cells that can more quickly respond to previously encountered antigens, both in relation to effector cytokine production and proliferation. 

In general, the number of T cells expressing CD45RA in a person’s immune system decreases with age, while the number of T cells expressing CD45RO increases with age. That said, certain subgroups of T cells have been found to co-express both CD45RA and CD45RO, particularly T cells actively undergoing cellular division.

CD45 as a Marker for Lymphocyte Cell Separation

To study the characteristics and function of cells, those cells must first be isolated from the rest of the cells in a blood sample, leukopak, or peripheral blood mononuclear cells (PBMCs). Cell surface protein antigens can easily serve to distinguish between cell types and cell subsets in a sample, and thus allow targeting undesired cells for removal.

For example, because subsets of T cells express differing levels of CD45 isoforms, the presence or absence of those isoforms can be used to differentiate wanted T cell subsets from unwanted cells.

CD45 for T cell Isolation with Akadeum’s Microbubbles

Developed by Akadeum Life Sciences, Buoyancy-Activated Cell Sorting (BACS) with microbubbles is a promising, innovative method for removing unwanted cells from whole blood, PBMCs, or leukopaks to deliver high-purity, highly viable populations of specific cell types or cell subsets for downstream analysis or research applications. Akadeum’s BACS products offer an affordable, easy-to-use alternative to traditional cell sorting procedures that require extensive training or expensive equipment to conduct. Moreover, cell separation with BACS can quickly process high volumes of samples, while maintaining the health and functionality of cells in the sample.

First, biotinylated antibodies specific for surface antigens on the unwanted cell population(s) are mixed into a biological sample, and the antibodies bind to their target antigens. Then, streptavidin-coated microbubbles are introduced to the cell suspension, and the streptavidin on these microbubbles binds with high affinity to the biotin on the antibodies. The buoyant microbubbles and their unwanted cellular cargo then float gently to the surface of the sample to be removed, leaving a high-purity sample consisting of the desired cell population(s) at the bottom of the sample tube—untouched, healthy, and ready for further analysis or a variety of downstream applications.

The CD45R isoform, also known as B220, is a highly expressed protein antigen expressed on the surface of most mouse B cells. It can be used to remove B cells from blood, PBMCs, or other sources of immune cells. The biotinylated anti-CD45R is used in the antibody cocktails in Akadeum’s Mouse T cell Isolation Kit, Mouse Naive T cell Isolation Kit, and Mouse Naive CD4+ T cell Isolation Kit to remove B cells while leaving the T cells, which express different CD45 isoforms, untouched.