Flow cytometry for CD4+ and CD8+ T cells

What are CD antigens?

Cluster of Differentiation (CD) molecules are found on the surface of leukocytes (white blood cells). They are useful for the identification of different cell types, and mediate a wide variety of immune functions. To date, over 350 CD molecules have been characterized through a series of international forums known as the Human Leukocyte Differentiation Antigens (HLDA) workshops. The first HLDA workshop was held in 1982, with the aim of bringing clarity to the considerable number of antibodies against leukocyte markers which were in existence at that time. With so many new antibodies being produced, it was difficult to know which of these recognized the same molecule. To resolve this, multiple research groups carried out blind testing of the antibodies, and the statistical procedure of cluster analysis was then used to give a CD designation to antibodies thought to be detecting the same molecule, as well as to the molecule itself. The most recent HLDA workshop, the tenth, was held in 2014.

What are Major Histocompatibility Complex molecules?

Major Histocompatibility Complex (MHC) molecules are glycoproteins whose role is to bind peptide fragments and display them on the cell surface for recognition by T-lymphocytes, stimulating an immune response when necessary. MHC molecules can be divided into two classes:

MHC class I

MHC class II

Found on almost every cell in the body Found on Antigen Presenting Cells (APC) such as macrophages, dendritic cells and B-lymphocytes
Bind endogenous antigens which originate from the cytoplasm; these include self-proteins (to which T-lymphocytes do not normally react) as well as foreign proteins that are produced within the cell (e.g. proteins required for viral replication) Bind exogenous antigens which originate extracellularly (e.g. from bacteria)
Following degradation of the antigen, peptide fragments are transported to the endoplasmic reticulum where they bind to MHC class I proteins before moving via the Golgi apparatus to the cell surface The APC degrades the pathogenic protein into peptide fragments, and sequesters these into the endosome for binding to MHC class II proteins before transportation to the cell surface
Present antigen to cytotoxic T-lymphocytes Present antigen to helper T-lymphocytes
The presence of foreign or over-abundant self-antigens targets the cell for destruction The presence of foreign antigens stimulates antibody production, and attracts immune cells to the area of infection

What is the significance of CD4 and CD8?

T-lymphocytes originate from hematopoietic stem cells in the bone marrow, then move to the thymus for development. During this process, they mature into two major subsets – CD4-positive helper T-lymphocytes, and CD8-positive cytotoxic T-lymphocytes – which then enter the peripheral blood and tissues. Upon encountering an antigen, further cellular differentiation occurs to produce effector T-lymphocytes with a wide range of specific functions.
T-lymphocytes are only capable of recognizing an antigen when it is presented by MHC molecules on the surface of another cell. Specific T-cell receptors on the surface of the T-lymphocyte are used to bind the antigen associated with the MHC molecules, and the subsequent cellular response is mediated by signaling events initiated by the T-cell receptor complex and the CD4 or CD8 co-receptor. CD4 positive cells recognize antigens which are presented by MHC class II molecules, while CD8 positive cells recognize antigens that are presented by MHC class I.

CD4 and CD8 for flow cytometry

Flow cytometric cell sorting of T-lymphocytes (CD3-positive) into CD4-positive and CD8-positive cell populations is commonly employed within multicolor T-lymphocyte marker panels. The inclusion of antibodies against additional CD molecules allows further characterization of each sub-population through quantification and evaluation of different T-cell subsets. For example, depletion of a specific cellular population can be used as a marker of disease progression, while an increase in another population may be indicative of a response to treatment.

Fluorometric detection for flow cytometry

Antibody binding within a flow cytometry experiment typically relies on fluorometric detection and, as the number of readouts increases, it is often beneficial to use directly labeled primary antibodies for detection. These offer several key advantages over labeled secondary antibodies:

Non-specific binding is avoided since secondary antibodies are not used
Multiplexing is possible with antibodies from the same species
Faster since there is no secondary antibody incubation step and therefore fewer wash steps
Data quality is improved through assay simplification
However, despite the benefits of direct detection, many immunoassays still rely on the use of labeled secondary antibodies. The main reason for this is that direct labeling of primary antibodies with detection moieties such as fluorescent dyes can be a complicated and time-consuming process, which requires specialist knowledge.

Lightning-Link® for direct antibody labeling

Lightning-Link® from Expedeon is an innovative technology that enables quick and easy antibody labeling. To produce the conjugate, the antibody is simply pipetted into a vial of lyophilized Lightning-Link® mixture containing the label of interest, incubated, and is then ready for use. With no washing or separation steps, antibody recovery is 100%. Furthermore, the technology is fully scalable, allowing easy transfer from R&D to manufacturing.

The Lightning-Link® conjugation process.

The Lightning-Link® conjugation process

The Lightning-Link® product range includes almost 40 fluorescent labels, for which we have compiled the maximal absorption and emission wavelengths, Stokes shift data and extinction co-efficients into our fluorescence table, to allow easy selection of a diverse panel of reagents.
For further information, why not download our free guide or watch our webinar? And if you have any questions, please contact us.