Introduction of easily Ignored Thymus Lymphocytes - CD4 and CD8 Co-expressing Cells

Experimenters who have done flow cytometry experiments are usually familiar with CD4 and CD8 indicators. Detection of CD4+ T cell and CD8+ T cell typing is one of the most common experiments in flow cytometry.

Typically, we detect CD3 first to circle total T lymphocytes, and then use CD4 antibodies and CD8 antibodies respectively to distinguish CD4+ T lymphocytes (helper or regulatory T cells) and CD8+ T lymphocytes (cytotoxic T cells).

Figure 1. Neonatal mouse spleen cells

The above figure shows the results of CD3-PE, CD4-FITC, CD8-APC trichromatic flow cytometry of Neonatal mouse spleen cells. As can be seen from the figure 1, most of the cells in the spleen of Neonatal mice are non-co-expressed singleton cells, of which about 2/3 are CD4+CD8- cells and 1/3 are CD4-CD8+ cells.

In mature T lymphocytes, CD4 and CD8 are mutually exclusive indicators, so it is unlikely to make mistakes in stages of early panel design, adjustment compensation and data analysis. Under normal conditions, the two indicators do not co-express. However, thymic lymphocytes, the CD4+CD8+ co-expression cells are often ignored by flow cytometry novices.

T cells differentiate, develop and become mature in thymus. Thymocytes are T cells in different differentiation stages. Lymphocyte-like progenitor cells differentiated in the bone marrow enter the thymus through the blood circulation, where they complete the development of T cells and become mature T cells. Then these mature T cells enter the peripheral lymphatic organs through the blood circulation.

Figure 2. The development process of T cells (Picture source: Medical Immunology)

The development of T cells will go through the stages of lymphoid progenitor cells → progenitor T cells → pre-T cells → immature T cells → mature T cells, and the antigens expressed by T cells in different stages are different. According to the expression of CD4 and CD8, T cells in the thymus can be classified into double negative cells (DN cells), double positive cells (DP cells) and single positive cells (SP cells).

1. Pre-T cells and cells in preceding stages are all DN cells that express neither CD4 nor CD8. After induction, the pre-T cells proliferated rapidly and began to express CD4 and CD8.

2. After proliferation, the cells enter the DP cell stage. At this point, cells co-express CD4 and CD8 and stop proliferating.

3. After positive selection, Immature T cells further differentiate into SP cells, namely CD4+ or CD8+ single positive cells. About two thirds of cells differentiate into CD4+ single positive cells and one third differentiate into CD8+ single positive cells, and 95% of the cells undergo apoptosis during this process.

4. After negative selection, SP cells become mature T cells and enter the peripheral immune organs through the blood circulation.

Figure 3. Neonatal mouse thymus cells

The above figure shows the flow cytometry results of CD4-FITC and CD8-APC in the thymus of neonatal mice. It can be seen from the figure that in the thymus of newborn mice, most cells co-express CD4 and CD8.