**5. Analysis of cellular functions of immune cells**

#### **5.1 Cell proliferation**

Severe combined immunodeficiencies (CIDs) including T-B + NK-, T-B-NK+, T-B-NK- and T-B + NK+ and/or isolated T cell deficiencies are severe forms of PIDs due to important roles of T lymphocytes to combat directly or indirectly protein and viral antigens [55]. T lymphocytes have specific subsets to achieve their superior roles on specific antigenic determinant. Their deficiencies due to specific molecular defects affect their activation, receptor editing, functions and proliferative capacity cause critically ill disease phenotype. They need to re-regulate their receptors and proliferate to expand agent-specific clones such an army to combat during various specific-infections. Therefore detecting cell proliferation is significant for the diagnosis and/or the course of the disease. Non-radioactive cell tracking dyes such as CFSE (carboxyfluorescein succinimidyl ester) has been started to use for the assessment of cell proliferation in flow cytometry. CFSE is a non-fluorescent dye and becomes permeable through its two acetate groups and passing through the cell membrane. After entering the cells, following the separation of acetate groups via esterases, it becomes fluorescent and its permeability is decreased. Succinimidyl group of CFSE reacts with amino groups of mostly from lysine residues of intracellular molecules such as cytoskeletal proteins and forms stable covalent bonds. In

#### **Figure 12.**

*Comparison of CD3+ T lymphocyte proliferation between a patient with SCID and a healthy control individual. Normal proliferation in the healthy control sample (top) and loss of CD3+ T lymphocyte proliferation in the patient with SCID (below).*

each cell division its fluorescent density is decreased and this decrease in cells is evaluated in flow cytometry [56–58]. Severely affected lymphocyte proliferation in a patient with severe combined immunodeficiency is shown in **Figure 12**. See the CFSE cell staining protocol in Section 5.1.1.
