*3.1.3 FOXP3 expression in IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome*

A further example of intracellular protein testing by flow cytometry in patients with PIDs involves screening for patients with possible immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (the gene encoding FOXP3 protein is located on the X chromosome, therefore only males are affected by the disease while females are carriers). This diagnosis is confirmed in male patients whose CD4+CD25+ regulatory T cells demonstrate absence or decreased expression of FOXP3 [28].

See the intracellular staining protocol in Section 3.1*.*

### **3.2 Immediate early activation markers**

The activation of T lymphocytes is a complex and finely regulated process of events resulting in the expression of cytokine receptors, the production and secretion of cytokines and increased cell surface molecules that facilitate the immune response. The most important activation molecules expressed on T lymphocytes can be classified as early activation markers, such as CD69 and CD25, and late activation markers, such as CD62L and HLA-DR. Additionally, very late activation markers, such as VLA-1 have been described, playing a role in lymphocyte adhesion and extravasation [29].

**CD69** is a signaling membrane glycoprotein involved in the induction of T cell proliferation. CD69 is expressed at very low levels T cells at rest in PBMCs (<5– 10%) and is one of the first evaluable activation markers, being rapidly increased in T cells within the first hour of TCR. Expression of CD69 peaks at 16–24 h and then decreases. The inability to upregulate CD69 following TCR activation may be associated with T cell dysfunction [30].

**CD40 ligand (CD40L)**, also called CD154, is a member of the TNF-receptor superfamily, primarily expressed on activated T cells, and functions as a co-stimulatory molecule by binding CD40 which is constitutively expressed on antigen presenting cells (APCs). The CD40L-CD40 ligation is a fundamental interaction for B lymphocytes proliferation, formation of germinal centers, Ig class switch, antibodies maturation, and B lymphocyte differentiation to plasmatic cell and to memory B lymphocyte. Deficiency of CD154 is an X-linked disorder that only males are affected and females are the carriers, also known as Hyper IgM syndrome [31]. Expression of CD40L on resting T cells from healthy donors is very low (<1%) and is quickly upregulated within 1–2 h after TCR stimulation via the transcription factors NFAT and AP-1. CD40L expression peaks near 6 h after stimulation and declines by 16–24 h (**Figure 6**) [32].

In summary, CD69 and CD40L are both rapidly induced following T cell activation and both exert important functions in T cell biology. Expressions of these markers have both been shown to be altered in patients with different PIDs.

Immediate early activation markers staining protocol:


**37**

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

3.Stain with appropriate mix of antibodies of interest.

*(left) and stimulated (right) where a normal expression of CD40L and CD69 is observed.*

7.Discard the lysing solution after 5 min of centrifugation at 1500 rpm.

*The expression of CD40 ligand is studied by stimulating PBMCs with PMA plus ionomycin for 4 h, and then the expression of a cell activation marker such as CD69 is analyzed. Dotplots of PMBCs without stimulation* 

10.Add 100 μL of PBS and the sample is ready to be acquired by flow cytometry.

(The same protocol is useful for all the different activation marker techniques,

The assessment of lymphocyte function typically refers to the study of T cell function through cell proliferation in response to polyclonal nonspecific and specific stimuli; however, this concept is more extensive and refers to any method that evaluates any of the various effectors or regulatory aspects of subset of lymphocytes [33]. As we have seen previously, these studies are of particular importance when the functional impact of a mutation is unknown; in such cases, flow cytometry provides a number of different immune function assessment techniques, including evaluation of specific immune

9.Discard the PBS after 5 min of centrifugation at 1500 rpm.

changing mitogens, and incubation time depending on each case).

cell line functions, cell activation, proliferation, and cytokine production.

Lymphocyte phenotype studies should be accompanied by laboratory tests assessing the functional immunity of T cells when a primary immunodeficiency is

4.Incubate for 20 min at RT and in the dark.

6.Incubate for 10 min at RT and in the dark.

8.Wash with 2 mL of PBS.

**Figure 6.**

**4. Evaluation of cellular function**

**4.1 Lymphocyte proliferation assay**

5.Lyse the red cells using 2 mL of lysing solution.

*DOI: http://dx.doi.org/10.5772/intechopen.89743*

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.89743*

### **Figure 6.**

*Innovations in Cell Research and Therapy*

*enteropathy X-linked) syndrome*

**3.2 Immediate early activation markers**

associated with T cell dysfunction [30].

and extravasation [29].

with genetic mutation evaluation, and it is characterized by high negative predictive value. The lack of intracellular perforin expression in NK cells is characteristic of

A further example of intracellular protein testing by flow cytometry in patients with PIDs involves screening for patients with possible immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (the gene encoding FOXP3 protein is located on the X chromosome, therefore only males are affected by the disease while females are carriers). This diagnosis is confirmed in male patients whose CD4+CD25+ regulatory T cells demonstrate absence or decreased expression of FOXP3 [28].

The activation of T lymphocytes is a complex and finely regulated process of events resulting in the expression of cytokine receptors, the production and secretion of cytokines and increased cell surface molecules that facilitate the immune response. The most important activation molecules expressed on T lymphocytes can be classified as early activation markers, such as CD69 and CD25, and late activation markers, such as CD62L and HLA-DR. Additionally, very late activation markers, such as VLA-1 have been described, playing a role in lymphocyte adhesion

**CD69** is a signaling membrane glycoprotein involved in the induction of T cell proliferation. CD69 is expressed at very low levels T cells at rest in PBMCs (<5– 10%) and is one of the first evaluable activation markers, being rapidly increased in T cells within the first hour of TCR. Expression of CD69 peaks at 16–24 h and then decreases. The inability to upregulate CD69 following TCR activation may be

**CD40 ligand (CD40L)**, also called CD154, is a member of the TNF-receptor super-

family, primarily expressed on activated T cells, and functions as a co-stimulatory molecule by binding CD40 which is constitutively expressed on antigen presenting cells (APCs). The CD40L-CD40 ligation is a fundamental interaction for B lymphocytes proliferation, formation of germinal centers, Ig class switch, antibodies maturation, and B lymphocyte differentiation to plasmatic cell and to memory B lymphocyte. Deficiency of CD154 is an X-linked disorder that only males are affected and females are the carriers, also known as Hyper IgM syndrome [31]. Expression of CD40L on resting T cells from healthy donors is very low (<1%) and is quickly upregulated within 1–2 h after TCR stimulation via the transcription factors NFAT and AP-1. CD40L expression peaks near 6 h after stimulation and declines by 16–24 h (**Figure 6**) [32]. In summary, CD69 and CD40L are both rapidly induced following T cell activa-

tion and both exert important functions in T cell biology. Expressions of these markers have both been shown to be altered in patients with different PIDs.

1.Stimulate heparin whole blood or peripheral blood mononuclear cells

2.Incubate the appropriate time depending on the activation marker of your

Immediate early activation markers staining protocol:

(PBMCs) with the appropriate stimuli or mitogen.

familial hemophagocytic lymphohistiocytosis type 2 [26, 27]. See the intracellular staining protocol in Section 3.1.

See the intracellular staining protocol in Section 3.1*.*

*3.1.3 FOXP3 expression in IPEX (immunodysregulation polyendocrinopathy* 

**36**

interest.

*The expression of CD40 ligand is studied by stimulating PBMCs with PMA plus ionomycin for 4 h, and then the expression of a cell activation marker such as CD69 is analyzed. Dotplots of PMBCs without stimulation (left) and stimulated (right) where a normal expression of CD40L and CD69 is observed.*


(The same protocol is useful for all the different activation marker techniques, changing mitogens, and incubation time depending on each case).
