**3. Analysis of surface molecules in PIDs**

#### **3.1 Evaluation of molecules which are constitutively expressed on cell surface**

In the diagnosis of suspicious patients for PID, flow cytometry is frequently applied to detect specific molecules which are expressed on specific subset of immune cells in clinical immunology research laboratory [2, 3]. It is used for immunophenotyping as well as in the detection of specific protein expression in cells. In the evaluation of constitutively expressed proteins on cell surface, activation with specific stimulus is not required. CD40 and CD55 deficiencies are the examples which are described in detail in Section 3.1.1. and 3.1.2 for the surface protein expression analysis in PIDs.

In the staining of surface proteins, fixation and permeabilization steps are not needed. Therefore staining protocol is easier and faster than intracellular staining of the proteins which is described in Section 4. Following staining protocol is used to detect surface protein expressions in PIDs:


## *3.1.1 CD40 deficiency in hyper IgM syndrome*

CD40 is a costimulatory molecule which is expressed on antigen presenting cells such as B cells, macrophages and dendritic cells. CD40 interacts with CD40L on T cells in GC zones and is activated in the maturation of B cells and isotype switching [33, 34]. Similar to CD40L deficiency, CD40 deficiency is investigated for suspicious Hyper IgM syndromes. Decreased or unfunctional CD40 expression on B lymphocyte as well as CD40L expression defects on T cells in suspicious patients for Hyper IgM syndrome is related to disease occurrence [35, 36]. See the Section 3.1. for the staining protocol.

### *3.1.2 CD55 expression in CHAPLE syndrome*

Decay-accelerating factor (DAF) or CD55 is an inhibitor molecule of complement system and it is related to various diseases and a recently described PID which is named as (CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE) CHAPLE syndrome. Because CD55 acts as an inhibitor of complement system, low or loss of expressions due to mutations in its encoding gene, complement system is more active in patients than healthy individuals [37–39] (see the Section 3.1. for the staining protocol).

#### **3.2 Analysis of the expression of induced surface proteins in PIDs**

#### *3.2.1 CD40L expression in T lymphocytes in hyper IgM syndrome*

CD40L, also known as CD154, is expressed on T cells and responsible for the interaction with CD40 which is expressed on antigen presenting cells such as B cells. CD40L is a member of TNF-receptor superfamily and its interaction with CD40 on B cells is associated with Ig class switching, affinity maturation and GC formation. In most of the patients with CD40L deficiency, loss or decreased CD40L protein expression on T cells are associated with increased levels of soluble IgM levels and decreased IgG and IgA levels are investigated [35, 36]. Expression of CD40L protein on T cell surface is very low and increased by activation using Phorbol Myristate Acetate (PMA) and ionomycin inducing transcriptional activity of NFAT and AP-1 transcription factors in T cells following T cell receptor stimulation. Following 3 hours of activation of PBMCs, CD69 which is an early activation marker and CD40L expression are detected on T cell surface (**Figure 4**). Staining protocol of CD40L and CD69 on CD3+ CD8- T cells are as in below:


*Flow Cytometric Approach in the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.96004*

#### **Figure 4.**

*Gating strategy for CD40L and CD69 expression on CD3+ CD8- T cells in unstimulated and stimulated samples from a healthy control (top) and a patient (below).*


## *3.2.2 CD70 expression*

CD27/CD70 signaling pathway is significant for the immune response to Epstein–Barr virus (EBV) infections. CD27 is expressed on T lymphocytes as well as B lymphocytes and whereas its ligand, CD70, is limited to induced T and B lymphocytes and dendritic cells. CD27-CD70 signaling is responsible for T cell survival, Treg activity, B cell differentiation and proliferation. Due to CD27-CD70 partnership in immune response against to EBV, similar clinical characteristics are monitored in patients with CD27 and CD70 deficiencies [40–42]. EBV-associated lymphoproliferative disorder, lymphoma, hypogammaglobulinemia and autoimmune manifestations are generalized clinical symptoms in both deficiencies [41, 42]. Therefore, analyzing of CD27 and CD70 proteins in PBMCs using flow cytometry due to its rapid and quantitative analysis guide to clinicians as a first step molecular diagnosis of patients with these clinical manifestations before sequencing. **Figure 5** shows the gating strategy for CD70 staining. Staining protocol for CD27 is as in Section 3.1.

#### **Figure 5.**

*A representative image of CD70 expression on CD19+ B lymphocyte in a healthy control and a patient.*

CD70 activation and staining protocol is as below:

	- Peripheral blood mononuclear cells (PBMCs) are separated by ficoll density gradient protocol from 1 to 2 ml of whole blood in tube with EDTA
	- Wash PBMCs with Phosphate Buffer Saline (PBS) buffer, centrifuge at 300 g for 5 min and discard the supernatant and resuspend the cells with serum free media
	- Prepare two flasks for each sample to analyze unstimulated and stimulated samples
	- Put the appropriate number of cells to culture flask and add 2,5 ug/ml phytohemagglutinin (PHA) in the completed culture media
	- Incubate the cells in humidified incubator for 72 hours
	- After 72 hours add appropriate volume of IL-2 to the cells
	- At the day of 8, wash the cells with PBS
	- Centrifuge at 500 g for 5 min and discard the supernatant
	- Add appropriate volume of CD70 antibody and incubate for 30 min at room temperature
	- Wash the cells with PBS and Centrifuge at 500 g for 5 min and discard the supernatant
	- Resuspend the cells with 300 ul PBS and analyze at flow cytometer.

*Flow Cytometric Approach in the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.96004*
