*Innovations in Cell Research and Therapy*

functioning of a signaling pathway in particular. When studying STAT phosphorylation, three essential factors must be taken into account: (A) the cell lineage in which STAT phosphorylation will be studied. In the study of PID patients, it is a limitation since it is usual that not all cell lineages are present; (B) choosing the correct recombinant cytokine is crucial as cytokine receptors are expressed differently in distinct lymphocyte lineages; (C) and selecting the appropriate antibodies against the STAT phosphorylation sites [30]. These studies can also be performed by WB, which is a very sensitive but more time-consuming technique and delays results [38].

STAT phosphorylation staining protocol:


(The same protocol is useful for the assay of different STATs. Mitogens/ cytokines and antibodies may change depending on the STAT you are studying. Note that different commercial kits can be used to assess phospho-proteins, so the protocol can be slightly different in each case).

**41**

**Figure 8.**

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

*4.2.1 STAT1 phosphorylation for the diagnosis of chronic mucocutaneous* 

STAT1 is a transcription factor which in humans can be activated by several ligands such as interferon alpha (IFNα), interferon gamma (IFNγ), epidermal growth factor (EGF), platelet derived growth factor (PDGF) or interleukin 6 (IL-6). Mutations in the STAT1 molecule can be gain of function (GOF) or loss of function (LOF). Both of them can cause PID with different phenotypes and

**STAT1 GOF dominant mutations** were first discovered in patients with chronic mucocutaneous candidiasis (CMC). This disease typically shows symptoms such as persistent infections of the skin, oral/genital mucosa and nails caused mainly by *Candida albicans*. CMC may very often result from primary immunodeficiency. To assess the presence of a STAT1 GOF defect, isolated PBMCs are stimulated *in vitro* with INFγ. In order to know if there is a gain of function, we perform different experimental conditions, such as unstimulated, stimulated with INFγ, and with INFγ plus *Staurosporine*. *Staurosporine* is an inhibitor for Ca2+/calmodulin-dependent protein kinase II inhibiting STAT1 phosphorylation [40]. The use of *Staurosporine* is justified to clarify the difference between control subjects (STAT1 WT alleles) and the CMC patients (STAT1 GOF alleles). STAT1 phosphorylation is assessed in CD14+ monocytes [41]. In a healthy donor, we can observe that STAT1 phosphorylation is reduced with the addition of Staurosporine, while in a patient with STAT1 GOF, phosphorylation is maintained despite the use of Staurosporine (**Figure 8**) [42]. Regarding **STAT1 LOF autosomic recessive mutations**, there are two genetic deficiencies that compromise the response to interferons type I and III. On the one hand, there is autosomal recessive deficiency (RA), partial or even complete STAT1. These types of defects are diagnosed in patients with an increase in intracellular or viral bacterial infections, with impaired responses to IFN α, β, γ, and IL27. In complete recessive form, there is a very low response to anti-viral and antimycotical treatments. On the other hand, partial STAT1 deficiency can also be autosomal dominant (AD); phenotypically causes impaired IFNγ responses and patients suffer with selective intracellular bacterial diseases such as Mendelian susceptibility to mycobacterial diseases (MSMD). To assess if there is a STAT1 LOF, isolated PBMCs

*The STAT1 phosphorylation and de-phosphorylation study for the diagnosis of function gain mutations (GOF) is evaluated in CD14+ monocytes. In a healthy donor (left), we can see that STAT1 phosphorylation is reduced with the addition of staurosporine (gray) while in the patient (right), phosphorylation is maintained despite* 

*the use of staurosporine (black: no staurosporine, white: control of isotypes).*

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

*candidiasis*

symptoms [39].

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

*Innovations in Cell Research and Therapy*

interest.

3.Wash with 2 mL of PBS.

tions per minute (rpm).

and in the dark.

antibody.

cytometry.

STAT phosphorylation staining protocol:

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

6.Incubate for 20 min at 37°C and in the dark.

8.Incubate for 15 min at 37°C and in the dark.

14.Incubate for 30 min at RT and in the dark.

15.Wash with 1 mL of PBS with FBS at 10%.

protocol can be slightly different in each case).

7.Lyse the red cells using 1 mL of lyse and fix solution.

10.Wash with 1 mL of PBS with fetal bovine serum (FBS) at 10%.

11.Discard the PBS + FBS 10% after 5 min of centrifugation at 1500 rpm.

12.Add 500 μL of permeabilization buffer and incubate 30 min on ice or 4°C

13.Add the appropriate volume of intracellular anti-phosphorylated-STAT

16.Discard the PBS + FBS 10% after 5 min of centrifugation at 1500 rpm.

(The same protocol is useful for the assay of different STATs. Mitogens/ cytokines and antibodies may change depending on the STAT you are studying. Note that different commercial kits can be used to assess phospho-proteins, so the

17.Add 100 μL of PBS + FBS 10% and the sample is ready to be acquired by flow

5.Add 100 μL of appropriate stimuli.

functioning of a signaling pathway in particular. When studying STAT phosphorylation, three essential factors must be taken into account: (A) the cell lineage in which STAT phosphorylation will be studied. In the study of PID patients, it is a limitation since it is usual that not all cell lineages are present; (B) choosing the correct recombinant cytokine is crucial as cytokine receptors are expressed differently in distinct lymphocyte lineages; (C) and selecting the appropriate antibodies against the STAT phosphorylation sites [30]. These studies can also be performed by WB, which is a

1.Stain 100 μL of heparin whole blood with appropriate mix of antibodies of

9.Discard the lyse and fix solution after 5 min of centrifugation at 1500 revolu-

very sensitive but more time-consuming technique and delays results [38].

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

**40**
