*4.2.3 STAT5 phosphorylation for the diagnosis of severe combined immunodeficiency (SCID)*

X-linked severe combined immunodeficiency (X-SCID) is an inherited disorder of the immune system caused by inborn errors in the gene IL2RG, which encodes interleukin IL-2 receptor common γ chain. The γc is a transmembrane protein shared by different cytokine receptors IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In the absence of an effective γc function, early lymphoid progenitor cells are not able to respond properly to these cytokine signals leading low or absence of T and NK cells and hypogammaglobulinemia despite a normal B cell count [47]. T−B+NK− phenotype is characteristic for X-SCID and JAK3 deficiencies. T−B+NK+ SCID include IL-7Rα, CD3δ, CD3ε, and CD3ζ deficiencies. X-SCID represents close to half of all SCID patients. Patients with X-SCID are prone to severe, recurrent, and persistent infections caused usually in their first 6 months of life. The disease is lethal within the first 2 years of life, unless they undergo hematopoietic stem cell transplantation (HSCT) or gene therapy [48, 49].

In addition to the more deleterious mutations, which cause the most severe phenotype of the disease, some other several patients with a milder form of combined immunodeficiency, often referred to as "leaky" or "hypomorphic" SCID. Most of these cases of "leaky" SCIDs are due to hypomorphic mutations. Due to their poorly defined clinical and immunological phenotype, patients with the atypical forms are diagnosed later in childhood or even in adulthood [50].

**43**

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

See STAT phosphorylation staining protocol in Section 4.2.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition characterized by persistent macrophage/lymphocyte activation and a multisystemic hyperinflammatory syndrome. The clinical diagnosis of HLH is challenging and requires a composite of several findings. According to the updated criteria of the Histiocyte Society [51], the diagnosis is currently established by fulfilling 5 of 8 clinical and laboratory criteria, or the finding of a genetic defect consistent with HLH. Defects associated with HLH are generally associated with abnormal NK cell function. The characterization of these patients requires genetic and functional studies, among the latter are the study of cytotoxic capacity and degranulation. In *cytotoxicity assays*, we measure the functional capacity of natural killer cells. In this assay, PBMCs or purified NK cell are co-incubated at different ratios with a target HLA-null cell lines (usually K562) known to be sensitive to NK cell-mediated cytotoxicity [52]. After an incubation period, dead target cells are identified by nucleic acid staining, which specifically penetrates the dead cells and is measured by flow cytometry [53]. Chromium-51 (51Cr) release assays are also commonly used for accurate and precise quantification of cytotoxicity. The main disadvantages of this method are the use of radioactivity, which can be hazardous to health, and are impractical and costly due to the short half-life of 51Cr and to the need for radio-

cells/mL concentration in a final vol-

cells/mL concentration in a final volume

cells/mL.

cells/mL.

6.Make a serial dilution of the patient's PBMCs in FACS tubes, adding the same

**4.3 NK cell cytotoxicity and degranulation assay evaluation**

logical safety training and authorization requirements [54].

3.Incubate both cell types separately at 37°C over night.

4.Count and adjust patient PBMCs at 2.5 × 106

concentration of K562 cell line in each tube.

5.Count and adjust K562 cells at 0.2 ×106

Cytotoxicity assay protocol:

ume of 5 mL of RPMI.

of 5 mL of RPMI.

1.Count and adjust patient PBMCs at 106

2.Count and adjust K562 cells at 2 × 105

A study of expression of the common gamma chain (CD132), expressed broadly on T and B lymphocytes, NK cells, monocytes, and granulocytes, can be performed by flow cytometry [3]. However, as mentioned above, the presence of protein does not rule out a possible functional defect. It is therefore advisable to evaluate the signaling of the IL2-IL2RG-JAK3-STAT5 pathway, studying the phosphorylation of STAT5. For this purpose, whole blood is stimulated with IL-2 and STAT5 phosphorylation is evaluated in lymphocytes. It should be noted that a patient with X-SCID will not have T lymphocytes or NK cells, therefore the test should be performed on B lymphocytes and it is important to consider the inclusion of specific B cell lineage markers. STAT5 phosphorylation is clearly reduced in patients with a common gamma chain deficiency compared to a healthy control [39]. This assay can be performed under suspicion of mutations in the following genes: IL2RG, JAK3,

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

STAT5 or IL-7Rα.

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

*Innovations in Cell Research and Therapy*

with a healthy donor [43].

phenotypes [44].

are stimulated with INFγ and phosphorylation is assessed in CD14+ monocytes. In these cases, STAT1 phosphorylation in monocytes is clearly diminished comparing

Hiper IgE Syndrome (HIES) is a PID defined as an association of atopy in a context of very high serum IgE levels, characteristic bacterial and fungal diseases, low-level clinical and biological inflammation, and various non-hematopoietic developmental manifestations. Somewhat arbitrarily, three disorders were successively put forward as the underlying cause of HIES: autosomal dominant (AD) STAT3 deficiency, the only disorder corresponding to the original definition of HIES, and autosomal recessive (AR) DOCK8 and PGM3 deficiencies, in which atopy and high serum IgE levels occur in a context of manifestations not seen in patients with typical HIES. Indeed, these three disorders disrupt different molecular pathways, affect different cell types, and underlie different clinical

Phenotype tests discussed in previous chapters, demonstrating low number of Th17 lymphocytes, may guide in-depth study of a defect in STAT3 signaling. These are the analysis of the ability to produce IL-17 by stimulation with PMA and ionomicin, adding a Golgi Plug such as Brefeldin and subsequent intracellular staining against IL17. And the STAT3 phosphorylation test in lymphocytes. Whole blood is stimulated with IL-6 and/or IL-21 and intracellular staining is performed to evaluate STAT3 phosphorylation. Different conditions are needed, such as: not stimulated, stimulated, and both with STAT3 antibodies or with an isotype control to have internal controls. Phosphorylation is clearly decreased in a patient with

X-linked severe combined immunodeficiency (X-SCID) is an inherited disorder of the immune system caused by inborn errors in the gene IL2RG, which encodes interleukin IL-2 receptor common γ chain. The γc is a transmembrane protein shared by different cytokine receptors IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In the absence of an effective γc function, early lymphoid progenitor cells are not able to respond properly to these cytokine signals leading low or absence of T and NK cells and hypogammaglobulinemia despite a normal B cell count [47]. T−B+NK− phenotype is characteristic for X-SCID and JAK3 deficiencies. T−B+NK+ SCID include IL-7Rα, CD3δ, CD3ε, and CD3ζ deficiencies. X-SCID represents close to half of all SCID patients. Patients with X-SCID are prone to severe, recurrent, and persistent infections caused usually in their first 6 months of life. The disease is lethal within the first 2 years of life, unless they undergo hematopoietic stem cell transplantation

In addition to the more deleterious mutations, which cause the most severe phenotype of the disease, some other several patients with a milder form of combined immunodeficiency, often referred to as "leaky" or "hypomorphic" SCID. Most of these cases of "leaky" SCIDs are due to hypomorphic mutations. Due to their poorly defined clinical and immunological phenotype, patients with the atypical forms are

See STAT phosphorylation staining protocol in Section 4.2.

STAT3 deficiency compared to a healthy control [45, 46].

*immunodeficiency (SCID)*

(HSCT) or gene therapy [48, 49].

diagnosed later in childhood or even in adulthood [50].

See STAT phosphorylation staining protocol in Section 4.2.

*4.2.3 STAT5 phosphorylation for the diagnosis of severe combined* 

*4.2.2 STAT3 phosphorylation for the diagnosis of Hiper IgE Syndrome (HIES)*

**42**

A study of expression of the common gamma chain (CD132), expressed broadly on T and B lymphocytes, NK cells, monocytes, and granulocytes, can be performed by flow cytometry [3]. However, as mentioned above, the presence of protein does not rule out a possible functional defect. It is therefore advisable to evaluate the signaling of the IL2-IL2RG-JAK3-STAT5 pathway, studying the phosphorylation of STAT5. For this purpose, whole blood is stimulated with IL-2 and STAT5 phosphorylation is evaluated in lymphocytes. It should be noted that a patient with X-SCID will not have T lymphocytes or NK cells, therefore the test should be performed on B lymphocytes and it is important to consider the inclusion of specific B cell lineage markers. STAT5 phosphorylation is clearly reduced in patients with a common gamma chain deficiency compared to a healthy control [39]. This assay can be performed under suspicion of mutations in the following genes: IL2RG, JAK3, STAT5 or IL-7Rα.

See STAT phosphorylation staining protocol in Section 4.2.
